HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Krishek, B. J. Articles by Smart, T. G. Search for Related Content PubMed PubMed Citation Articles by Krishek, B. J. Articles by Smart, T. G.

Proton sensitivity of rat cerebellar granule cell GABA_A receptors: dependence on neuronal development

Belinda J. Krishek and Trevor G. Smart

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

1. The effect of GABA_A receptor development in culture on the modulation of GABA-induced currents by external H⁺ was examined in cerebellar granule cells using whole-cell and single-channel recording.

2. Equilibrium concentration-response curves revealed a lower potency for GABA between 11 and 12 days in vitro (DIV) resulting in a shift of the EC₅₀ from 10.7 to 2.4 M.

3. For granule cells before 11 DIV, the peak GABA-activated current was inhibited at low external pH and enhanced at high pH with a pK_a of 6.65. For the steady-state response, low pH was inhibitory with a pK_a of 5.56.

4. After 11 DIV, the peak GABA-activated current was largely pH insensitive; however, the steady-state current was potentiated at low pH with a pK_a of 6.84.

5. Single GABA-activated ion channels were recorded from outside-out patches of granule cell bodies. At pH 5.4-9.4, single GABA channels exhibited multiple conductance states occurring at 22-26, 16-17 and 12-14 pS. The conductance levels were not significantly altered over the time period of study, nor by changing the external H⁺ concentration.

6. Two exponential functions were required to fit the open-time frequency histograms at both early (< 11 DIV) and late (> 11 DIV) development times at each H⁺ concentration. The short and long open time constants were unaffected either by the extracellular H⁺ concentration or by neuronal development.

7. The distribution of all shut times was fitted by the sum of three exponentials designated as short, intermediate and long. At acidic pH, the long shut time constant decreased with development as did the relative contribution of these components to the overall distribution. This was concurrent with an increase in the mean probability of channel opening.

8. In conclusion, this study demonstrates in cerebellar granule cells that external pH can either reduce, have no effect on, or enhance GABA-activated responses depending on the stage of development, possibly related to the subunit composition of the GABA_A receptors. The mode of interaction of H⁺ at the single-channel level and implications of such interactions at cerebellar granule cell GABA_A receptors are discussed.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter present in the mammalian CNS, a role that relies upon activation of the 'type A' GABA receptor. These receptors are hetero-oligomers composed of different subunits selected from six families designated as: (1-6), (1-3), (1-3), (1), (1) and ð (1) (Sieghart, 1995; Davies et al. 1997; Bonnert et al. 1999). Whilst neuronal GABA_A receptors are likely to be composed of various combinations of subunits, receptor expression studies indicate that , and subunit co-assemblies form functional ion channels exhibiting many aspects of the neuronal GABA_A receptor's pharmacological repertoire (Sigel et al. 1990; Verdoorn et al. 1990).

Due to their ease of isolation and well-documented development, granule neurones of the cerebellum are an attractive cell type in which to study the expression, organisation and developmental regulation of GABA_A receptors (Wisden et al. 1996). Cerebellar granule cells are known to possess at least six subunit genes encoding 1, 6, 2, 3, 2 and subunits, which leads to the possibility that they express several distinct GABA_A receptor subtypes (Wisden et al. 1996). Although the assembly pathway used to discriminate between different receptor subunit combinations remains unknown, a number of different GABA_A receptor subunit combinations derived from both pharmacological and biochemical analyses of recombinant receptors have been postulated: current views accommodate 12/32, 62/32, 162/32, 162/32, 12/32 and 62/3 GABA_A receptors (Korpi & Luddens, 1993; Mertens et al. 1993; Carucho & Costa, 1994; Khan et al. 1994, 1996; Quirk et al. 1994; Caruncho et al. 1995; Korpi et al. 1995; Pollard et al. 1995). Moreover, there appears to be a specific association between the 6 and subunits, with the removal of the 6 protein in knockout animals causing a selective loss of the subunit (Jones et al. 1997).

Interestingly, it has been shown that protons endogenously modulate both invertebrate muscle GABA (Takeuchi & Takeuchi, 1967; Smart & Constanti, 1982) and vertebrate neuronal GABA_A receptors (Kaila et al. 1993; Robello et al. 1994; Krishek et al. 1996; Pasternack et al. 1996; Huang & Dillon, 1999). Moreover, modulation by external H⁺ is dependent upon the receptor subunit composition (Krishek et al. 1996) and this has implications for native GABA_A receptors that differentiate throughout development. The granule cell contains GABA_A receptor subunit genes which undergo developmental changes in levels of expression presumably commensurate with the different roles played by GABA in the neonate and adult. The 1 subunit mRNA is detected very early postnatally, whereas the 6 and subunit mRNAs are only observed after postnatal day 6 (P6) and P12, respectively (Laurie et al. 1992). Furthermore, there is a parallel increase in both the 1 and 6 subunit mRNAs at later stages between P14 and P21, with a peak for both subunits at P21 (Bovolin et al. 1992; Zheng et al. 1993, 1994). The relative abundance of these subunit mRNAs does not differ between P21 and adult cerebellum, whereas abundance for 1 message is double that of the 6 mRNA (Bovolin et al. 1992; Zheng et al. 1993). The aim of the present study was to investigate the pharmacological implications of cerebellar granule cell maturation by ascertaining GABA_A receptor sensitivity to external H⁺ using both whole-cell and single-channel analyses. We demonstrate that for a single cell type, at different stages of development, GABA-activated responses exhibit differential sensitivity to external H⁺.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Cell preparation: rat cerebellar granule cells

