Modulation by extracellular pH of the activity of GABAA receptors on rat cerebellum granule cells

0306-4522(94)E0147-V

h’eurrxcience Vol. 61, NO. 4, pp, 833-837, 1994 ElsevierScienceLtd Copyright 0 1994 IBRO Printed in Great Britain. All rights reserved 0306~4522/94 $7.00 + 0.00

MODULATION BY EXTRACELLULAR pH OF THE ACTIVITY OF GABA, RECEPTORS ON RAT CEREBELLUM GRANULE CELLS M. ROBELLO,*t *Dipartimento

P. BALDELLI* and A. CUPELLOS

di Fisica, Universiti di Genova, Via Dodecaneso 33, 16146 Genova, Italy $Centro di Neurofisiologia Cerebrale, C.N R., Geneva, Italy

Abstract-The Cl- currents activated by GABA via GABA, receptors in rat cerebellum granule cells in culture were studied by whole-cell patch-clamp. These currents were measured at various extracellular pH. The currents activated by 100 PM GABA, both the peak and the steady-state component, increase at acidic pH’s and decrease at basic pH’s. The transition point being at around 7.7. Interestingly, passing from pH 7.4 to 6.4 the GABA dose response curve indicates that the increases in the peak current are relateh to an augmented maximal current. The increases in the steady-state component are mainly due to a higher affinity of the receptors for the neurotransmitter and disappear at saturating [GABA]. The study of the I-Vcurves for the GABA activated peak Cl- currents at pH 6.4, 7.4 and 8.4 reveals linearity in the latter instance. However, an outward rectification is present at the two more acidic pH’s. This fact suggests that the protonation of basic amino acids at the acid pH does involve rectification of Cl- channel conductance. Overall, the data indicate that slight changes in in situ extracellular pH may have profound influences on GABA, receptor function.

tiondeprotonation events, the net charge present in such cases is heavily influenced by pH. In other words, it appears very likely that pH may influence in opposite ways the function of cationic versus anionic ionotropic receptors. Quite recently, data have been presented showing that N-methyl-D-aspartate glutamate receptors are modulated by H+ .“.13

The concept that ion channels activated by a neurotransmitter are part of a superfamily of ionotropic receptors has emerged a few years ago.’ This idea derived from the evaluation of the primary sequences of the subunits for the GABA* receptor and their comparison with those of the glycine and nicotinic acetylcholine (ACh) receptors. Experiments have revealed that at the mouths of these ionic channels there is a concentration of amino acids giving charges opposite to those of the permeating ions. Thus, at the mouth of the Cl- channel activated by GABA there is an accumulation of lysine and arginine, which are likely to give positive charges by protonation at neutral PH.‘,” On the contrary, at the mouth of the pore opened by ACh at the nicotinic receptor there is an accumulation of amino acids such as glutamate and aspartate, which give a negative charge.6 It seems logical to infer that accumulation of such amino acid residues which give a charge opposite to the one of the permeating ion has been favoured by evolution. This condition would favour a local accumulation of the permeating ion thereby increasing the driving force for its permeation.6 It is obvious that since the presence or absence of charges at these amino acid residues is due to protona-

EXPERIMENTAL

PROCEDURES

Cell cullure Granule cells were prepared from eight-day-old Wistar rat (Morini, Italy) cerebella following the procedure of Levi et a/., as previously described.9 Cells were plated at a density of 2 x IO6 per dish on glass coverslips placed in 35-mm plastic dishes and kept at 37°C in a humidified 95% air 5% CO, atmosphere. Experiments were performed between days 5 and 12 after plating. Electrophysiology Voltage-clamp recording from the dissociated neuron was carried out in whole-cell mode at room temperature. Patch pipettes were prepared from borosilicate glass capillaries (Type 1406129 Hilgenberg, Malsfeld, F.R.G.) using a programmable Sachs and Flaming puller model PC-84. The resistances of the patch pipettes filled with the internal solution varied from 5 to 10 MR. Ionic currents were recorded with a patch-clamp amplifier EPC-7 (List-Electronic, Darmstadt, F.R.G.) and filtered at 3 kHz. Both stimulation and data acquisition were performed with a Labmaster D/A, A/D converter driven by pClamp software

jTo whom correspondence should be addressed. Abbreviukms: ACh, acetylcholine; EGTA, ethylene glycolbis (amino ethyl-ether) tetra-acetate; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid. 833

