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C H A P T E R

8
Gramicidin Perforated Patch
Norio Akaike

o u t l i n e

I. Introduction 141 . Effect of Neuronal Trauma


D 144
E. Effect of H2O2 Treatment 144
II. Application of Gramicidin Perforated Patch
F. Effect of Furosemide on Intracellular [Cl]i 145
to CNS Neurons 142
A. Getting the Patch 142 III. Conclusions 145
��������
B. GABA ���������
Responses 142
References 146
C. Developmental Change in Glycine and
GABA Responses 143

metabotropic receptors (Korn and Horn, 1989; Kurachi


I.  Introduction et al., 1989; Sims et al., 1991; Ueno et al., 1992; Wendt
et al., 1992; Ye and Akaike, 1993); G-protein and second
Developments and improvements of the conven- messenger mediated responses (Harata and Akaike,
tional whole-cell patch technique during the last 1993; Shirasaki et al., 1994; Uneyama et al., 1993);
decades yielded many insights on various aspects and intracellular Ca2 buffering systems (Akaike and
of membrane physiology (Akaike et al., 1978; Hamill Uneyama, 1994; Mistry and Hablitz, 1990; Omura et al.,
et al., 1981; Kostyuk et al., 1975; Lee et al., 1979). The 1993). These disadvantages of the conventional whole-
patch clamp has permitted quantitative analysis of a cell patch recording configuration were overcome by
variety of voltage-gated- and ligand-gated-ion chan- the development of the ‘‘perforated patch’’ recording
nels, the electrogenic Na/K pump and the Na/ configuration using pore-forming antibiotics that are
Ca2 exchanger to mention a few examples. While selectively permeable to certain ions. These antibiot-
whole cell patch technique allows one to control the ics include nystatin (Akaike and Harata, 1994; Horn
composition of the intracellular milieu by equilibrat- and Marty, 1988), amphotericin B (Rae et al., 1991) and
ing it with that of the artificial pipette solutions, the gramicidin (Abe et al., 1994; Akaike, 1996; Ebihara et al.,
dialysis or the dilution of intracellular components dis- 1995; Kakazu et al., 1999, 2000; Nabekura et al., 2002;
turbs the native cytoplasmic biochemical environment Rhee et al., 1994; Tajima et al., 1996; Wake et al.,
which contains substances that are key for regulation 2007). Nystatin forms small aqueous pores (radius
and function of various transporters, ion channels and 0.4 nm) on the cell membrane that are permeable

Physiology and Pathology of Chloride Transporters and Channels in the Nervous System 141 © 2009, Elsevier Inc.
142 8.  Gramicidin Perforated Patch

