Académique Documents
Professionnel Documents
Culture Documents
8
Gramicidin Perforated Patch
Norio Akaike
o u t l i n e
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 58 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- (23 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
(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, 3 105 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. 105 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 (3 105 M; thick horizontal bars), recorded at a VH of 50 mV, before and at different times during H2O2 (5 105 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
Mistry, D.K. and Hablitz, J.J. (1990). Nystatin-perforated patch Tajima, Y., Ono, K., and Akaike, N. (1996). Perforated patch-clamp
recordings disclose NMDA-induced outward currents in cul- recording in cardiac myocytes using cation-selective ionophore
tured neocortical neurons. Brain Res. 535, 318–322. gramicidin. Am. J. Physiol. 271, C524–C532.
Myers, V.B. and Haydon, D.A. (1972). Ion transfer across lipid mem- Thompson, S.M. and Gahwiler, B.H. (1989). Activity-dependent dis-
branes in the presence of gramicidin A. II. The ion selectivity. inhibition. II. Effects of extracellular potassium, furosemide, and
Biochim. Biophys. Acta 274, 313–322. membrane potential on ECl in hippocampal CA3 neurons. J.
Nabekura, J., Ueno, T., Okabe, A., Furuta, A., Iwaki, T., Shimizu- Neurophysiol. 61, 512–523.
Okabe, C., Fukuda, A., and Akaike, N. (2002). Reduction of Ueno, S., Ishibashi, H., and Akaike, N. (1992). Perforated-patch
KCC2 expression and GABAA receptor-mediated excitation method reveals extracellular ATP-induced K conductance in
after in vivo axonal injury. J. Neurosci. 22, 4412–4417. dissociated rat nucleus solitarii neurons. Brain Res. 597, 176–179.
Olsen, R.W. and Sieghart, W. (2009). GABA A receptors: subtypes pro- Uneyama, H., Uneyama, C., and Akaike, N. (1993). Intracellular
vide diversity of function and pharmacology. Neuropharmacology mechanisms of cytoplasmic Ca2 oscillation in rat megakaryo-
56, 141–148. cyte. J. Biol. Chem. 268, 168–174.
Omura, T., Munakata, M., and Akaike, N. (1993). Nystatin-perfo- van den Pol, A.N., Obrietan, K., and Chen, G. (1996). Excitatory
rated patch recordings disclose KA-operated outward currents actions of GABA after neuronal trauma. J. Neurosci. 16, 4283–4292.
in rat cortical neurons. Brain Res. 627, 345–348. Wake, H., Watanabe, M., Moorhouse, A.J., Kanematsu, T., Horibe,
Rae, J., Cooper, K., Gates, P., and Watsky, M. (1991). Low access S., Matsukawa, N., Asai, K., Ojika, K., Hirata, M., and Nabekura,
resistance perforated patch recordings using amphotericin. B. J. J. (2007). Early changes in KCC2 phosphorylation in response to
Neurosci. Methods 37, 15–26. neuronal stress result in functional downregulation. J. Neurosci.
Rhee, J.S., Ebihara, S., and Akaike, N. (1994). Gramicidin perforated 27, 1642–1650.
patch-clamp technique reveals glycine-gated outward chloride Wendt, D.J., Starmer, C.F., and Grant, A.O. (1992). Na channel kinet-
current in dissociated nucleus solitarii neurons of the rat. J. ics remain stable during perforated-patch recordings. Am. J.
Neurophysiol. 72, 1103–1108. Physiol. 263, C1234–C1240.
Shirasaki, T., Harata, N., and Akaike, N. (1994). Metabotropic gluta- Ye, J.H. and Akaike, N. (1993). Calcium currents in pyramidal neu-
mate response in acutely dissociated hippocampal CA1 pyrami- rons acutely dissociated from the rat frontal cortex: a study by
dal neurones of the rat. J. Physiol. 475, 439–453. the nystatin perforated patch technique. Brain Res. 606, 111–117.
Sims, S.M., Lussier, B.T., and Kraicer, J. (1991). Somatostatin acti-
vates an inwardly rectifying K conductance in freshly dis-
persed rat somatotrophs. J. Physiol. 441, 615–637.