Article

Cell Biochem Biophys (2008) 52:5984
DOI 10.1007/s12013-008-9027-2
REVIEW PAPER
Polyunsaturated Fatty Acid Modulation of Voltage-Gated Ion

Channels
Linda M. Boland Michelle M. Drzewiecki
Published online: 2 October 2008

Humana Press Inc. 2008
Abstract Arachidonic acid (AA) was found to inhibit the

function of whole-cell voltage-gated (VG) calcium currents
nearly 16 years ago. There are now numerous examples
demonstrating that AA and other polyunsaturated fatty
acids (PUFAs) modulate the function of VG ion channels,
primarily in neurons and muscle cells. We will review and
extract some common features about the modulation by
PUFAs of VG calcium, sodium, and potassium channels
and discuss the impact of this modulation on the excitability of neurons and cardiac myocytes. We will describe
the fatty acid nature of the membrane, how fatty acids
become available to function as modulators of VG channels, and the physiologic importance of this type of
modulation. We will review the evidence for molecular
mechanisms and assess our current understanding of the
structural basis for modulation. With guidance from
research on the structure of fatty acid binding proteins, the
role of lipids in gating mechanosensitive (MS) channels,
and the impact of membrane lipid composition on membrane-embedded proteins, we will highlight some avenues
for future investigations.
Keywords Ion transport Voltage-gated channel
Phospholipase A2 Monoacylglycerol-lipase
Diacylglycerol-lipase Arachidonic acid
Docosahexaenoic acid Polyunsaturated fatty acid
LTP Kv4 Membrane stretch Voltage sensor
L. M. Boland (&) M. M. Drzewiecki

Department of Biology, The University of Richmond, B-100
Gottwald Science Center, Richmond, VA 23173, USA
e-mail: lboland@richmond.edu
Introduction
As integral membrane proteins, voltage-gated (VG) ion
channels are nestled within a lipid barrier that separates
two aqueous compartments. It has become increasingly
important to consider the function of the plasma membrane
as a dynamic reservoir of lipid signaling molecules. Specific lipid messengers can be enzymatically cleaved from
the plasma membrane upon stimulation by neurotransmitters, neurotrophic factors, and cytokines. Membrane lipid
composition and the availability of diffusible lipids thus
change on the time scale of cellular signaling events.
Combined with long-term changes in membrane composition due to dietary intake, disease, development, or aging,
the short-term availability of lipids such as PIP2, fatty acid
esters of CoA, lysophosphatidic acid (LPA), and free fatty
acids, allows the membrane habitat to be a source of
change in cellular activity.
Voltage-gated ion channels are ideally suited to receive
the messages encoded by lipid molecules. The transmembrane domains of channels are surrounded by the precursors
of lipid signaling and channel gating requires considerable
movement of the protein within this hydrophobic environment. Lipid signals that affect VG ion channel function will
transiently alter the duration and magnitude of the ionic
fluxes across the membrane, providing a direct mechanism
for lipids to regulate excitability, synaptic transmission,
rhythmicity, and contractility. Thus, in neurons and cardiac
muscle, lipid messengers add to the diversity of mechanisms for fine-tuning the electrical properties of the cell. In
this article, we will present and evaluate the evidence for
direct modulation of VG ion channels by polyunsaturated
fatty acids (PUFAs). We have known about this form of ion
channel modulation for at least 16 years now [13] and
there are numerous examples for a variety of VG channels.
60
However, our understanding of the molecular mechanisms

and structural basis of this modulation is limited. We will
discuss current research to localize molecular sites of action
and general strategies used to assess the mechanisms of
PUFA modulation. To provide some clues for future
investigations, we will also review how fatty acids bind to
other proteins, how they promote the opening of mechanosensitive (MS) channels, and how the lipid composition
of the membrane impacts the dynamic proteins that are
embedded within it. We first consider the source and
structural nature of the partners in this form of ion channel
modulation and review evidence for the physiologic significance of PUFA modulation of VG channels.
EET
HETE
diHETE
HDHA
PLA2
Lyso-PAF
DHA
Lyso-PAF
transferase
PAF
PAF acetyl hydrolase
AA
5-LOX
Leukotrienes
PGH2
Other
prostaglandins
TXA2
Lyso-PAF
Ethanolamine
or Choline
Inositol
Sources of PUFAs as Modulatory Agents
The Fatty Acid Nature of the Plasma Membrane

PLC
A lipid bilayer with embedded proteins is the basic

membrane architecture. The lipids are diverse in structure,
comprising glycerophospholipids, cholesterol, and sphingolipids, yet they all share the common feature of polar
regions of the lipid facing the aqueous compartments and
non-polar regions comprising the interior region of the
bilayer. Built upon the three carbon backbone of glycerol,
membrane glycerophospholipids (hereafter, phospholipids)
have a polar phosphate group (phosphatidic acid) or
phosphate plus a polar, hydrophilic group in the sn-3
position (ethanolamine, choline, inositol, or serine) and
two fatty acid tails (Fig. 1). The fatty acid tail esterified to
the first carbon of the glycerol backbone (sn-1 position) is
generally a saturated fatty acid, lacking carboncarbon
double bonds. The tail esterified to the second carbon (sn-2
position) is generally an unsaturated fatty acid with one
(monounsaturated) or more (polyunsaturated) carboncarbon double bonds (Fig. 1). Phosphatidylcholines and
phosphatidylethanolamines are zwitterionic and carry one
negative and one positive charge; whereas phosphatidic
acid, phosphatidylserine, and phosphatidylinositol each
carry a single negative charge. Negative charges in the
head groups of membrane lipids are important for VG
channel function, based on recent evidence from the KvAP
bacterial VG channel [4]. Furthermore, phospholipids are
not symmetrically distributed within the bilayer of the
plasma membrane. For example, in the mammalian plasma
membrane, phosphatidylcholine is more concentrated
in the outer leaflet than in the inner leaflet, whereas
phosphatidylserine and phosphatidylinositol are more
concentrated in the inner leaflet than in the outer one [5, 6].
This asymmetry may impact the nature of lipid interactions
with charged residues in transmembrane regions of channel proteins.
PLD
AA
IP3
DAG
PA
AEA
AA-Lyso PA
PLA2
COX-2
12-LOX
2-AG
MAG lipase or FAAH
Lyso PA 12-OHPGE2
AEA
EA
glycerol
AA
DAG lipase
Fig. 1 Biochemical pathways for release of PUFAs from membrane

phospholipids. Schematic illustrations of the pathways that elicit
arachidonic acid (AA) and docosahexaenoic acid (DHA) from membrane phospholipids. Actions of the enzyme phospholipase A2 are
shown in the top panel. Actions of the enzymes phospholipase
phospholipase C (PLC) and phospholipase D (PLD) are shown in the
bottom panel. To illustrate the phospholipid structure, the three carbon
backbone of glycerol is depicted as a solid horizontal line and the polar
head groups are depicted as boxes. Note that anandamide (AEA) is also
released by hydrolysis of phospholipid precursors. Additional details
about the biochemical pathways for lipid signaling and the structure
and function of the various metabolites are described in recent reviews
[20, 22, 182, 183]. Abbreviations: PAF = platelet activating factor,
EET = epoxy-eicosatrienoic acid, HETE = hydroxyeicosatetraenoic
acid, 5-LOX = 5-lipoxygenase, PGH2 = prostaglandin H2, TXA2 =
thromboxane A2, IP3 = inositol triphosphate, DAG = 1, 2 diacylglycerol, 2-AG = 2-arachidonoylglycerol, MAG = monoacylglycerol,
PA = phosphatidic acid, PLA2 = phospholipase A2, 12-LOX = 12lipoxygenase, COX2 = cyclo-oxygenase 2, 12-OH-AEA = 12hydroxyl anandamide, PGE2 = prostaglandin E2
Among the lipid constituents of the membrane, the fatty

acid nature of the phosphoplipids is an important characteristic that influences both membrane structure and lipid
signaling pathways. The length of the fatty acyl tail and the
degree of saturation influences lateral mobility and fluidity
of lipids and proteins [7], as well as the composition of the
fatty acid reservoir that can be utilized, upon demand, for
cellular signaling. Most naturally occurring unsaturated
fatty acids have double bonds in the cis configuration in

which adjacent carbon atoms are on the same side of the
double bond. Fatty acids with multiple double bonds have
highly flexible structures, as determined by molecular
dynamic simulations [8, 9]. As we shall review, the presence and degree of unsaturation of the fatty acyl chain are
generally important for PUFA modulation of VG channels.
This implies that fatty acids with more flexibility are better
able to modulate VG channel function, although a structural explanation for this is not yet clear.
The major fatty acid constituents of mammalian membrane phospholipids are long chain x-3 and x-6 PUFAs. In
x-3 fatty acids, the first carboncarbon double bond is
located between the third and fourth carbon atom counting
from the methyl end of the fatty acid whereas in x-6 fatty
acids, the first double bond is located between the sixth and
seventh carbon atom. Mammals, including humans, can
synthesize saturated and some monounsaturated fatty acids
[10]. But certain long chain x-3 and x-6 PUFAs are
considered essential since they must be obtained from the
diet (fish oils and vegetable oils, respectively) or synthesized from fatty acid precursors [11]. In humans, the x-3
fatty acids, eicosapentaenoic acid (EPA, C20:5) and
docosahexaenoic acid (DHA; C22:6), are the major products of desaturation and elongation of the precursor,
a-linolenic acid (ALA, C18:3). The x-6 fatty acid, arachidonic acid (AA; 20:4) is the major product of the
precursor, linoleic acid (LA; C18:2). AA and DHA are
major constituents at the sn-2 position of mammalian
plasma membrane phospholipids (Figure 1).
Retinal and neuronal membranes are particularly enriched in AA and DHA [12] which may seem surprising
since neurons lack the desaturase and elongase enzymes
needed to synthesize these PUFAs from their fatty acid
precursors. However, endothelial cells and astrocytes
manufacture AA and DHA and release them into neural
tissue [13]. As will be discussed later, x-3 fatty acids
(EPA, DHA) are protective for cardiac arrhythmia and their
presence in cardiac myocyte plasma membranes is
increased by the consumption of fish oil [1417] or certain
fish, such as cod and salmon [18]. Among other roles, DHA
appears to be required for normal development and function of the retina [19]. Since PUFAs are abundant in the
plasma membranes of excitable cells, the membrane
phospholipids can serve as a ready supply of AA and DHA
for various cellular functions [20].
Release of PUFAs from the Membrane
Membrane release of AA is perhaps the best studied
mechanism for regulating the availability of any PUFA. The
activation of certain G-protein coupled metabotropic
receptors initiates an enzyme-mediated hydrolysis of
61
membrane phospholipids, which liberates a non-esterified