834

M. Robello et rtl.

(Axon Instrument, Burlingame, CA). Data were fitted to models using the software Asystant (Asyst Software Technologies, Rochester, NY). S&lions In all experiments the internal solution contained (in mM): 142 KCI, 4 MgCI?, IO HEPES, 2 EGTA, 3 ATP. The pH was adjusted to 7.3 with a Trizma base.

The external solution contained (in mM): 135 NaCI, 5.4 KCI. 1.8 CaCI,, 1 MgCI,, 5 HEPES, IO glucose. The pH was adjusted using NaOH. Only in I/V experiments 3 x lO_‘M tetrodotoxin was added to the external solutions and the following internal solution was used (in mM): 124 CsCI, 1 CaCl,, IO HEPES, I I EGTA, 4 MgCl,, 3 ATP. The pH was adjusted to 7.3 with a Trizma base.

GABA _

6

pH=6.4 4

1

100

10

GABA

(yM)

pH=7.4

80 :

GABA 40

20

0

I

I

I

1

10

100

GABA

(pM)

Fig. 1. Left panels--currents activated in whole-cell recording by 10m4 M GABA in a granule cell voltage clamped at -80 mV at three different pH’s of the external bathing solution. Time-course of desensitization can be fitted by Eqn 1 (dashed lines) using the following parameter values: pH 6.4 (I, = 1784 pA, r, = 2 ms. I, = 574 pA, 7z = 60 ms); pH 7.4 (I, = 1415 pA, I, = 2 ms, I2 = 670 PA, rz = 70 ms); pH 8.4 (I, = 1320 pA, time-constants as a function of I, = 1.6 ms, I2 = 420 pA, r2 = 56 ms). Right: r, and rz desensitization GABA concentration at pH = 6.4 (e), 7.4 (A) and 8.4 (m). Points are the mean value of two experiments.

GABA,

835

receptors and pH

GABA

L...i~.__..J.~_._--------L-.3 IC

9

8

7

5

5

PH

s 2

GABA was dissolved in distilled water (10 mM) and then diluted with the external solution at the desired concentration just before experiments were conducted. Once diluted GABA was applied together with the external solution to the cell bath by steady perfusion i3 ml/min gravity flow). All chemicals were purchased from Sigma Chemical Co., St Louis, MO. RESULTS GABA induced Cl _ currents as a function I$ extra-

cellular pH Application granule

of 10m4M GABA

to the exterior of

cells evoked a Cl- current which displays a

peak (I,) and a steady-state (I,,) (Fig. 1). The pH of the external medium varied between 9.5 and 5.5, and the GABA activated Cl- current determined (I, and I,,). Figure la shows GABA currents at three different pH’s: 6.4, 7.4 and 8.4. lP and 1, are plotted in Fig. 2 as a function of pH. In both cases the current increased at lower pH, with a sigmoidal trend. The transition pH was around 7.7 in both cases. Fitting the data in a Hill plot gave an n value of around I both for I, and I,,. The ratios I,,, (at pH 5.5)/&, (at pH 9.5) were 1.51 for Ip and 1.41 for I,,. Dose-response

curves at various pH

The dose-response curves for GABA activated Clcurrents at pH 6.4, 7.4 and 8.4 are reported in Fig. 3a,b for I, and I,,, respectively. Since, as quoted

300

zoo

Fig. 2. Semilogarithmic plot of peak amplitude (&,)(a) and steady-state current (I_) (b) as a function of the external solution pH. Theoretical fitting is obtained from the equation: I = (I_ -I,,,) C”/C” + Ki + I,,,,, using the parameters: n = 0.98, K,,= 7.7pH units, im, = 2420 pA, I,,, = 1600 pA for the f, and n = 0.84, K,, = 7.7 pH units, I,, = 380 pA, I,,, = 270 pA for the I,. Each cell was voltage clamped at -8OmV and perfused by 10m4M GABA at various pH of the external solution. Each point is the average of n = 10 - 20 experiments i: S.E.