Whole cerebellar tissues were removed from P4 Sprague-Dawley rats (killed by decapitation) and mechanically dissociated by expulsion from a 5 ml syringe through a sterile nylon mesh with a 210 m weave. The dissociated cells and microexplants were grown on poly-L-lysine in minimum essential medium (Gibco) with Earle's salts supplemented with 10% v/v fetal calf serum, 10% v/v horse serum, 2 mM glutamine, 0.6% w/v glucose, 100 units ml^-1 penicillin-G and 100 g ml^-1 streptomycin. After 24 h, 10% v/v fetal calf serum was removed and 20 mM KCl added. Thereafter, the culture medium was replenished every 3-4 days and neurones were incubated at 37°C in humidified 95% air-5% CO₂. Granule neurones possessing membrane potentials of -40 to -70 mV, were used after 3 days in vitro (DIV) and up to 10/11 DIV for 'early-time' recordings and from 12 to 17 DIV for 'late-time' recordings. The experiments were carried out at room temperature (21-23°C).

Whole-cell and single-channel recording

Experiments on cultured neurones were performed using Axopatch-1C and List EPC7 amplifiers. For whole-cell recording patch electrodes (1-5 M) were filled with a solution containing (mM): 120 KCl; 1 MgCl₂; 1 CaCl₂; 10 Hepes; 11 EGTA; and 2 adenosine triphosphate; pH 7.1. Sylgard-coated patch pipettes (10-15 M) for single-channel recording contained (mM): 130 NMDG; 20 TEA-Cl; 0.24 CaCl₂; 10 glucose; 10 Hepes; 5 EGTA; and 2 adenosine triphosphate; pH 7.3. The cells were continuously superfused with a Krebs solution containing (mM): 140 NaCl; 4.7 KCl; 1.2 MgCl₂; 2.5 CaCl₂; 5 Hepes; and 11 glucose. Whole-cell voltage clamp and single-channel current data were filtered at 1.5 kHz (-3 dB, 8 pole Butterworth filter, 48 dB per octave) and recorded on a Gould WindoGraf 40-8474-00 pen recorder and DTR-1201 digital tape-recorder, respectively.

Analysis of GABA-activated membrane current

GABA-induced peak whole-cell membrane currents (I) were measured and normalised (I_N) to the peak response induced by EC₅₀ concentrations of GABA ranging from 10 M (early times) to 2.5 M (late times). These data were used to construct equilibrium concentration-response relationships for GABA and were fitted with the Hill equation:

I_N/I_N,max = 1/(1 + (EC₅₀/[A])ⁿ),

where I_N and I_N,max represent the normalised GABA-induced peak current at a given concentration and the maximum peak current induced by a saturating concentration of GABA, respectively. EC₅₀ defines the concentration of GABA (A) which induces 50% of the maximum response and n is the Hill coefficient.

pH model

The pH titration data for early and late time recordings were fitted (as appropriate) according to the following function using a non-linear least-squares Marquardt-Levenburg routine:

I = f{(K_a1(nK_a2 + 2m[H⁺]) + l[H⁺]²)/([H⁺]² + K_a1(2[H⁺] + K_a2))}.

This function provided estimates of the pK_a values. The terms l, m and n weight the relative contribution each form of the receptor protein (P) makes to the overall titration curve, where l weights the undissociated receptor protein (PH₂), m the monovalent anion (PH^-) and n the divalent anion (P^2-) for a receptor protein exhibiting two pK_a values (Krishek et al. 1996). I represents the change in GABA-induced current.

Single-channel analysis

Single GABA channel currents were analysed in excised outside-out membrane patches from rat cerebellar granule cell bodies. Patches were accepted for kinetic analysis if there appeared to be only one active channel, or the number of multiple channel openings never exceeded 2% of all detected openings. Data were replayed from tape, digitised at 20 kHz and analysed using Strathclyde Electrophysiological Software (SES). Gaussian curves were fitted to the amplitude distributions defining the mean current, peak current and standard deviation using a non-linear least-squares routine. The single-channel conductance was calculated from the mean unitary current determined from the Gaussian curve fits. Separate open and shut durations were measured using a 50% threshold cursor to the main single-channel current in each patch. The transition detection of open and shut events was then used to form an idealised record of the digitalised data. Frequency histograms were constructed from the measured individual open and shut durations and analysed by fitting exponential functions. Using a Levenburg-Marquardt non-linear least-squares routine the area under the exponential curve, the time constants and standard errors of the mean were determined.