free fatty acid. Three different phospholipase enzymes are
known to catalyze hydrolysis at different sites of action
within the phospholipid and yield different products (see
Fig. 1). Phospholipase A2 (PLA2) can release PUFAs in a
single step by catalyzing the hydrolysis of the sn-2 acylester
bond of phospholipids, yielding AA and a lysophospholipid
(Fig. 1, top). Mammalian cells contain several different
PLA2 enzymes, each capable of releasing AA (or DHA)
from the sn-2 position of membrane phospholipids [21].
Activation of phospholipase C (PLC) or phospholipase
D (PLD) also produces precursors of AA. The fatty acid
can then be released by the actions of a second enzyme,
a diacylglycerol- or monoacylglycerol-lipase (Fig. 1,
bottom).
Once released, free AA has several possible fates. AA
may diffuse within the membrane and has been proposed to
diffuse outside the cell, acting as a retrograde messenger
[22, 23], although its limited solubility in aqueous solution
would be expected to minimize its presence outside a lipid
environment. AA can be further metabolized by a variety
of enzymes (cyclooxygenases, lipoxygenases, and epoxygenases) to produce prostaglandins, leukotrienes, and
thromboxanes, which collectively are called eicosanoids
(Fig. 1, top [24]). The eicosanoids have many modulatory
functions of their own, including roles as pro-inflammatory
agents [25, 26], but, with rare exceptions, they are not
modifiers of VG channel activity. EPA and DHA oxidation
also yield prostaglandins and leukotrienes, which differ in
structure from the AA metabolites and have opposing, antiinflammatory functions [24]. The oxidized products of
EPA and DHA have not been tested as modulators of VG
channels. However, as we shall discuss, there are many
examples showing that AA modulates the activities of VG
ion channels without a requirement for further metabolism
of the fatty acid. Another fate of AA release from the
membrane is the downstream activation of protein kinases
and signal transduction cascades. While many channels are
targets of phosphorylation by certain kinases, this mechanism is also not a general explanation for the effects of AA
on VG channels.
Several other products of phospholipid hydrolysis share
structural similarity with PUFAs and when tested, are often
found to mimic the effect of AA on the modulation of VG
ion channels. The endogenous ligands of the G-protein
coupled cannabinoid receptors [27], 2-arachidonoylglyercol (2-AG) and anandamide (arachidonoylethanolamide or
AEA), have been isolated from brain and other tissues and
can be produced locally by a series of enzymatic modifications of membrane phospholipid precursors (see Fig. 1,
top [20]). Although their best known functions are neurobehavioral, through cannabinoid receptor activation, 2-AG
and AEA may also directly affect cellular excitability by
62
the modulation of VG channel function, in the absence of

further metabolism. One physiologic role for this modulation is exemplified by AEA-mediated inhibition of
sensory neuron Na? current, which may contribute to this
lipids analgesic properties [28, 29].
Structural Features of Voltage-Gated Channels and the

Impact of Lipids
Voltage-gated ion channels are those specialized for
channel opening as a function of changes in membrane
potential. Several related channel types are activated by
membrane voltage changes in coordination with another
signal and will also be included in this discussion.
In all of these cases, the channel contains a number of
transmembrane domains which interact closely with the
lipid environment of the membrane, accessory proteins,
and domains of the channel-forming protein itself.
Channels Gated Directly by Membrane Voltage
Changes
Channels opened by membrane depolarization include
calcium (Cav), sodium (Nav), and potassium (Kv) channels.
The structure of a VG channel can be studied by reviewing
the membrane topology of a Kv channel [30, 31]. The
channel is comprised of four (4) pore-forming a subunits,
each of which has six (6) transmembrane segments (S1S6)
and intracellular amino- and carboxy-termini. A segment
between the fifth (S5) and sixth (S6) transmembrane
domains contributes to the extracellular side of the pore
region (P) while part of S6 and the S4S5 linker contribute
to the inner mouth of the pore. The a subunits of Cav and
Nav channels are comprised of four domains, each of which
resembles a single Kv subunit. So, one a subunit is all that
is needed for a functional Cav or Nav channel. The poreforming subunits of VG channels may also co-associate
with one or more accessory subunits, some of which may
be necessary or ubiquitous partners. Some of the accessory
subunits are cytoplasmic and others have one or more
transmembrane domains. This adds to the complexity of
the channel structure and the possible sites for interaction
with membrane lipids. Accessory subunits have a general
role to enhance membrane trafficking of the synthesized
protein and often affect channel gating [32].
The most important feature that distinguishes VG
channels from other channel types is that the VG channel
senses a transmembrane voltage change and transduces
this into mechanical rearrangements of the channel protein
so that the conducting region opens a pore. The major
constituent of the voltage sensor is the S4 region, an
unusual transmembrane region in which positively
charged arginine or lysine residues are regularly spaced

from one another by two hydrophobic residues [31, 32].
S4 moves in response to transmembrane voltage changes
and its motion induces the S4S5 linker helices to open
(or close) the S6 inner helices which gate the pore. When
the pore is in the open conformation, it is surprising to see
that two of four conserved arginine residues in S4 face the
inhospitable lipid environment [33]. The S4 helix, together with the C-terminal half of the S3 helix, is part of a
helix-turn-helix structure referred to as a voltage-sensor
paddle and this is held together by hydrophobic interactions [34] and has a number of possible sites for lipid
protein interaction [33].
The pore of VG ion channels has a hydrophilic interior
and most VG channels have high ionic selectivity, which is
due to highly conserved residues in the pore region [34,
35]. While the opening and closing of the pore is determined by changes in membrane potential, depolarization
may also lead to inactivation of the channel, a condition
which renders the channel no longer capable of conducting
ions, even in the presence of favorable voltage differences.
For example, some K? channels are inactivated by an Ntype ball and chain mechanism, where a tethered,
cytoplasmic N-terminal domain blocks the pore after
channel opening. This traps the channel in an open but
inactivated state. Hydrophobic residues in the N-terminus
are essential for interactions within the channel and electrostatic interactions mediated by charged residues in the
N-terminal region must interact directly with the pore to
occlude conduction [31, 36].
Channels Gated by Voltage in Combination with
Regulatory Stimuli
The Cav, Nav, and Kv channels are all activated by depolarizing stimuli and other stimuli are not necessary for full
opening of the pore region, under physiologic conditions.
However, some channels are regulated by both membrane
voltage and internal calcium (BK) or membrane voltage
and cyclic nucleotides (HCN). BK channels, also referred
to as Maxi-K or Slo channels, are big conductance, voltage- and Ca2?-activated K? (KCa) channels. The BK a
subunit has a core structure that is similar to that of the Kv
channels except it contains an extra transmembrane domain
(S0) and so the N-terminus of the subunit is externally
located. The BK a subunit also contains a large C-terminus
with additional hydrophobic domains (S7S10). Within the
S7S10 domains are sites for Ca2? binding including
conserved aspartate residues within a Ca2? bowl region in
S10 and acidic residues within the RCK domain (regulator
of K? conductance), a region between the C-terminus of S6
and C-terminus of S7. BK a subunits assemble as tetramers
to form channels.
The BK channels contain an S4 region and voltage is the

primary stimulus for activation because it occurs even at
nM concentrations of Ca2? [37] and even when the Ca2?
bowl and RCK domains are deleted [38]. Ca2? and voltage
appear to act independently to open the channel, but within
the physiologic range of these stimuli, regulation likely
occurs by allosteric mechanisms [37].
The structure of the bacterial Ca2? activated MthK
channel, crystallized in the Ca2?-bound and open state
[34], shows that each of the four subunits of the channel
contributes two RCK domains which form a gating ring.
Ca2? binding displaces the RCK domains, expanding the
gating ring [39]. A linker between S6 and RCK is influenced by the Ca2? binding, which forces the S6 inner
helices to open a pore. These types of molecular rearrangements may also contribute to the Ca2?-dependent
gating mechanism of BK channels [40].
Accessory b subunits of BK channels have two transmembrane domains and co-association of a and b subunits
makes the channel more sensitive to Ca2? than for a subunits alone [41, 42]. Recent evidence suggests that the b1
subunit (but not b2) enhances Ca2? sensitivity by altering
conformational changes in S4 [37]. Also, the b2 or b3
subunits (but not b1) introduce a cytoplasmic, N-terminal
form of inactivation [37]. The interaction between a and b
subunits may also be important in the sensitivity of BK
channels to AA modulation [43].
Other Ca2?-activated K? channels with an intermediate
(IK1, also called SK4) or small conductance (SK1SK3)
are gated by Ca2? binding to calmodulin, which is constitutively associated with the C-terminal end of the
channel [44, 45]. SK channels are more sensitive to Ca2?
than BK channels but they are not VG, despite a topology
similar to Kv channels and a modified S4 region with 3
instead of 7 (Kv) positive charges [32, 44, 46]. Due to
relative lack of information about SK channel sensitivity to
modulation by PUFAs and their lack of voltage-dependent
gating, we will limit our discussion (see Structural
Basis) to one key example (IK1) which may inform us
about the molecular localization of PUFA effects on ion
channels [47].
Hyperpolarization-activated cyclic nucleotide-gated
(HCN) channels form a family of non-selective cation
channels which share the basic membrane topology of Kv
channels. HCN channels are particularly important in the
pacemaking activity of spontaneously active cells in the
sinoatrial node of the heart and thalamocortical relay
neurons [48]. Despite the fact that they contain a positively
charged S4 region, HCN channels are activated by membrane hyperpolarization rather than depolarization [49].
Cysteine accessibility approaches have shown that in HCN
channels, membrane depolarization causes an outward
translocation of S4 which closes the gate, whereas
63
hyperpolarization causes inward movement which opens

the gate [5052]. Thus, the movement of S4 in HCN
channels has the same dependence on the polarity of the
voltage change as for Kv channels although it has an
opposite effect on gating. Cyclic AMP binds to a C-terminus cyclic nucleotide binding domain (CNBD) on each
of the four subunits of the HCN channels. Binding of
cAMP introduces a conformational change in the C linker,
a region that connects the CNBD to the pore, which
relieves a resting inhibition of the gate. This adjusts the
sensitivity of the voltage sensor, much as Ca2? does for the
BK channels.
Examples of PUFA Modulation of Voltage-Gated