(PM)

100

Fig. 3. Dose-response curves. Semilogarithmicplot of peak (a) and steady-state current (b) as a function of GABA concentration at pH ~6.4 (0). 7.4 (A) and 8.4 (i). Theoretical fitting is obtained from the equation: I =I,,,C”/C” + Kz, using the values of the parameters shown in Table 1. Each point is the average of n = 10 - 20 experiments i: S.E. Same experimental conditions as in Fig. 1.

above, the transition point is at pH 7.7, the decreases due to basic pH (8.4) are more evident than the increases at the acid one (6.4). The decreases at basic pH for r, appear to be due to both a lower i,,,,, and a higher K,, in the respect of the pH close to neutrality (Table 1). GABA, receptor desensitization at various pH’s The Cl- currents activated by GABA can be fitted in their temporal behaviour (see, for example, Fig. 1% left column) by the equation: f(t) = I,e-“rl + [2e-W,

(1)

Table 1. Parameter values obtained fitting the peak and the steady-state current activated by GABA at pH 6.4, 7.4 and 8.4 to the Hill equation (see Fig. 3) 1,

1, PH

n

imax

%

n

Jlza,

6.4 7.4 8.4

0.8 0.8

2450pA 2370pA 1870 pA

7pM 7.5~M 10pM

0.6 0.5 0.5

290pA 310pA 240pA

1

Kd

l.lpM 3.7pM 3rcM

836

M. Robe110el nl. Table 2 EKecectof lo-’ M flunitrazepam on the chloride peak current activated by 10 PM GABA at pH 6.4, 7.4 and 8.4 PH

I,, (PA) GABA 10fiM

Ip @A) Flunitrazepam 100nM

Increment

6.4 1.4 8.4

1400+ 280 (n = 16) 1250& 210 (n = 12) 960 + 180 (n = 16)

1510* 250 (n = 7) 1390+200 (n = II) 1120* 180 (n =9)

11% 17%

In this equation two desensitization time-constants r, and r2 appear for the rapid and the slow process. The data in the column on the right in Fig. I show that pH in the 6.4-8.4 interval does not influence significantly the dependence of T, and cl on [GABA].

8%

phenomenon is evident at a more acidic pH 6.4. In fact, at this pH there is an outward rectification of the Cl- current at positive V inside.

DISCUSSION

Eflect of ,jlunitrazepam at various pH’.s The effect of 100 nM tlunitrazepam on the Cl-~ current activated by 10 PM GABA at pH 6.4, 7.4 and 8.4 is reported in Table 2. The potentiation by this benzodiazepine is only slight but increases steadily from more acid to more basic pH. I-V plots .jbr GABA-activated JJH’.F

currents at various

The I-V plots are reported in Fig. 4. They show the trend already described above by which the Clcurrent is increased at acid pH, whereas the opposite happens at the basic pH. The additional information is that this applies to the whole V range studied (- 80 to +80 m V inside) implying that the effect is independent of the actual direction of Cl- fluxes (inward at positive and outward at negative intracellular potentials). However, a distinct difference of the curves at the three different pH’s is that no rectification is present at pH 8.4 whereas such a (j”1)

JO””

I

l

1

Fig. 4. Typical whole-cell I-V relations during application of 10 p M GABA at pH = 6.4 (@), 7.4 (A) and 8.4 (m). The membrane potential was changed ramp-wise from -80 to +80 mV, duration I s. Leakage current measured in the absence of GABA was subtracted. The solutions used in these experiments were such to minimize currents other than the Cl- one (see Experimental Procedures).