to small monovalent cations with the following selec- gramicidin A, B and C, into methanol at 10 mg/ml.
tivity sequence: Rb  K  Cs  Na  Li, and to After ultrasonication for a few seconds, the stock
anions such as Cl. When inserted into patch pipettes, solution is directly dissolved into the patch pipette
the permeability properties of nystatin allow electrical solution, resulting in a final concentration 0.1 mg/ml.
access to the cell while preserving relatively large intra- Patch pipettes are fabricated from glass tubes
cellular molecules such as cAMP and ATP. Thus, the (Narishige, G-1.5) on a two-stage vertical pipette
nystatin-perforated patch technique allows the study puller. The pipette tips are initially filled with grami-
of functional channel responses in their native envi- cidin-free internal solution by immersion, and then
ronment, without altering the cellular contents which the remainder of the pipette is backfilled with the
includes key second messenger molecules (Akaike and same patch pipette solution containing gramicidin.
Harata, 1994). However, since the nystatin pores are The resistance of a pipette having an internal diam-
permeable to monovalent anions, the intracellular Cl eter of about 1 m is 58 M. As gramicidin diffuses
concentration ([Cl]i) is determined by the Cl con- to the pipette tip and enters the cell membrane, a
centration in the patch pipette solution, and thus the time-dependent change in the capacitative current
native [Cl]i is altered. is observed (Fig. 8.1A). In small neurons dissociated
Cl is one of the major and most important constitu- from rat CNS, where the soma diameter is 10–15 m,
ents of living cells; it participates in a number of func- the membrane perforation by gramicidin requires
tions such as cell volume and pHi regulation (Chapters more than 30 min after making a G seal. However,
15 and 4, respectively), salt secretion and absorption when patch pipettes having larger internal diameter
(Chapters 16 and 29), G-protein-coupled signal trans- (23 M resistance) are applied to larger cell bod-
duction (Higashijima et al., 1987) and modulation of ies (diameter 30 μm) the current recording is initi-
network excitability via GABA and glycine receptor- ated some 20 min after the G seal formation. Once
channel complexes (e.g. Chapters 14, 19 and 24 in this gramicidin ionophores are formed on the patch cell
volume). Considering the functional significance of membrane, the ion conducting channels can be con-
Cl, a method that allowed electrophysiological mea- stantly maintained for 2 h. It should be noted that
surements of membrane currents carried by Cl while gramicidin in the patch pipette solution loses its activ-
preserving native [Cl]i was required. These needs ity within 2 h. Thus the gramicidin pipette solution
prompted the development of the gramicidin-perforated should be refreshed frequently.
patch recording technique (Akaike, 1996). Gramicidin
is a polypeptide antibiotic that forms small pores in
the cell membrane similar to nystatin, but allows only
B.  GABA Responses
the movement of monovalent cations excluding anions
(Hladky and Haydon, 1972). The cation selectivity It is well established that both GABA and glycine
sequence of gramicidin in cell membranes is similar are the most important inhibitory neurotransmit-
to that measured in lipid bilayers: H  NH4  Cs  ters in the mammalian CNS (Lynch, 2004; Olsen and
 Rb  K  Na  Li (Myers and Haydon, 1972; Sieghart, 2009). Application of the gramicidin-perfo-
Tajima et al., 1996). Gramicidin is not permeable to Cl rated patch technique to mammalian CNS neurons
in lipid bilayers or in living cells (Myers and Haydon, makes possible the recording of native GABA- or
1972; Tajima et al., 1996). Thus, by adding gramicidin glycine-gated currents (Abe et al., 1994; Ebihara et al.,
to the patch pipette solution, we can observe native 1995; Kakazu et al., 1999). Figure 8.1B shows the
responses from electrically and chemically excitable GABA-induced hyperpolarization observed in a neu-
cells, while maintaining native [Cl]i, independently of ron dissociated from rat substantia nigra pars reticu-
the Cl concentration in the patch pipette solution. lata (SNR) recorded under current-clamp by means
of a gramicidin-perforated patch pipette. The rest-
ing membrane potential (Em) in this cell was 58 mV
(Ebihara et al., 1995). Under voltage-clamp condi-
II.  Application of gramicidin tions, GABA elicited an outward current at a hold-
perforated patch to cns ing potential (VH) of 50 mV. Since Cl is an anion,
neurons the outward current implies Cl flux into the cell. To
change the recording mode to conventional whole
cell patch configuration the patched membrane was
A.  Getting the Patch
ruptured by increasing suction in the pipette interior.
The stock solution of gramicidin is prepared by dis- This resulted in a clear-cut change in the direction of
solving gramicidin D (Sigma), which is a mixture of the GABA-induced current from outward to inward
iI.  Application of gramicidin perforated patch to cns neurons 143

A B GABA(10−5M) Em=−58mV

0.8
100mV

10s
C GABA(10−5M)
0.6

Current amplitude (nA)


VH=−50mV
0.4
Rupture

1 min ~10mM Cl−


150mM Cl−
12.5
0.2 22.5
32 200pA
40
5ms
200pA
0
40 80 120 10s
Time (min)
Figure 8.1  Gramicidin-perforated patch recording from substantia nigra pars reticulata (SNR) neurons. A. Relationship between the time
after establishment of a G seal and the peak capacitative current amplitude. Transient capacitative currents were elicited by 10 ms hyper-
polarizing voltage steps from 60 to 70 mV every 30 s. The inset shows the capacitative currents 1–40 min after forming the G seal. The
holding potential (VH) was 60 mV. B. Recording of membrane potentials using gramicidin-perforated patch technique under current-clamp
conditions. GABA (105 M) was exogenously applied during the period indicated by the horizontal bar. Resting membrane potential (Em) was
58 mV. C. GABA-induced outward (upper panel) and inward (lower panel) currents at a VH of 50 mV before and after the rupture of the
patch membrane, respectively. The upper panel shows GABA-induced outward current recorded by the gramicidin-perforated patch tech-
nique. Under these conditions, the intracellular Cl concentration ([Cl]i) was not disturbed by the 150 mM Cl concentration in the patch
pipette solution. The lower panel illustrates GABA-induced inward current in the same neuron after rupture of the patch membrane by
increasing the negative pressure of the patch pipette interior. The patch pipette Cl rapidly diffused into the cell interior within 5 min of the
rupture and the [Cl]i reached 150 mM. (Reproduced from Ebihara et al., 1995, with permission.)