Channels
Ca2? Channels
In general, PUFAs inhibit VG Ca2? currents and often
modulate the time course of inactivation. The low voltageactivated T-type channels are the most rapidly inactivating
channels among the diverse family of Ca2? channels.
Extracellular application of AA or DHA inhibited the native
T-type currents recorded in neuroblastoma cells [53, 54]
and cells from the adrenal medulla [55]. AEA mimicked the
inhibitory effect of AA on T-type currents [53] and, in
addition, accelerated the time course of inactivation [54]. A
3-fold enhancement in the rate of recovery from inactivation was also observed in the medullary cells [55].
Neurons and muscle cells express several Ca2? channel
types and thus it is useful to study recombinant channels
expressed in isolation from other channel types. This is
especially important since toxins, blockers, or agonists are
often used in whole-cell recordings to isolate a pharmacological component of the mixed Ca2? current and the
impact of these agents on the mechanism(s) of PUFA
modulation is not known. Chemin and colleagues, in
important and thorough work, expressed each of the three
cloned T-type channels, Cav3.1, 3.2, and 3.3, and observed
inhibitory modulation by AA or AEA [54, 56]. In each
case, inhibition of the peak inward Ca2? current was
accompanied by a left-ward shift in the steady-state inactivation curve and an acceleration of inactivation kinetics.
These features, plus a slowed recovery from inactivation,
were also observed by Talavera and colleagues [57].
Modulation in cell-free inside-out patches supports a direct
action of the lipid modulator [56]. Evidence also supports a
dependence of the modulation on fatty acid chain length
and degree of unsaturation [56]. An interesting result is
that, compared to wild-type Cav3 channels, mutant channels with slow inactivation had an increased affinity for AA
modulation. This supports the notion that structures that
64
determine inactivation gating are important in AA modulation [56]. A lack of state-dependence for the AA
modulation also suggested that transitions between inactivated states, rather than open-inactivated transitions may
be sensitive to AA. Overall, these results strengthen the
evidence for a modulation of Ca2? channel inactivation by
PUFAs.
Inhibition of neuronal N-type Ca2? currents by AA has
been well studied at the whole-cell and single-channel level
by Rittenhouse and colleagues. Using cell-attached patches
from rat superior cervical ganglion (SCG) neurons, AA
inhibited N-type current by increasing the probability of
null sweeps which is explained as an increased occupancy
of closed gating states [58]. In related work using the same
cell type, AEA also inhibited the N-type current [59].
There are no studies on PUFA modulation of recombinant
N-type (Cav2.2) channels nor investigation of the impact of
the accessory subunits on this form of channel modulation.
However, important experiments that advance our understanding of native signaling pathways showed that the AAmediated modulation of SCG N-type current required an
M1 muscarinic receptor coupled to the Gq-type of G-protein and both PLC and PLA2 [60, 61].
Polyunsaturated fatty acids inhibit the dihydropyridinesensitive L-type Ca2? channel current studied in neurons
and muscle cells. DHA inhibited the L-type current in
cardiac myocytes [62] and tracheal smooth muscle cells
[63]. PUFAs also inhibited the high-voltage activated Ca2?
current in Muller cells of the retina and a component of this
current may be L-type [64]. In neonatal and adult ventricular myocytes, EPA and other PUFAs reduced plasma
membrane L-type current and elicited a small, but significant, left-ward shift of the steady-state inactivation curve
[65]. Monounsaturated and saturated fatty acids did not
inhibit this current [65]. A rare finding is that PUFAs
increased whole-cell Ca2? current in cardiac myocytes [3].
In this case, however, the pharmacological identity of the
affected current was not known and the structural specificity of fatty acid modulation differed from other studies.
Both long chain unsaturated and, surprisingly, saturated
fatty acids, caused a multifold increase of Ca2? current
amplitude although short-chain fatty acids and fatty acid
esters were not effective [3]. There are no reports on PUFA
modulation of recombinant L-type (Cav1.x) channels nor
tests of the impact of the b, c, and d accessory subunits.
Na? Channels
Evidence for direct effects of PUFAs on VG Na? channel
function is best supported by research done in muscle cells.
There has been limited exploration of PUFA modulation of
neuronal Na? currents. PUFAs inhibited Na? currents
studied in cardiac [6568], skeletal [69], and smooth [70]
muscle cells and in most cases, induced a left-ward shift of

the steady-state inactivation curve [65, 66, 69]. Acceleration of the time course of fast inactivation was rarely
reported [69]. A right-ward shift of the activation curve
[66] and a reduction of Na? channel gating currents,
without an impact on single-channel conductance, suggest
a change in the availability of Na? channels [69]. This
explains the important observation that PUFAs increased
the threshold for action potential firing in cardiac myocytes
[67, 68]. As was found for Ca2? channel currents, saturated
and monounsaturated fatty acids were ineffective modulators of Na? currents [66]. Consistent with their effects on
whole-cell Na? currents, PUFAs also inhibited human
cardiac Nav1.5 channels [65, 7173], induced a left-ward
shift in the inactivation curve [65, 71, 72], and delayed the
recovery from inactivation [65, 72]. A curious effect of
monounsaturated fatty acids on recombinant Na? channels
was prevented upon b subunit co-expression [65] and will
be discussed under Structural Basis.
Potassium Channels
Some of the same features noted for PUFA inhibition of
Ca2? and Na? channels are also found for K? channels,
despite the incredible diversity of K? channel functions.
Most often, PUFAs and AEA inhibit K? currents (Table 3)
although there are some examples of enhanced K? currents
[43, 7476]. K? current inhibition by PUFAs is generally
mimicked by the non-metabolizable eicosatetraynoic acid
(ETYA) [7783], not blocked by inhibitors of AA oxidative metabolism [43, 82, 84, 85], and not mimicked by
saturated and monounsaturated fatty acids [43, 81, 86]. An
exception is a case in which enhancement of KCNQ1
(KvLQT1 or Kv7.1) current by DHA was mimicked by the
saturated fatty acid, lauric acid (C14:0) [74]. Both rapidly
inactivating and slowly inactivating or non-inactivating K?
currents in native cells are modulated by AA or other
PUFAs. There is no cellular specificity to the modulation;
channels natively expressed in neurons, microglia, endocrine cells, and cardiac myocytes are all modulated.
From the research on recombinant K? channels, there
are two cases in which modulatory effects of PUFAs were
altered by the co-expression of a and b subunits of the
channel. Kinetic modulation of inactivation in the fastinactivating Kv4 channels required the co-expression of
KChIP subunits, whereas the inhibition of the peak outward current did not [83]. The co-association of Kv4 a and
b subunits re-capitulates the likely molecular structure of
native A-type channels and a similar kinetic effect of AA
on the A-type current in hippocampal neurons was previously reported [87]. AA also reversibly increased the
amplitude of BK currents but an additional effect,
slowing of the inactivation time course, depended upon
co-expression with b2 or b3 subunits [43]. These results

suggest an explanation for modest differences that are
sometimes reported for recombinant channels and their
native cellular currents (Tables 13) since native channels
may include one or more types of accessory subunits.
In a seminal paper on channel modulation, AA or AEA
caused a potent and rapid inactivation of Kv3.1 channels
which otherwise lack N-type inactivation [88]. AA (4 lM)
was equally effective when applied to the outside or the
inside surface of isolated macropatches and the effects on
inactivation were reversible. AA did not compete with the
open-channel blocker TEA, so the fatty acid may work by
creating an altered conformational state rather than
inducing the same structural effects of open-state inactivation. However, the story becomes even more interesting
because a different lipid, PIP2, selectively removed the fast
inactivation of Kv1.1/b1.1 or AA-modulated Kv3.4 currents. Once the inactivation was removed, it could be
restored again by application of AA or AEA. The PIP2
effect on Kv1.1/b1.1 depended on the lipids negatively
charged head group, as if the lipid could interfere with
positive charges in the inactivation domain. Indeed, PIP2
inhibited the inactivating function of synthetic ball peptide
applied directly to the inside-out macropatch. Although
PIP2 may act by interfering with the endogenous inactivation mechanism, AA does not appear to confer
inactivation by acting like an inactivation domain since the
effect depended on the species of the permeating ion. It
was suggested, therefore, that AA-induced inactivation is a
different mechanism than open-channel block [88]. This
research further supports a direct role for PUFAs in altering
the conformational states of channel proteins.
In summary, the examples of PUFA modulation of VG
channels share a number of features. PUFAs generally
inhibit currents, although HCN channel gating is facilitated
by PUFAs [89]. The effects tend to be reversible. Ionic
current inhibition is often accompanied by a left-ward shift
in inactivation gating, and often speeds up the inactivation
time course. Different types of VG channels have unique
characteristics with respect to their sensitivity to membrane
voltage and gating kinetics, among other features. Changes
in these properties will impact how these channels participate in their important roles in action potential firing,
transmitter release, synchronous activity, muscle contraction, and regulation of the heartbeat, among other
functions. We will consider several important physiologic
outcomes of PUFA modulation of VG channels.
Physiologic Roles for PUFA Modulation of VG
Channels
Lipid messengers released from membrane phospholipids
have many possible physiologic and pathological roles in
65
nervous tissue [22] and cardiac muscle [65] and it is worth

asking, what functions might be attributed specifically to
PUFA modulation of VG channels?
In nervous tissue, a role for PUFA modulation of VG ion
channels is suggested in synaptic plasticity, development,
and as an outcome of ischemia, seizures, and perhaps other
pathological conditions [19, 20]. Over the past 20 years,
perhaps the strongest evidence has accumulated to support
a role for PUFA modulation of fast-inactivating Kv4
channels in long-term potentiation (LTP) in the hippocampal CA1 region. As a lasting enhancement of synaptic
communication, LTP is often considered a cellular mechanism for learning and memory. Early studies in individual
neurons showed that application of AA inhibits the neuronal A-type current, now ascribed to Kv4 channels plus
their KChIP subunits [82, 87, 90]. An example is shown in
Fig. 2 for Kv4/KChIP channels where application of AA
inhibited the peak K? current and induced a faster inactivation time course (Fig. 2b).
Kv4 channels are ideally positioned to influence LTP.
These channels and their KChIP accessory subunits are
highly localized to post-synaptic somatodendritic membranes [9193] where they regulate the integration of
high frequency trains of synaptic activity and suppress
enhanced excitability [80, 94]. The importance of Kv4
channels can also be tested in a computer-simulated hippocampal neuron in which a reduction in the availability of
Kv4 channels results in an increased firing rate (Fig. 2c).
This result is consistent with the enhancement of action
potential firing in hippocampal neurons upon experimental
inhibition of A-type currents [92, 94, 95]. Therefore,
PUFA inhibition of Kv4 channels may be permissive for
LTP and several additional lines of evidence support this
hypothesis.
In population recordings done in hippocampal slices, the
application of phospholipase blockers inhibited CA1 region
LTP [96]. Importantly, the blockers were required to be
present simultaneously with a high frequency stimulation
(tetanus) of Schaffer collateral afferent fibers, suggesting
that the pre-synaptic tetanus induces the release of PUFAs
which is necessary for LTP[96]. Block of LTP by the PLA2
inhibitor was reversed by direct application of DHA or AA
during, but not after, the tetanus [97], indicating that
exogenous PUFAs were sufficient to trigger LTP formation. Maintenance of LTP was not affected by AA [97], but
a co-requirement for the tetanus and PUFAs suggests that
post-synaptic activity is important. PUFAs also induced
LTP elicited without a tetanus in a low Mg2? solution
(which relieves block of the NMDA-type of post-synaptic
glutamate receptors) [98]. This suggests that NMDA
receptors play a role specifically (e.g., through Ca2? influx)
or generally (e.g., through post-synaptic depolarization) in
the mechanism of PUFA effects on LTP.
Inhibition
Effect on current or channel
AA
DHA
Neonatal rat SCG neurons
Guinea pig cardiac ventricular

myocytes
HEK 293
HEK 293
HEK 293
TsA201
Cav 3.2
Cav 3.1, 3.2, and 3.3
Cav 3.1
Cav 3.1, 3.2, and 3.3
AEA and AA but saturated

fatty acids are not effective
AA
AEA
AA
Modulatory agent
[55]
Voltage-dependent inhibition
Inhibition
Inhibition
Inhibition and left shift of current-voltage curve
Inhibition and leftward shift of inactivation curve
Inhibition, acceleration of inactivation, slowed recovery from inactivation,

and hyperpolarizing shift in steady-state inactivation
Inhibition and acceleration of inactivation kinetics
Abbreviations: PUFA = polyunsaturated fatty acid, AA = arachidonic acid, DHA = docosahexaenoic acid, EPA = eicosapentaenoic acid, ETYA = 5,8,11,14-eicosatetraynoic acid,
AEA = arachidonoylethanolamide (anandamide), SCG = superior cervical ganglion, Alternative channel names: Cav3.1 is a1G, Cav3.2 is a1H, Cav3.3 is a1I
[56]
[57]
[54]
[187]
References
[65]
[62]
[58, 60]
[63]
[59]
Inhibition and increased probability of null sweeps [58, 60]

in single-channel recordings
Inhibition and acceleration of recovery from

inactivation
Inhibition. Acceleration of inactivation kinetics, and left-ward shift of inactivation curve
Slow inactivation mutants were also tested and had increased affinity for inhibition by AA
Examples of modulation of whole-cell and recombinant Ca2? channel currents
Expression system
[53]
[186]
[64]
[3]
[2]
[1]
References
Inhibition and acceleration of inactivation kinetics [54]
Inhibition
Enhancement, but inhibition with higher

concentrations
Neonatal rat heart cells and adult rat Variety of PUFAs (Saturated and monounsaturated Inhibition. Left-shifted inactivation curve
ventricular myocytes
fatty acids were not effective)
EPA and DHA
Guinea pig tracheal myocytes
AEA
DHA and AA
Bovine adrenal zona fasciculata
Rat SCG neurons
AEA
NG10815 neuroblastoma
AA
AA (Oleic and myristic acid were less effective)
NG10815 neuroblastoma
Neonatal rat SCG neurons
AA
Long chain unsaturated and saturated fatty acids