The influence of net charges at the entrance of an ionic pore on pore conductance has been thoroughly described by Dani.’ It appears logical that the presence of a net charge opposite to that of the permeating ion species is instrumental in determining an augmented concentration of the ion and an increase in the driving force for permeation. In the case of an asymmetricity of the net charge across the pore with, say, the charge present at one pore entrance and not at the other one, the ionic conductance from the side of the charge to the other one would be higher than vice versa. This has been predicted theoretically4 and found experimentally for the nicotinic ACh receptor.6 It is thus clear that pH plays an important role in modulating conductance across neurotrdnsmitter-activated ionic pores. This appears especially true taking into account that ionic channels activated by neurotransmitters present an accumulation of basic” or acid6 amino acid residues at the ionic pore mouth. The former case involves anionic channels for Cl- and the latter cationic channels for Na’ and K +. In the present approach we have studied by whole cell patch-clamp the function of cerebellar granule cell GABA, receptors at various pH’s of the extracellular medium. It has been demonstrated previously that the currents measured under the present conditions are GABA, receptor mediated Cl- currents.‘” The data show an augmented function of GABA, receptors at acid pH and a lowered one at basic pH. Working with 100 PM GABA, the pH dependence of both I, and I,, is described by a sigmoid with a transition pH close to neutrality, 7.7. Since for all the three pH’s studied (6.4, 7.4 and 8.4) 100 p M [GABA] is in the saturation range (Fig. 3), this transition represents, mainly, effects on the maximal Cl-- current obtainable. Considering the data in Table I referring to the I,, it is evident that minor changes in I,,,, and Kd occur at acid pH. However, at basic pH (8.4) there is both a marked decrease in I,,,, and a clear reduction in affinity. This implies that partial deprotonation of basic amino acid on the receptor extracellular side, affects both the GABA recognition site and the Cl- channel conductance. Speaking of

GABA,

receptors and pH

837

the I,, component, the data suggest a decrease in ISSmnr Apart from the information about the nature of the GABA, receptors of the granule cells, which has been at pH 8.4 vs pH 7.4. However, passing from 7.4 to 6.4 there is instead an increased affinity for GABA. We discussed above, interesting physiological hints derive from the present results. Firstly, it has been demonhave previously discussed r, and I, as repre~nting strated that GABA, receptor activation in GABAtwo distinct populations of GABA, receptors with, results in muscles* and neurons’ acceptive respectively, a high and a low rate of desensitizaintracellular acidification and extracellular alkalinizdtion.” Evidently, two such populations differ in the tion. Thus, one can suggest that the decreased funcdistribution of basic amino acids on their extracellution of GABA, receptors upon extracellular lar side. For instance, the GABA recognition site for alkalinization is a built-in mechanism for a Ip is more sensitive to removal of protons at basic homeostatic control of GABA postsynaptic inhipH’s than to their addition at acid pH’s. The opposite bition. In other words, in cases of an excessive is true for the is, component. The desensitization process for granule cell GABA action there would be an extracellular alkalinization so as to decrease further GABA inhibition GABA, receptors is independent of pH (Fig. lb), whereas the facilitatory effect by flunitrazepam is at the GABA, receptor. Secondly, under certain pathological conditions such as during epileptic slightly higher at basic pH (Table 2). seizures there is acidosis in the brain tissue.’ Thus, The exami~tion of the I-i/ curves for lp at the an increase in inhibitory activity at the GABA, three pH’s studied reveals that alkalinization from pH 7.4 to 8.4 reduces the Cl- current evoked by receptor may participate, among other things, in the termination of seizure activity m the brain. 10e4 M GABA and renders the I-V plot linear in the Finally, a key role is played by fast GABA, mediated whole range of inside voltages studied (-80 to inhibition m controlling excitatory events in +80 mV). neurons and in particular in preventing M-methyl-DHowever, at more acidic pH of 6.4 besides a higher aspartate receptor activation.’ Thus, local extraceliuCl- current throughout the whole voltage range the lar pH variation may modulate step by step GABA activated current presents a clear outward rectification at positive inside voltages (Fig. 4). This inhibitory events in neurons and their interaction with excitation. This is especially true around rectification suggests the importance of net positive charges by protonation of basic amino acids at the the physiological pH 7.3’, where the change in extracellular mouth of the Cl-- pores. This in fact the GABA, receptor activity is particularly steep (Fig. 2). results in an easier out-in passage for Cl-- anions.

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