(Fig. 8.1C). These results clearly indicate that Cl from auditory information from both the contralateral side
the patch pipette solution (150 mM Cl ) diffuses into as inhibitory glycinergic inputs and the ipsilateral
the neuronal cell body of SNR following rupture of side as excitatory glutamatergic inputs. This arrange-
the membrane patch. The resulting increase in [Cl]i ment is important for sound localization. In immature
shifts ECl toward more depolarized values than VH LSO neurons (P0–P2), glycinergic synaptic inputs or
causing Cl efflux through GABAA receptor-Cl exogeneous application of glycine induces membrane
channel complexes. Several observations have dem- depolarization in these neurons (Kakazu et al., 1999;
onstrated that Cl cannot permeate the gramicidin- Kandler and Friauf, 1995). The depolarizing response
formed pores, strongly suggesting that native [Cl]i is turns into a hyperpolarizing one with maturation
unaltered under gramicidin patch conditions (Akaike (Fig. 8.2A). Using gramicidin perforated patch record-
and Harata, 1994; Hladky and Haydon, 1972). Further ing it was possible to infer the native [Cl]i of LSO
validation of the method has been provided by using neurons at each developmental stage, from the rever-
independent measurements of [Cl]i by means of sal potential (EGly) of the glycine responses measured
fluorescent indicators. These reports show that [Cl]i from current/voltage (I/V) curves before and during
inferred from the reversal of GABA responses mea- glycine application under voltage-clamp conditions
sured through gramicidin patches is close to that (Ehrlich et al., 1999; Kakazu et al., 1999). In 50%
measured with fluorescent dyes (see Chapter 7 in this of LSO neurons at P0–P2 and P6–P8, EGly was more
volume and Achilles et al., 2007). depolarized than the resting membrane potential (Em).
At P13–P15 EGly became more hyperpolarized than Em
in 90% of LSO neurons. The differences in the mean
C.  Developmental Change in Glycine and values of EGly observed between P0–P2 and P13–P15
and between P6–P8 and P13–P15 were statistically
GABA Responses
significant (p  0.05) (Fig. 8.2B). The glycine-induced
The lateral superior olive (LSO) neurons in rat responses in rat LSO neurons were likely to result
brainstem, one of the central auditory nuclei, receives from Cl gradients favoring the flux of Cl through
144 8.  Gramicidin Perforated Patch

Figure 8.2  Developmental changes in glycine responses in lateral superior olive (LSO) neurons. Aa. At P0, 3  105 M glycine (solid bar)
induced a depolarization that generated an action potential. The record was obtained though a gramicidin-perforated patch under current-
clamp configuration. Ab. At P15, glycine induced a hyperpolarization, resulting in blockage of action potentials. Resting membrane potentials
(Em) were 53 and 60 mV in a and b, respectively. The reversal potentials of the glycine-induced currents (EGly) measured from the current/
voltage (I/V) relationships in the LSO neurons of P0 and P15 rats were 35 and 70 mV, respectively, at a VH of 50 mV. B. Developmental
changes in intracellular Cl concentration ([Cl]i) in LSO neurons. Histograms of the percentages of LSO neurons are indicated as a function of
EGly (a) and [Cl]i (b) at P0–2, P6–8 and P13–15 rats. (From Kakazu et al., 1999, reproduced with permission.)