Enhancement without kinetic modulation
(Short-chain fatty acids and fatty acid esters were
not effective)
AA (mimicked by ETYA)
Inhibition without effect on voltage-dependence
AA (Saturated and trans-unsaturated fatty acids were Inhibition

not effective)
EPA and DHA
Modulatory agent
Rat osteoblasts
Recombinant T-type
Native L-type currents
Native N-type currents
Native T-type currents
Acutely isolated retinal Muller glia
Smooth muscle of rabbit ileum
Neonatal rat cardiac myocytes
Low-voltage activated and

high-voltage activated
current
current
Guinea pig cardiac myocytes
2?
Cellular preparation
Whole-cell Ca2? current
Whole-cell Ca
Whole-cell Ca
2?
Native currents, mixed types
Table 1 Fatty acid modulation of voltage-gated calcium channels
66
HEK293t
* This is the human cardiac Nav a subunit: SCN5A = H1 = Nav1.5
** Slow inactivation mutants had increased affinity for inhibition by PUFAs
Inhibition. Left-shifted inactivation curve
Inhibition. Voltage-dependent and hyperpolarizing

shift of inactivation curve
Inhibition, but becomes resistant to the PUFA within

one min
Enhanced activation at low test potentials but

inhibited peak activation. Left-shifted activation
curve (prevented by a cyclooxygenase inhibitor
and not mimicked by ETYA) and inactivation
curves (not impacted by a cyclooxygenase
inhibitor and not mimicked by ETYA)
Inhibition. Depolarizing shift of activation curve but

left-shifted inactivation curve. Fatty acids that
inhibited the current also showed an increase in
membrane fluidity as measured by fluorescence
anisotropy
Inhibition. Enhanced kinetics of fast inactivation.

Hyperpolarizing shift of inactivation curve.
Reduced gating currents. No change in singlechannel conductance
Inhibition of current and reduced binding of

radiolabeled toxin by EPA
Increased threshold for action potential firing
Inhibition. Reduced channel availability

and hyperpolarizing shift in activation curve
Inhibition.** Left-shifted inactivation curve.

Enhanced inactivation and delayed recovery
from inactivation
Inhibition. Prevented by mutation N406 K
Examples of modulation of whole-cell and recombinant Na? channel currents. For abbreviations, please see Table 1
DHA, EPA
EPA. (Saturated or monounsaturated

fatty acids were also modulators but
co-expression with b subunits
eliminated this)
Human Nav 1.5*
EPA and other PUFAs
EPA, DHA
Human bronchial smooth muscle

cells
Whole-cell Na? current
EPA, DHA
HEK293t
Cultured rat ventricular myocytes
AA, DHA
HEK 293
Rat olfactory receptor neurons
AA
Human Nav 1.5*
Rat dorsal root ganglion
TTX-sensititive and
TTX-resistant
DHA, EPA, LA (Linoleic acid (C18:2) and

saturated or monounsaturated fatty acids
were ineffective or less effective)
Human Nav 1.5*
Cardiac muscle
AA and oleic acid (Palmitic acid (C16:0)

oleic acid methyl ester were not effective)
Modulatory agent
Rat skeletal and cardiac muscle
EPA, DHA and other PUFAs, as well as

ETYA (Saturated or monounsaturated fatty
acids were not effective)
Modulatory agent
Expression system
Cultured rat cardiac myocytes
Recombinant
Native currents
Table 2 Fatty acid modulation of voltage-gated sodium channels
[71]
[65, 72]
[73]
References
[70]
[65]
[188]
[113]
[66]
[69]
[67, 68]
References

67
Rat olfactory receptor neurons
Whole-cell K? current
AA, ETYA
DHA (through peroxidation
products)
AA
Chick ciliary ganglion neurons
Rat hippocampal neurons
Rat neurons, CA1 hippocampal slice
Rat pituitary melanotrophs
Bovine corneal epithelial cells
Rat CA1 hippocampal pyramidal cells
Rat ventricular myocytes
Hair cells of chick cochlea
IA
IA
IA (but not IK)
Fast-inactivating K?
current
Fast-inactivating K?
current
IA
ITO
Cultured mouse and rat cardiomyocytes
Pineal gland
Rat pulmonary myocytes
Neocortical neurons
Cultured microglial cells
Pancreatic islet beta cells and

INS1 insulinoma cells
Delayed-rectifier
IK
IK
Sustained K? current
Outwardly rectifying K?
current (predominantly
Kv 1.3 in these cells)
Delayed rectifier
Native, slowly inactivating
IA
AA, palmitoleic acid (C16:1)
Rat hippocampal neurons
IA
AA
AA
DHA
AA
DHA, EPA (monounsaturated fatty

DHA, AA
AA, ETYA (AA effects were more

variable)
AA (pM conc., intracellular)*,

ETYA. Enhanced by
intracellular glutathione and
prevented by extracellular
ascorbate
AA
AA, ETYA
AA
Pineal gland
DHA, EPA (monounsaturated fatty

AA, DHA
Modulatory agent
IA
Native, rapidly inactivating
Cellular
preparation
K? current or channel type
Table 3 Fatty acid modulation of voltage-gated potassium channels
Inhibition. Accelerated rate of inactivation
Inhibition. Modest increase in inactivation kinetics
Inhibition
Inhibition of late outward current and acceleration of

inactivation**
Inhibition. Induced time-dependent decay
Inhibition. Accelerated inactivation. Left-shifted

inactivation curve
Inhibition
Inhibition and concomitant activation of a steadystate current
Inhibition
Inhibition
Inhibition. Enhanced rate of inactivation. Leftshifted inactivation curve and slowed recovery
from inactivation
Inhibition. Left-shifted inactivation curve. Did not

alter inactivation rate
Inhibition. Enhanced the amplitude of backpropagating dendritic action potentials
Inhibition
Inhibition. Enhanced rate of inactivation and slowed

recovery from inactivation. Broadened interictal
spikes recorded in elevated [K?]o model of
epilepsy
Inhibition. Increased rate of inactivation
Inhibition. Slowed inactivation without effect on

activation or deactivation
[192]
[84]
[191]
[85]
[86]
[81]
[190]
[114]
[80]
[189]
[79]
[78, 116]
[90]
[77]
[87]
[86]
[104]
References
68
EPA, DHA, ALA
LA
AA, ETYA
DHA
Mammalian fibroblasts
Xenopus oocytes
(macropatches)
CHO cells
Stably transfected fibroblast

cell line
CHO cells
HEK 293
Xenopus oocytes
CHO cells
Xenopus oocytes
Bovine adrenal medullary

chromaffin cells
Rabbit arterial smooth
muscle
Kv 1.2 and Kv
3.1a
Kv 3.4 and Kv
1.1/b1.1
Kv 1.5
Kv 1.5
Kv 1.5 and Kv
2.1
Kv 2.1
Kv 4 (but not Kv
1, Kv 2, nor Kv
3)
Kv 4.3
Kv 4.2
Native Ca2?
activated K?
currents
BK
BK
Inhibition. Increased rate of activation and inactivation. Left-shifted

activation curve
Inhibition but only when applied from extracellular side, not in patch
pipette
AA or activation of a Ca2?
dependent PLA2
DHA
Sf9 cells
Kv 1.1
AA (lipoxyenase
activity required)
AA, other fatty acids and other
charged lipids
Modulatory agent
AA, ETYA, ETI
AA
AA, DHA, ETYA

(OA was not effective)
AA, AEA
Modulatory agent
Expression system
Recombinant,
Kv-type
Current inhibition but through

lipoxygenase metabolites
Fatty acids and negatively charged lipids with [8 carbons enhanced
current (NPo in single channels); positively charged lipids reduce
current; these effects occur only when the modulators are applied to
the extracellular side of the patch. Neutral and short-chain lipids
have no effect. Not explained by surface charge effects
Inhibition of peak current. Accelerated the inactivation time course,

but only when co-expressed with KChIP subunits
Inhibition
Inhibition. Increased in rate of recovery from inactivation but no

change in rate of activation of inactivation
Inhibition. Accelerated rate of inactivation
Inhibition. Increased rate of activation and left-shifted activation

curve. LA was only effective when applied extracellularly and not
in the patch pipette. Methyl ester of LA affected activation only
Inhibition. ALA left-shifted activation curve, accelerated activation

kinetics and slowed deactivation. EPA and DHA, but not ALA,
reduced steady-state levels
Inhibition. Accelerated inactivation. Left-shifted inactivation curve.

Required extracellular application (no modulation when added to
whole-cell recording pipette)
Inhibition and inactivation of Kv 3.4 and of Kv 1.1/b1.1 currents when

native inactivation had been removed by PIP2
Increased current (DHA). Response depended on co-expression with

KCNE1 accessory subunit
DHA, lauric acid (C14:0). Oleic

acid (C18:1) was less effective
and EPA was ineffective
Xenopus oocytes
Kv 7.1 (KCNQ1)
Modulatory agent
Cellular
preparation
K? current or
channel type
Table 3 continued
[76, 197]
[112]
References
[83]
[196]
[82]
[192]
[195]
[194]
[81]
[88]
[191]
[193]
References
[74]
References

69
1A
100ms
B 125
100
% of control
Abbreviations: IA = Transient or rapidly inactivating, A-type current which may be encoded by the Kv4 gene family plus associated subunits such as KChIPs or other subunits (e.g.,
Kv1.1 ? b1.1); IK = delayed rectifier current which can be encoded by different Kv gene families (but not Kv4); ITO = calcium-independent transient outward potassium current in heart which
is encoded by the Kv4 gene family plus associated subunits; IK1 = intermediate conductance calcium-activated K? channel; BK = big conductance calcium-activated K? channel, also known
as maxiK and slo (Slopoke); ETI = 5,8,11-eicosatriynoic acid (C20:3); LA = linoleic acid (C18:2); ALA = a - linolenic acid (C18:3); OA = oleic acid (C18:1). For additional abbreviations,
please see Table 1
** AA also increased peak current and accelerated activation, but pharmacological evidence suggested this effect was due to PKC activation
* Extracellular AA also inhibited current, but required lM concentrations
Examples of modulation of whole-cell and recombinant K? channel currents. Only channels with voltage-dependent or voltage- and calcium-dependent activation were considered
[43]
Increased current (NPo in single channels). When co-expressed with
b2 or b3 (but not b4) or tested with intracellularly applied free
synthetic b2-ball peptide, the PUFAs also slowed inactivation, but
only when applied from the intracellular side
AA, DHA, OA (Saturated fatty
acids were not effective.)
BK
Xenopus oocytes
[75]
Increased channel activity. Did not require cannabinoid receptors/Gproteins
AEA and methandamide
BK
HEK 293
References
Modulatory agent
Expression system
Recombinant, Ca2? activated
Table 3 continued
70
75
50
25
0
slow
fast
C normal
reduce Kv4
20 mV
20 ms
Fig. 2 PUFA modulation of a Kv4 current and a test of physiologic

impact. (A) Kv4.2/KChIP1b currents were recorded in oocyte voltage
clamp recordings (90 to ?40 mV; the methods are published [184].
AA (30 lM, arrow) inhibits peak current and enhances inactivation
(M. Drzewiecki and L.M. Boland, unpublished data). The inset
highlights the initial 200 ms in the absence and presence (arrow) of
AA (scaled to the peak of the control current). The results are
consistent with the current inhibition and modulation of inactivation
kinetics shown by other labs [82, 83, 87, 90]. (B) The effect of 30 lM
AA on fitted slow and fast time constants for the inactivation time
course of Kv4.2/KChIP currents at ?40 mV. Error bars indicate SEM,
n = 6. (C) Computational simulation using a neuronal compartment
model, based on the parameters of Traub and colleagues [185]. We
modeled a somal compartment with six ionic conductances (VG
channels and calcium-activated channels plus a leak conductance; C.
Maley, A. Varghese, and L.M. Boland, unpublished data). To
simulate the impact of inhibition of Kv4-type conductances on
neuronal firing, we introduced a 10-fold reduction in the contribution
of the Kv4 (IA) conductance, while all other parameters remained the
same. A single 50 ms, 1 nA current injection was used at the soma.
The increased firing frequency reproduces the effect on hippocampal
neuron firing activity in the presence of the Kv4-specific heteropodatoxin, as shown by Liss and colleagues [94]
The mechanism by which a tetanus may result in PUFA

inhibition of Kv4 channels and permit induction of LTP has
yet to be directly linked to one of the many signaling
pathways that could couple to enzymatic release of PUFAs.
However, one hypothesis is that post-synaptic glutamate
receptors couple to activation of a calcium-independent
form of PLA2 which locally increases PUFA concentration
near the somatodendritic Kv4 type channels, leading to

inhibitory modulation of these fast-inactivating K? channels. As predicted by this hypothesis, pharmacological
block of the intracellular, calcium-independent form of
PLA2 prevented LTP induction [96] and tetani that induce
pre-synaptic glutamate release also induce AA release from
hippocampal slices [99]. Endogenous AA release from
neurons also follows the direct application of glutamate to
slices, is prevented by glutamate receptor antagonists [99],
and occurs following strong stimulation of both ionotropic
and metabotropic glutamate receptors [100]. Glial cells
provide another possible source of PUFAs. AA and DHA
can be released from cultured rat brain astrocytes upon
stimulation of P2Y purinergic receptors in these cells [101]
Based on sensitivity to specific enzyme inhibitors, the
transmitter-stimulated DHA release from astrocytes also
appears to require calcium-independent PLA2 [101], just as
found for stimulation of neuronal afferents. However,
unlike neurons, transmitter-stimulated AA release from
astrocytes may require a calcium-dependent form of PLA2
[101].
To complete this story, we need information on the
impact of a tetanus on Kv4 channel function and identification of the G-protein-coupled receptor pathway that
yields the PUFAs. Despite a missing link, this active area
of research substantiates an important physiologic role for
PUFA modulation of Kv4 channels. Recent studies further
implicate a role for PUFAs in behavioral learning and
memory. Stereotaxic injection into rat CA1 hippocampus
of an inhibitor of calcium-independent PLA2 (but not an
inhibitor of calcium-dependent PLA2) impaired the acquisition of short-term and long-term memory in an inhibitory
avoidance task [102]. These effects occurred in the absence
of cell death. There is also a potential link between PUFAs
and age-associated memory impairments. Aged mice fed a
diet deficient in the x-3 precursor, ALA, demonstrated
impaired spatial learning and visual function, while aged
mice fed a DHA-rich diet showed improved visual function
[103]. The membrane concentration of AA within phospholipids is reduced in aged rats compared with young rats
and the concentration of AA correlates with the ability to
induce LTP in hippocampal slices [104]. This suggests that
events or conditions that reduce PUFA availability or
release (e.g., by reduction in receptor activation, efficacy of
coupling to PLA2, or PLA2 activity) may play a role in
memory deficits. Consistent with this notion, a recent study
showed that dietary supplementation with AA and DHA
improved immediate memory in human subjects with mild
cognitive impairment or organic brain lesions, but not
Alzheimers disease [105]. This leads to the suggestion that
impaired synaptic plasticity may be ameliorated by the
effects of PUFAs on LTP induction, but perhaps impairments due to cell death cannot be overcome by this
71
mechanism. Further research is needed to determine the

specific mechanisms by which PUFAs may influence
complex behavioral processes such as memory formation
and retrieval, especially in light of the many other physiologic effects of PUFAs and their metabolites, including
effects on inflammation and apoptosis [26].
Other physiologic effects of PUFA modulation of VG
channels in neurons include a role in regulating excessive
excitability. High concentrations of AA are released from
brain tissue as a result of seizure or ischemia [106]. PUFA
inhibition of Ca2? and Na? channels would help limit
excitability and may be protective in the event of pathological hyperexcitability [107]. However, PUFA inhibition
of K? currents would oppose these protective effects,
raising the issue that the duration of fatty acid availability,
the local concentration of fatty acids, and the spatial constraints of these lipid modulators and their channel targets
will determine whether actions on VG channel function
results in a net increase or decrease in excitability. In
addition, the effects of PUFAs on other channel types are
important to consider when assessing the role of PUFAs in
cellular excitability. PUFA-mediated activation of the K2P
channels TRAAK and TREK-1 may be neuroprotective
[108] as the opening of background leak K? channels
would tend to depress cellular excitability.
In heart tissue, a role for PUFA modulation of VG ion
channels is firmly linked to changes in myocyte excitability
and this has been recently reviewed [15]. In brief, evidence
supports a positive cardiovascular effect of x-3 PUFAs to
prevent fatal arrhythmias and reduce sudden cardiac death
[109]. The x-3 and x-6 PUFAs increase action potential
firing threshold in cardiac myocytes and lengthen the
refractory period [67, 110]. The stabilizing effects of
PUFAs on cardiac excitability are largely ascribed to
inhibitory modulation of Na? and L-type Ca2? channels
[62, 6567, 71]. Plasma concentrations of free AA may
also increase during ischemia and cardiac reperfusion
[111], which could add to inhibitory modulation of these
VG channels.
A Consideration of Mechanisms
While the importance of PUFA modulation of VG channels
has become apparent, an understanding of the molecular
mechanisms remains to be clarified. A schematic overview
of hypothetical mechanisms (Fig. 3) can be used in conjunction with our discussion. First, we will briefly assess
experimental strategies that have been used so far and their
implications.
To determine the direct or indirect nature of channel
modulation by AA, inhibitors of lipoxygenase and cyclooxygenase pathways can establish whether metabolism of
72

electrostatic
interactions with
channel
protonation of
channel residues
binding
internal
acidosis
oxidation of
channel residues
redox
PUFA
metabolism
oxidative
metabolites
membrane
curvature
membrane
stretch
binding
hydrophobic
mismatch
hydrophobic
interactions with
channel
membrane
compression/expansion
Fig. 3 Schematic illustration of mechanisms for PUFA modulation

of voltage-gated channels. Solid arrows highlight possible mechanistic explanations for changes in the functional properties of VG
channels, induced by direct application or physiological release of
PUFAs. Dashed arrows represent mechanisms that may be linked.
Each mechanism could result in changes in the conformational states
of the channel protein. See text for details
AA is required for the modulation. In addition, commercially available non-metabolizable analogs of AA, such as
ETYA, are useful in assessing whether metabolism of AA
is required for a modulatory effect. When these tools have
been used, the inhibitors generally fail to block AA-mediated effects and ETYA generally mimics the effect of
externally applied AA. Independent assessment that the
inhibitors block the formation of leukotrienes and prostaglandins and allow AA concentrations to remain elevated
during its application to cells has not been reported.
However, in most cases, evidence supports the conclusion
that the modulatory agent is the PUFA itself. Several
exceptions exist [112114]. In rat dorsal root ganglion
neurons, AA application enhanced VG Na? currents and
induced a left-ward shift in the activation curve but these
effects were prevented by a cyclooxgenase inhibitor and
not mimicked by ETYA [113]. Also, in bovine adrenal
chromaffin cells, AA inhibition of BK current was blocked
by inhibitors of lipoxygenases [112].
Since the free cellular concentrations of AA and other
PUFAs are largely unknown, it is difficult to assess the
physiologically relevant concentrations of free fatty acids
in the vicinity of their molecular targets. The range of
130 lM is often effective for experimental modulation of
VG channels and this concentration range includes the
values reported for plasma concentrations of free, unesterified AA (513 lM) [115]. It is important to note, however,
that the local concentration of AA upon cleavage from
excitable membranes is unknown and thus, the relationship
between channel modulation achieved by experimental
application of AA and that achieved by physiologic stimuli
is undetermined. In some preparations, lower concentrations of AA are effective. The most dramatic example is for
IA inhibition by picomolar concentrations of AA applied
intracellularly, although modulation by extracellular AA

required micromolar concentrations [78, 116]. This may
suggest an intracellular/internal leaflet site for modulation
or that elevation of the concentration of internal AA following external application is limited, thus explaining why
a higher concentration is required when the PUFA is applied
externally. However, these results also differed from many
other reports on PUFA modulation of IA or Kv4 type currents since the inhibition was not accompanied by an
increase in the inactivation time constants and pharmacological evidence supported a role for oxidative products of
AA, rather than direct effects of AA [78].
An important experimental issue concerns the side of the
membrane to which the PUFAs are applied during electrophysiologic studies. The membrane side of application
could influence the results if PUFA transport into or across
the membrane is limited or if a required site of action is
exposed to only one solution interface. Whereas most
studies have used extracellular application of PUFAs, a
direct test from both sides shows no difference in the
modulation of K? channel currents based on membrane
side of application [88]. Some studies demonstrate VG
channel modulation when PUFAs are applied extracellularly but fail to observe modulation when they are added to
the patch pipette [81, 86]. This method of application,
however, may limit access and is not as reliable as direct
application to the inside-out patch. So, these results alone
do not rule out the possibility of modulatory actions which
are independent of the side of membrane application.
A test of membrane solubility of the modulators is also
desirable. Since fatty acids have limited solubility in water
and are even transported in aqueous environments bound to
protein carriers, it is reasonable to predict that their mechanism of action requires solubility in the membrane. To
make the lipid insoluble in the membrane, one can add a
hydrophilic group such as coenzyme A (CoA) to the fatty
acid and test its ability to modulate channel function. This
can simultaneously test the requirement for membrane side
of application since extracellular application of AA-CoA
would prevent access to the internal leaflet of the membrane,
whereas intracellular application would prevent access to
the external leaflet. However, a limitation of this approach is
that the large CoA moiety may reduce access of the fatty acid
to intimate spaces that are needed for its action. Thus,
molecular size may be an equally important explanation for
a possible failure of AA-CoA to modulate channel function
in the same way as observed for a free fatty acid.
The specificity of action of free fatty acids involves
assessing the importance of carbon chain length and degree
of unsaturation of the molecule. The concept of structural
specificity relies on the prediction that some fatty acids will
be ineffective modulators. Generally, this is supported by
evidence that saturated fatty acids are not effective
modulators of VG channels (see Tables 13). Although there