glycine-operated Cl channels, and thus EGly was D.  Effect of Neuronal Trauma
considered to be equal to ECl. Under this assumption
it was possible to calculate [Cl]i for each individual When rat embryonic 18-day hypothalamic neu-
neuron using the Nernst equation. Figure 8.2B also rons are cultured for 24–35 days, GABA application
shows significant differences in [Cl]i between P0– induces a hyperpolarization. In neurons traumatized
P2 and P13–P15 (p  0.01), and between P6–P8 and by neurite transection, or osmotic challenges or excess
P13–P15 (p  0.01). These results clearly indicate that heat, synaptically released or exogenously applied
glycine’s effect switches with maturation from depo- GABA was depolarizing (Fig. 8.3A) and this depolar-
larizing to hyperpolarizing due to a fall in [Cl–]i in ization produced an increase in [Ca2]i (van den Pol,
LSO neurons during the second week after birth. 1996). The depolarization after trauma lasted longer
Age-related changes in [Cl]i were also observed in than a week.
the nucleus basalis of Meynert which is populated by
large cholinergic neurons and is a major source of cho-
linergic input to the cerebral cortex. The Meynert neu-
E.  Effect of H2O2 Treatment
rons receive GABAergic inputs. The EGABA measured During application of H2O2 for 1–6 h GABA-
from I/V curves became more negative with matu- induced outward currents decreased with time and
ration. The mean [Cl]i calculated from the Nernst finally reversed to inward currents after 9 h (Fig.
equation using both the known [Cl]o and the mea- 8.3Ba). This phenomenon appears to be due to loss of
sured EGABA had the following values: 34.8, 22.3 and tyrosine phosphorylation of KCC resulting in func-
13.1 mM for P0, 2-week- and 6-week-old rat neurons, tional down-regulation of this cotransporter and
respectively (Akaike, 1996). consequent increase in [Cl]i. Accordingly, during
III.  Conclusions 145

Figure 8.3  Change in polarity of GABA response induced by trauma or H2O2. Aa. 105 M GABA-induced membrane hyperpolariza-
tion from 60 to 72 mV in a rat hypothalamic neuron cultured for 33 days. b. In a traumatized neuron GABA depolarized the cell mem-
brane from 60 to 38 mV and produced an action potential (AP). All recordings were made using gramicidin-perforated patch configuration.
(Reproduced with permission from van den Pol et al., 1996). B. Changes in EGABA in primary cultured hippocampal neurons during continuous
incubation with H2O2. a. Representative whole-cell membrane currents recorded using the gramicidin-perforated patch technique, in response
to application of GABA (3  105 M; thick horizontal bars), recorded at a VH of 50 mV, before and at different times during H2O2 (5  105 M)
incubation as indicated. The GABA-evoked outward currents gradually decreased in amplitude, and eventually after 9 h incubation with H2O2
they changed to an inward current. b. Averaged EGABA measured in neurons incubated with H2O2 for up to 9 h as indicated. H2O2 incuba-
tion caused EGABA to rapidly (1 h) shift to a more depolarized level at a VH of 50 mV. ** p  0.01. (From Wake et al., 2007; reproduced with
permission.)

continuous H2O2 incubation EGABA is shifted towards (Thompson and Gahwiler, 1989). The [Cl]i inferred
more positive potentials and finally the GABA- from Egly indicated that [Cl]i increased approach-
induced current becomes inward (Fig. 8.3Ba). A more ing the values that would have if it was passively
gradual depolarizing shift in EGABA also occurred after distributed across the plasma membrane (Fig. 8.4B).
successive application of H2O2 (Fig. 8.3Bb), presum- These results are in agreement with the notion that a
ably corresponding to a time when total KCC protein furosemide-sensitive Cl extrusion mechanism plays
levels are decreased (Wake et al., 2007). an important role in maintaining [Cl]i below elec-
trochemical equilibrium in mature CNS neurons.
The furosemide-sensitive mechanism may be KCC.
F.  Effect of Furosemide on Intracellular [Cl]i The electro-neutral operation of this cotransporter
precludes direct observation of ion translocation by
Since Cl cannot permeate the gramicidin pores, electrophysiological techniques. However, using the
the constancy of GABA- or glycine-induced currents gramicidin-perforated patch method it is possible to
reflect the existence of [Cl]i regulatory mechanisms indirectly assess its function by estimating [Cl]i.
that maintain a stable [Cl]i. One of these mechanisms
is KCC, which extrudes Cl actively under physiolog-
ical conditions (Chapter 17). As shown in Fig. 8.4A, in
dissociated neurons from the lateral superior olive of
III.  Conclusions
rats the glycine-induced Cl currents recorded with
gramicidin patches gradually decrease in amplitude The development of the whole-cell patch technique
in the presence of 1 mM furosemide (Kakazu et al., has contributed enormously to our current under-
1999, 2000), a finding that is in agreement with pre- standing of membrane physiology. However, the dis-
vious observations made in hippocampal neurons turbance of cell constituents by the artificial patch
146 8.  Gramicidin Perforated Patch

of the rat: new approach with gramicidin perforated patch-clamp


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