are some exceptions [3, 65, 72], across the family of VG
channels, a pattern emerges in which longer carbon chains
and greater numbers of carboncarbon double bonds
increase the magnitude of the modulatory effects of PUFAs.
This is consistent with specificity of action. However, other
lipid molecules with related structures are sometimes tested
and found to mimic the modulation by fatty acids. For
example, AA [58] and anandamide (AEA) [59] produce the
same inhibitory effects on N-type Ca2? channel currents in
rat SCG neurons. When it has been tested, AEA often mimics
the effect of AA on other VG channels (Tables 13). It would
be worthwhile to test if related lipids act through the same
mechanism to modulate channel function. Tests of functional occlusion may be limited, however, by the difficulty in
obtaining a maximal effect of either lipid applied alone.
The specificity of modulation by PUFAs also involves
assessing the specificity of channel type affected. This is
especially helpful in native neurons and muscle cells where
more than one VG channel type is expressed. The ability to
separate ion channel currents in the same cell by pharmacological methods improves the opportunity to identify the
affected channels. Combined with studies of recombinant
channels, the specific actions of PUFAs on different
channel types can be assessed.
Finally, the nature of the endogenous mechanism of
release of the PUFAs is also important. As experiments
with cloned VG channels studied in expression systems
now predominate, this is largely unstudied. Direct application of AA induces VG channel modulation, but it would
be helpful, in native cells, to test that AA occludes the
modulation produced by co-activation of G-protein coupled receptors that activate PLA2. This would invoke a
specific signal transduction pathway in the physiologic
modulation of the channel by AA. This has been done
thoroughly in only one study [61].
Structural Basis
Site-directed mutagenesis is a common approach to test the
functional importance of a specific amino acid residue in
the channel. Variations on this theme include deletion
mutagenesis to remove a group of residues and test for a
loss of a particular function, as well as chimeric swapping
of homologous but non-identical regions of channels and a
test of gain of function in an otherwise insensitive channel
protein. Sometimes, the co-expression of multiple subunits
is tested and results suggest that some channels accessory
subunits may be needed to confer a specific modulatory
effect of PUFAs on VG channels [43, 74, 83]. However,
the application of mutagenesis strategies to explore this
modulation have been limited and the results, so far, are
73
inconclusive regarding the structural basis of PUFA modulation of VG channels.

A number of labs have shown that PUFAs inhibit VG
Na? current in cardiac muscle (see Table 2). An early
suggestion that PUFAs may bind directly to the Na?
channels was suggested due to the inhibitory effects of
EPA on the binding of radio-labeled toxin to cardiac Na?
channels [67, 68]. However, the only mutagenesis study
has targeted the S6 region of domain I of the Nav1.5
channel in which an asparagine to lysine mutation disrupted PUFA modulation of the channels [117]. In another
type of channel, AA inhibition of the intermediate conductance calcium-activated K? channel (IK1 or SK4) was
largely prevented by a threonine to serine mutation in the
conserved pore region (T250S) and was completely prevented in double mutants that pair this mutation with
another in the S6 region (V275A) [47]. Substitution of the
IK1 (AA-sensitive) residues into SK2 (AA-insensitive)
conferred partial AA sensitivity to SK2, whereas mutations
introduced to several other regions of these channels did
not impair the modulation, indicating that these two sites
are necessary and sufficient for the modulation by PUFAs.
The specificity of these mutations notwithstanding, a
defined role for these residues in the modulation is not
clear. One possibility, although it seems unlikely, is that
these residues participate in a binding site for PUFAs. A
more plausible explanation is that these residues are
important in how the protein interacts with the lipid
environment.
A general effect of PUFAs on the fluidity of the lipid
environment [8] would seem to be an imprecise explanation
for modulation of VG channels since not all native channel
types present in a cell and not all recombinant channels are
targets for PUFA modulation [65, 78, 82]. A structural basis
for the impact of the lipid environment on VG channel
function has been proposed by MacKinnon and colleagues
[4]. Voltage-dependent gating of the bacterial channel
KvAP reconstituted into liposomes depends on a negatively
charged phosphodiester within a phospholipid head region.
It was found that positively charged arginine residues in the
voltage sensor of KvAP (equivalent to S4 in VG channels)
require the negatively charged lipid head groups for interaction during the gating process. Molecular dynamics
simulations are consistent with a role for positive charges in
the voltage sensor in stabilizing electrostatic interactions
with phospholipids [118, 119]. These results confirm that
specific lipidprotein interactions are vital to VG channel
function. Other evidence for electrostatic interactions
between lipid and protein comes from a study of BK
channel modulation by PUFAs in which the positively
charged, cytosolic inactivation domain of the b2 subunit
was implicated in an interaction with the carboxyl group of
AA [120].
74
In assessing a structural basis for PUFA interactions

with VG channel proteins, it may be useful to appreciate
how fatty acids interact with other proteins that bind and
transport them through an aqueous environment. Serum
albumin is the most abundant protein in plasma and functions to carry molecules of low water solubility such as
fatty acids, bile salts, and bilirubin. Curry and colleagues
[121] determined the crystal structure of human serum
albumin complexed with five molecules of the saturated
fatty acid, myristic acid (C14:0). The binding sites are
somewhat flexible and can adapt to fit several different
ligands. The anionic forms of fatty acids bind to albumin
using nonpolar interactions between the fatty acid hydrocarbon chain and uncharged amino acid side chains that
line the binding sites. Five of the seven fatty acid binding
sites of human serum albumin also have basic or polar side
chains that interact closely with the carboxyl group of the
bound fatty acid [121, 122]. Arginine and tyrosine residues
are important in one of the sites (site 4), and lysine is
important in another site (site 5) [123].
Another family of proteins, the intracellular fatty acid
binding proteins (iFABP), participates in the intracellular
trafficking of fatty acids and other lipids. The three-dimensional structures of several iFABPs have been determined by
X-ray crystallography and NMR (for review, see [124]). The
ligand specificity is fairly broad; a variety of hydrophobic
compounds will bind. The many members of this protein
family share little amino acid sequence identity, suggesting
that different structures may accommodate the ligands,
although all of the iFABPs share a three-dimensional b clam
structure [125127]. Like the situation for serum albumin,
the carboxylate of a fatty acid bound to an iFABP is buried
deep within the three-dimensional structure of the protein.
Liver FABP has a particularly large binding pocket that can
accommodate two fatty acids at once. One fatty acid is buried
deep in the pocket with the carboxyl group inaccessible to
solvent. This fatty acid assumes a U-shaped conformation in
the binding pocket and the carboxylate (COO-) interacts with
an arginine residue and also serine residues. The other fatty
acid is bound more superficially, has a more extended
structure, and has the carboxylate on the surface of the protein [128, 129].
In summary, in both serum albumin and iFABPs, the
carboxylate of a deeply buried fatty acid interacts with basic
or polar side chains of the protein. This provides a rationale
for testing for electrostatic interactions between the VG
channel protein and the negatively charged carboxylate of
PUFAs. Using the crystal structure of a Kv2.1/Kv1.2 channel
chimera, MacKinnons group has shown that the voltage
sensor domains are densely surrounded by lipids and, furthermore, that lipidprotein interactions stabilize charged
residues that are important for conformational changes
which couple voltage-sensing to pore opening [33].
However, effects of PUFAs on VG channel activation

kinetics and voltage-dependence of activation gating are not
commonly reported. Perhaps more likely candidates for
electrostatic interactions with the negatively charged carboxylate of PUFAs are channel regions involved in
inactivation gating since PUFAs commonly modulate inactivation kinetics and induce a voltage shift in the steady-state
inactivation curve (see Tables 13). New evidence suggests
that the positively charged, cytosolic inactivation domain of
the BK channel b2 subunit interacts with the carboxyl group
of AA [120] and this could be further explored by mutagenesis studies in other inactivating VG channels.
We also lack a structural explanation for why the length
of the hydrocarbon tail and the degree of fatty acid unsaturation are important in the modulation of VG channels.
The same pattern is apparent when studying the fatty acid
activation of tandem pore K2P channels where stimulation
requires at least one carboncarbon double bond and the
anionic (COO-) form of the fatty acid [130]. An explanation
for this is not found in the structure of FABPs since saturated and monounsaturated long chain fatty acids have
similar binding affinities on FABPs [123]. However,
molecular dynamic simulations have shown that the number
and location of CC double bonds and hydrocarbon chain
length impact fatty acid flexibility. DHA, a PUFA with a
22-carbon chain and six cis- double bonds, undergoes fast
conformational changes and has a highly flexible structure
[131]. These features may contribute to its ability to interact
with the G-protein coupled receptor, rhodopsin [131, 132].
Unesterified DHA is predicted to infiltrate certain spaces
between the transmembrane helices of rhodopsin [9, 133].
The dynamic changes in the protein during gating would
thus be influenced by the packing of DHA within these
spaces, which could explain how DHA facilitates conformational changes in rhodopsin upon activation [9].
Polyunsaturated fatty acids also modulate ligand-gated
channels [134137]. Recent evidence from Huettner and
colleagues [138] informs us about the structural determinants of PUFA inhibition of neuronal kainate receptors
[137, 139]. Guided by the phenotypic differences in GluR6
channels that had undergone RNA editing at the Q/R site in
the pore loop, Huettner and colleagues used an argininescanning approach to identify locations that are important
for PUFA block of the channels. Strong inhibition by
PUFAs required arginine substitution at pore loop residues
that are predicted to face toward the M1 and M3 helices
and the hydrophobic interior of the membrane but not the
cytoplasmic interface. This is generally consistent with the
results from the rhodopsin studies [9, 133]. Together, these
studies support the hypothesis that specific protein-PUFA
interaction sites modulate the function of integral membrane proteins during the dynamic rearrangements of the
protein.
Tests of other structural determinants, such as hydrophobic interactions between non-polar regions of the
channel and the hydrocarbon tail of fatty acids would be
useful given the degree to which the channels transmembrane domains interact with lipid [33, 35] and the
hydrophobic interactions of fatty acids and carrier proteins
[123, 124]. Perhaps PUFAs interact with hydrophobic
binding sites within the channel protein, similar to the
effects of local anesthetics [140, 141] or antiarrhythmic
drugs [142] on channels.
Mechanical Forces in the Membrane

Hydrophobic Mismatch
It should not be surprising that membrane-embedded proteins have important interactions with the lipid constituents
of the plasma membrane. In fact, some proteins are adapted
to take advantage of changes in membrane bending or other
mechanical forces generated within the lipid bilayer. MS
channels are perhaps the best example of this. They are
ubiquitous in cells and function universally to transduce
physical stimuli, such as membrane stretch and cell swelling,
into ionic fluxes. They are fundamentally important to life
and are present in all living organisms studied to date. At the
molecular level, we know the most about prokaryotic MS
channels which function to prevent cell lysis from hypotonic
stress. Despite the fact that MS channels lack structural
similarity with VG channels, they are excellent models in
which to study the impact of membrane lipids on the dynamic
structural rearrangements of membrane-embedded channel
proteins. Recent articles provide thorough reviews of the
structure and function of MS channels [143145] and so our
discussion is focused on specific features that may be
important to our understanding of how VG channel function
is modulated by lipids such as PUFAs.
Structural studies from X-ray crystals of a bacterial MS
channel of large conductance, MscL, show that the channel
is a homopentamer and each subunit is comprised of two
transmembrane domains, TM1 and TM2 [146]. Perozo and
colleagues used electron paramagnetic resonance (EPR)
and site-directed spin labeling of liposome-reconstituted
MscL to analyze the three-dimensional shape of the open
channel [147]. The open channel requires significant
expansion of the TM domains as they splay out laterally
and create an aqueous pathway. TM1 contains the porelining region of the channel, but both TM domains have
significant interactions with the lipid bilayer as their resting
positions must change during activation gating. Thus, as
might now be expected for perhaps all channel types, the
closed MscL channel is more compact and the open MscL
channel is more expanded. Since MS channels can be
75
activated in liposomes, the necessary components for

expansion of the protein during gating are present within
the lipid bilayer. Other proteins, including cytoskeletal
proteins, are not required [145]. While the normal physiologic stimulus for the channel is osmotic stress,
experimental studies have induced channel opening by
osmotic stimuli, as well as suction from a patch clamp
recording pipette. In either case, membrane tension changes would be expected to be translated into differences in
the lateral pressure induced by interactions at the lipid
protein interface [148, 149].
The concept of hydrophobic mismatch explains how
membrane tension is created when the length of an ion
channels hydrophobic transmembrane regions does not
match the length of the fatty acid tails of the phospholipid
molecules in the bilayer. A mismatch creates tension by
compression of the longer hydrophobic regions (thought to
be the fatty acid chains) so that the hydrophobic interactions
occur over a more equivalent distance across the bilayer. As
a consequence, the nature of lipidprotein interactions is
changed. To test this hypothesis as a mechanism for
mechanosensitive gating, Perozo and colleagues incorporated MscL channels into cell-free liposomes [147]. By
varying the acyl chain length, the liposome bilayer thickness could be altered. EPR and electrophysiologic evidence
support the prediction that thin bilayers favor channel
opening as the TM domains tilt towards the plane of the
bilayer in order to prevent the energetically unfavorable
exposure of hydrophobic residues to the polar environment
at the bilayer surface. Likewise, thick bilayers cause TM
domains to tilt more vertically, towards the axis perpendicular to the plane of the bilayer, in order to minimize the
interaction of the hydrocarbon tail of the fatty acid with
polar regions of the TM domains. Interestingly, hydrophobic mismatch alone did not induce MscL channel opening,
but instead shifted the pressure activation curve, as if it
altered the sensitivity of the channel to the stimulus of
membrane tension. Fatty acyl tail length is also predicted by
molecular dynamic simulations to influence MscL protein
lipid interactions, further supporting a role for hydrophobic
mismatch [150].
The possibility that hydrophobic mismatch could
explain PUFA modulation of ion channels was described
by Lundbaeck and Andersen [151] and has been tested by
Andersen and colleagues [152] and Leaf and colleagues
[73]. In support of this hypothesis, Leaf and colleagues
showed that application of non-ionic detergents mimicked
the inhibitory effect of PUFAs on Nav1.5 channels, despite
the fact that these compounds have no structural similarity.
One interpretation is that the detergents disrupted the
membrane tension which induced conformational changes
similar to the effect of PUFAs. A test of experimental
occlusion may be useful here to determine if the PUFAs
76
and non-ionic detergents are working through a common

mechanism for modulation of Nav channel function. In
cardiac myocytes, many ion channels can be modulated
simultaneously by AA or non-ionic detergents, which
supports a general role for membrane lipids in the function
of VG ion channels and other channel proteins. However,
not all ion channel types in cells are affected to the same
degree by PUFAs and some are not affected at all. For
example, Kv4 channels but not Kv1, Kv2, nor Kv3 were
modulated by AA or ETYA [82] and IK1, but not SK2
channels, were inhibited by AA [47].
The hydrophobic mismatch hypothesis predicts that the
experimental addition of PUFAs to one side of the membrane bilayer or the cleavage of fatty acids from their
positions in phospholipids on one side of the bilayer would
alter the membrane tension and thereby influence channel
conformation and function. This could be further tested in
liposomes or synthetic bilayers with reconstituted channels.
Hydrophobic mismatch might also be tested in cultured
cells fed a media rich in fatty acids of different chain
lengths, where chronic changes in length of the fatty acid
tails in membrane phospholipids may alter the magnitude
of the mismatch.
Membrane Curvature
In the cellular environment, membranes are not planar
bilayers, but undergo curvature, which is influenced by the
lipid composition of the membrane [7, 20, 153155].
Functional roles for membrane curvature abound in cell
biology as membrane fusion events, including synaptic
vesicle-mediated exocytosis of neurotransmitter, require
bilayer bending [156].
The hypothesis that membrane curvature is a gating
mechanism for MS channels was also tested by Perozo and
colleagues [157]. Asymmetric addition of lipids that have a
single fatty acid tail (lysophospholipids) was an activating
stimulus for McsL [158] because it altered the sensitivity of
the channel to pressure stimuli [157]. Lysophospholipids
have a conical geometry due to size differences between
the polar head group (wider) and the single acyl tail (narrower) [20]. According to the membrane curvature
hypothesis, a conical lipid will induce changes in membrane curvature if its concentrations in the two membrane
leaflets differ. Thus, the results indicate that forces needed
to change the conformational state of the MscL protein can
be induced by deformation of the lipid bilayer. A similar
result was found for the two-pore domain channels TREK1 and TRAAK, which were opened by asymmetric addition
of lysophospholipids to the cellular membrane [159, 160].
Polyunsaturated fatty acids are also conical in shape (in
the opposite direction from lysophospholipids), with the
hydrocarbon chain consuming a larger packing space than
the polar region of the molecule. In a monolayer, the asymmetry of PUFAs induces a negative curvature [20, 152, 161,
162]. Thus, an alternative hypothesis for the structural
mechanism of PUFAs on VG channel function is that a local
increase in PUFAs in one leaflet of the membrane alters
membrane curvature, which impacts the conformational
state of embedded channel proteins. Likewise, lipids that
induce positive curvature would be expected to reverse or
impair the channel modulation by PUFAs.
An impact of PUFAs on membrane curvature may
explain how they activate certain non-VG K2P channels
such as TREK-1 and TREK-2 [159, 163, 164]. In a similar
fashion, an alternative hypothesis for the structural mechanism of PUFAs on VG channel function is that an increase
in PUFAs on one side of the membrane alters membrane
curvature, which impacts the conformational state of
nearby channel proteins. This predicts that the effect of
PUFAs on VG channels would be opposed or reversed by
the application of lysophospholipids to the same side of the
membrane. To our knowledge, such an experiment has not
been reported, although lysophosphatidic acid (LPA) fails
to mimic the effect of AA on inhibition of whole-cell
N-type Ca2? currents [61].
A challenge for an explanation based on membrane
curvature is the ability to explain how the direct application
of AA to cells could induce the same modulatory effect as
upstream activation of PLA2 signaling pathways, a process
that should produce two asymmetric lipids of opposing
conical shapes. One possibility is that one of the products,
such as LPA, is inactive in producing the type or magnitude of change that is necessary for modulation of VG
channels or it cannot access a lipidprotein interaction
region that is required for modulation. Another possibility
is that asymmetric lipids accumulate in both membrane
leaflets, negating an effect on curvature when applied to
only one side of the membrane.
A role for PUFAs on membrane curvature should also
explain how modulation of VG channels is enhanced by
increasing hydrocarbon chain length and increasing number of double bonds in fatty acids. The impact of
asymmetric lipids on membrane bending would predict that
short chain or saturated fatty acids would have a smaller
effect on monolayer curvature and assume a more symmetric shape within a lipid bilayer. The concept of
membrane bending is difficult to assess because one would
like a direct measure of the membrane curvature and not
indirect measures, but the hypotheses have sound experimental predictions, based on our knowledge of the effects
of lipids on membrane geometry.
These explanations for the modulation of VG channels
by PUFAs are not mutually exclusive, nor do they detract
from the physiologic importance of PUFA modulation of
cellular excitability. Furthermore, specificity of action may
be attributed to the impact of specific changes in membrane

lipid composition or protein structure on specific conformational states of the protein. Mutagenesis studies would
be strengthened by the possibility of identifying channel
residues of importance to conformational states that can be
regulated by membrane tension or membrane curvature.
Another challenge is to identify the phospholipid composition of different native membranes and how this impacts
individual channel types.
Mechananosensitivity of VG channels
We have gained some insight about the importance of the
lipid constituents of the membrane on channels whose
physiologic stimulus is mechanical deformation of the
membrane. Since one hypothesis for PUFA modulation of
VG channels is that the fatty acids induce mechanical
deformations that impact channel function, it is worthwhile
to review the evidence that VG ion channels can indeed be
modified by mechanical changes in the tension of the
plasma membrane [155, 165].
Using excised and cell-attached patches to record Shaker and other Kv channel activity, Morris and colleagues
have shown that the primary mechanism of gating can be
further modulated by membrane stretch. Stretch facilitated
channel opening when the probability of opening due to
membrane depolarization was low, but impaired further
opening when the probability of opening was already high
[166]. Deletion mutagenesis of the S3S4 linker region of
Shaker K? channels slowed the inactivation time course,
allowing one to measure activation gating. Under these
conditions, membrane stretch produced a significant
acceleration of activation gating, suggesting that membrane tension can substitute for the stimulus of membrane
depolarization [167]. Both activation and inactivation
processes were stretch-sensitive [168, 169]. These results
reveal that the activation process of VG channels is
inherently stretch-sensitive even though, in wild-type
channels, activation gating utilizes voltage-sensing and
transduction of this stimulus into channel gating.
Movements of the channel protein during gating would
be expected to both induce changes in the lipidprotein
interface and be altered by the lipidprotein interface [33].
In Kv channels, both voltage-sensitive transitions such as
the closedclosed transitions during the activation of
individual subunits and voltage-independent transitions,
such as the final concerted activation step before channel
opening, have been identified as mechanosensitive [168,
169]. In each case, significant movement of the channel
protein within the bilayer may account for its sensitivity to
the membrane tension changes. However, different structural conformations would be expected to experience
different lateral pressure profiles [33, 170] which could
77
explain why certain gating transitions (e.g., closed to open,

open to inactivated) could be more or less sensitive to
mechanical forces. Fundamentally, a closed channel is a
more compact structure than an open channel. Thus, a
reasonable hypothesis is that membrane stretch distorts
lipidprotein interactions in such a way as to enhance
channel opening. An expectation of this hypothesis is that
any type of VG channel should be modulated directly by
changes in bilayer tension [165, 166].
To this end, the stretch-sensitivity of VG channels is not
confined to Kv channels. VG Na? [171], Ca2? [23, 172], and
cation-selective HCN channels have also been shown to have
mechanosensitive gating processes [173, 174]. Membrane
stretch speeded up the rate of inactivation of skeletal muscle
Nav a subunits when expressed alone. However, in the
presence of b subunits, which also speed up inactivation,
membrane stretch failed to further modulate this gating
process [175, 176]. The requirement for mechanosensitivity
in the absence of the b subunits suggests that bilayer tension
can substitute for the physiologic changes introduced by
structural interaction of the a and b subunits.
The opening of HCN channels was reversibly altered by
events that affect membrane stretch, such as cell swelling
[172] and membrane depletion of cholesterol [23]. The
mechanosensitivity of HCN channels may be particularly
important to their role as pacemaker channels in heart
where changes in membrane deformation during contractile events may provide an additional mechanism to
regulate HCN gating. In addition, the physiologic role of
HCN channels in the baroreceptor reflex of the aortic arch
[177] and osmosensory neurons of the magnocellular
supraoptic nucleus [178] may also be influenced by changes in blood pressure or cell volume which could alter
membrane stretch or compression.
An open question is whether the mechanical changes
that are required to influence voltage-sensing have a role
under normal physiologic conditions. Importantly, stretch
stimuli that modify the gating of cardiac muscle Nav1.5
channels were the same stimuli that activate TRPC1
stretch-activated currents [171]. This suggests that high
sensitivity of VG channels to transmembrane voltage gradients does not preclude the possibility of modulation of
conformational transitions by other stimuli. In addition,
certain conditions may create a greater impact on membrane tension or elasticity of the membrane, such as the
geometry of the membrane during cell division, cellular
migration, tissue compression due to tumor growth, or
tissue swelling due to inflammation [165]. Thus, evidence
for a modulatory mechanism for PUFAs which includes a
role for membrane stretch could have important physiologic and pathological implications.
Together, this research highlights a key structural issue,
that the VG channels may not require any specialized
78
mechanosensitive components of the protein structure in

order to retain the ability for conformational changes to
become altered by changes in the bilayer. Changes in the
bilayer could occur through depletion or enrichment by
certain lipids, alterations of lipid composition by diet,
biochemical changes in enzymatic activity, and alterations
of the physical properties that compress or expand the
bilayer and modify the proteinlipid interactions. Is it
possible that PUFA modulation of VG channels hijacks an
endogenous mechanism for altering the sensitivity of
voltage-dependent gating? The discussion presented above
argues in favor of this possibility. Furthermore, mechanical
stress does not change the amount of gating charge moved
nor introduce new conformations to VG channels [169].
This suggests that mechanical stress modulates existing
movements of the channel protein within the bilayer [171].
Generally though, the greatest effect of PUFAs on gating is
an acceleration of inactivation gating and a left-ward shift
of activation gating, in the range of 1020 mV with the
highest tested concentrations of AA (1030 lM). This is
essentially what PUFAs do to modulate VG channel
functionthey alter the threshold for gating processes,
however they interfere with certain processes more than
others. Structural mechanisms for PUFA modulation based
on mechanical stress must explain the specificity of the
modulation of inactivation gating as well as current
inhibition.
Mechanisms Involving Oxidation or Intracellular
Acidosis
The carboncarbon double bonds of PUFAs are subject to
oxidation and some evidence supports a role for fatty acid
oxidation products in the modulation of VG ion channels. In
isolated rat ventricular myocytes, DHA strongly inhibited
the transient outward K? current ITO, and also induced a
delayed outwardly rectifying K? current, referred to as IDHA
[114]. However, co-application of DHA with the antioxidant
a-tocopherol reduced DHA modulation of the VG currents.
In contrast, co-application of the oxidant, hydrogen peroxide, enhanced the effects of DHA [114]. Applied alone,
neither the antioxidant nor the oxidant altered the K? currents. These results tend to support a role for the oxidation
products of DHA in the modulation of certain cardiac K?
channels. It remains to be seen if the enzymatic oxidation of
DHA by cyclooxygenase or lipoxygenase enzymes occurs
following physiologic stimuli that also result in VG channel
modulation and if direct application of metabolites mimics
and occludes the effect of DHA.
There is evidence that the addition of free fatty acids to
planar lipid bilayers, liposomes, or other protein-free
environments can change the internal pH as fatty acids
move across cell membranes, without the need for a
transport protein [179]. Such pH changes have been shown

to occur only in the close vicinity of the membrane, while
the overall pH in the bulk volume remains unaffected
[180]. Thus, with respect to PUFA modulation of VG ion
channels, the external application of PUFAs may result in
internal acidification near the interface of the channel
protein and the inner leaflet of the membrane. There are
scarce reports that internal pH changes impact VG channel
function. Activation of the native HCN current, IH, is
regulated by internal protons, among other modulators and
activators [89]. Also, amino-terminal, N-type inactivation
of human Kv1.4 is regulated by internal pH in the physiologic range through ionization of specific amino acid
residues in the ball domain [181]. Since VG channel
inactivation kinetics and sensitivity of inactivation to
membrane voltage are often modulated by PUFAs, it may
be worthwhile to consider the impact of transient and
modest acidification near the internal leaflet of the
membrane.
Conclusions
The studies reviewed here support a role for direct PUFA
modulation of the function of several structurally related
but distinct ion channels in excitable cells. Important
advances have been made in understanding the lipid nature
of the plasma membrane and the source of different lipid
messengers. In several instances, we have a comprehensive
understanding of the physiologic impact of the PUFA
modulation of certain VG channels. In addition, the
numerous examples of native and recombinant VG channels that are affected by PUFAs includes insights on the
structural characteristics of fatty acids, a requirement for b
subunit co-association in certain cases, and the relationship
between PUFAs and channel inactivation. However, an
understanding of the structural basis for this modulation
remains elusive. We have reviewed experimental strategies
used to assess the mechanisms of PUFA modulation to date
and discussed recent results from mutagenesis studies
designed to assess a molecular site of action. A restlessness
with the current evidence suggests that we consider alternative explanations, including the mechanical forces that
alter lipidprotein interactions. We have reviewed critical
elements of this research and identified future experimental
strategies to fill the gaps in our understanding of the
mechanisms of PUFA modulation of VG channels.
Several additional questions remain unanswered. We do
not know the lifetime of AA as a signaling molecule following physiologic stimulation of its cleavage from
membrane phospholipids. Since AA is metabolized to
leukotrienes, prostaglandins, and thromboxanes (Fig. 1), its
lifetime may be short, and its duration of availability is
certainly important to its physiologic impact on cellular

excitability.
Some membranes are highly enriched in certain PUFAs
and present new opportunities to explore the functional
modulation of VG channels by physiologic cleavage of the
PUFAs from the membrane. For example, the mammalian
retina has the highest concentration of DHA-containing
phospholipids and could be a wealthy reservoir of DHA for
ion channel modulation in addition to its functions in
protection from inflammation and apoptosis [19].
In many cases, the mechanism of PUFA modulation of
VG channels is studied in model systems such as oocytes
or transfected cells. The upstream signaling pathways that
initiate PUFA release, under physiologic conditions, are
largely unexplored. More information about the possible
co-localization of important components of this modulation
(receptor, G-protein, phospholipids, phospholipase, channel) would be helpful. While it will be difficult to
reconstruct the entire signaling pathway from stimulus to
receptor to modulated channel, this must be addressed to
advance our understanding of the functional role of PUFAs
in the regulation of neuronal activity and behavior.
Is there a role for dietary PUFAs in brain and cardiac
function which is based on modulation of VG channel
function? A wealth of evidence suggests that a diet rich in
the x-3 fatty acids found in fish oils may be protective
against ischemia-induced, fatal cardiac arrhythmias.
PUFAs act, in the short-term, to prevent excessive excitability of cardiac tissue by decreasing inward Na? current,
suppressing L-type Ca2? current, shortening the action
potential, and inducing a positive shift in the action
potential threshold [65, 109]. However, the long-term
effects on channel function are largely unknown and could
involve changes in channel expression, association with
protein partners, as well as sustained modulatory effects
due to alterations in plasma membrane lipid composition.
The remaining questions allow for continued excitement
about research to uncover the molecular explanations for
how PUFAs modulate VG channels and alter the function
of excitable tissue.
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Acknowledgment The authors thank the Thomas and Kate Jeffress
Foundation and the Arts and Sciences Deans Office for research
support.
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Cell Biochem Biophys (2008) 52:5984

Polyunsaturated Fatty Acid Modulation of Voltage-Gated Ion

Published online: 2 October 2008

Abstract Arachidonic acid (AA) was found to inhibit the

L. M. Boland (&) M. M. Drzewiecki

Cell Biochem Biophys (2008) 52:5984

However, our understanding of the molecular mechanisms

Sources of PUFAs as Modulatory Agents

The Fatty Acid Nature of the Plasma Membrane

A lipid bilayer with embedded proteins is the basic

Fig. 1 Biochemical pathways for release of PUFAs from membrane

Among the lipid constituents of the membrane, the fatty

Cell Biochem Biophys (2008) 52:5984

fatty acids have double bonds in the cis configuration in

membrane phospholipids, which liberates a non-esterified

the modulation of VG channel function, in the absence of

Structural Features of Voltage-Gated Channels and the

Cell Biochem Biophys (2008) 52:5984

charged arginine or lysine residues are regularly spaced

Cell Biochem Biophys (2008) 52:5984

The BK channels contain an S4 region and voltage is the

hyperpolarization causes inward movement which opens

Examples of PUFA Modulation of Voltage-Gated

Cell Biochem Biophys (2008) 52:5984

muscle cells and in most cases, induced a left-ward shift of

Cell Biochem Biophys (2008) 52:5984

co-expression with b2 or b3 subunits [43]. These results

nervous tissue [22] and cardiac muscle [65] and it is worth

Effect on current or channel

Neonatal rat SCG neurons

Guinea pig cardiac ventricular

Cav 3.1, 3.2, and 3.3

Cav 3.1, 3.2, and 3.3

AEA and AA but saturated

Inhibition and left shift of current-voltage curve

Inhibition and leftward shift of inactivation curve

Inhibition, acceleration of inactivation, slowed recovery from inactivation,

Inhibition and acceleration of inactivation kinetics

Inhibition and increased probability of null sweeps [58, 60]

Inhibition and acceleration of recovery from

Inhibition. Acceleration of inactivation kinetics, and left-ward shift of inactivation curve

Effect on current or channel

Examples of modulation of whole-cell and recombinant Ca2? channel currents

Inhibition and acceleration of inactivation kinetics [54]

Enhancement, but inhibition with higher

EPA and DHA

Guinea pig tracheal myocytes

Bovine adrenal zona fasciculata

Rat SCG neurons

AA (Oleic and myristic acid were less effective)

Neonatal rat SCG neurons

Long chain unsaturated and saturated fatty acids

AA (Saturated and trans-unsaturated fatty acids were Inhibition

EPA and DHA

Native L-type currents

Native N-type currents

Native T-type currents

Acutely isolated retinal Muller glia

Smooth muscle of rabbit ileum

Neonatal rat cardiac myocytes

Low-voltage activated and

Guinea pig cardiac myocytes

Whole-cell Ca2? current

Native currents, mixed types