Drugs and solutions

Solutions were rapidly applied to neurones from a nearby (100-200 m) Y-tube. A complete exchange of solution around the cells was effected within 10-50 ms. All drug solutions were made using Krebs solution at the appropriate pH.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Developmental changes in GABA-induced currents

To characterise the GABA_A receptors in these cerebellar granule cells, concentration-response relationships were examined over a 3 week developmental time period in culture. Neurones at 13 DIV exhibited an increased sensitivity to GABA (0.5-100 M) compared to 9 DIV together with larger whole-cell currents (Fig. 1A and B). In addition, neurones at 13 DIV also exhibited an increased holding current noise in the absence of applied GABA. To establish whether the increased sensitivity to GABA occurred gradually over this period or whether there was a particular time during which the sensitivity to GABA 'switched', equilibrium concentration-response curves were constructed. Interestingly, these curves revealed an increased GABA potency occurring at 11-12 DIV resulting in a 4-fold significant (P < 0.05) decrease in the GABA EC₅₀ (Fig. 1C, Table 1).

		View larger version [in this window] [in a new window]
Figure 1 Developmental changes in GABA-induced currents in cerebellar granule cell GABAA receptors A and B, membrane currents evoked by rapidly applied 0.5-100 M GABA at 9 DIV (A) and 13 DIV (B). Current calibration of 100 pA corresponds to A and 200 pA to B. GABA was applied for the duration indicated by the continuous line. Records are from 2 neurones at pH 7.4 and recorded under whole-cell voltage clamp at -50 mV. C, left, normalised GABA-activated current (IN)-concentration curves constructed at 6 DIV (), 8 DIV (), 9 DIV (), 10 DIV (), 11 DIV (), 12 DIV (), 13 DIV (), 14 DIV (), 15 DIV () and 17 DIV (). Right, the same developmental data represented as two separate GABA concentration curves at 6-11 DIV () and 12-17 DIV (). All data were fitted to the Hill equation (n = 30) and EC50 values and Hill coefficients determined (Table 1).

Proton sensitivity of granule cell GABA_A receptors

To determine the sensitivity of granule cell GABA_A receptors to varying concentrations of external H⁺, GABA-activated membrane currents were recorded from cells before (early times) and after (late times) 12 DIV. In granule cells at 6 DIV, GABA-activated responses (peak and steady state) were clearly reduced at low pH (Fig. 2A); however, at high pH, the peak response to GABA was slightly enhanced, whereas the steady-state response was unchanged (Fig. 2B). The pH titration relationships for the peak GABA-activated current in cells recorded at 3, 6, 8 and 10 DIV revealed a sigmoidal plot which could be accounted for by assuming a site on the GABA_A receptor with a pK_a of 6.65 ± 0.21 (Fig. 2C and Table 1). The pH titration relationship for the steady-state GABA-activated response was described by a pK_a of 5.56 ± 0.17 (Fig. 2C and Table 1). In contrast, at 14 DIV, decreasing the pH from 7.4 to 5.4 resulted in either no or only a small enhancement in the peak response to GABA and a larger enhancement of the steady-state GABA response (Fig. 3A). Increasing the pH from 7.4 to 9.4 resulted in a small potentiation in the peak response to GABA and a subsequent reduction in the steady-state response suggestive of increased receptor desensitisation (Fig. 3B). The pH titration relationship for the peak GABA-activated current in cells recorded at 12, 14, 16, 17 and 20 DIV revealed a relative insensitivity to H⁺ (Fig. 3C). In contrast, the pH titration relationship for the steady-state GABA response exhibited an increased sensitivity to H⁺ over the experimental time period with a pK_a of 6.84 ± 0.26 (Fig. 3C and Table 1).

		View larger version [in this window] [in a new window]
Figure 2 Proton sensitivity of cerebellar granule cell GABAA receptors at early developmental times A and B, membrane currents activated by 10 M GABA at 6 DIV in granule cells at -50 mV holding potential. Cells were exposed to either pH 5.4 (A) or pH 9.4 (B) Krebs solution followed by a recovery (5 min) at pH 7.4. C, pH titration for the peak (left) and steady-state (right) GABA-activated inward currents at 3 DIV (), 6 DIV (), 8 DIV () and 10 DIV (). Data are normalised to the response obtained at pH 7.4 at each developmental time. All points are means ± S.E.M. (n = 12). The data were fitted with the pH model (see Methods) and pKa values determined (Table 1).

		View larger version [in this window] [in a new window]
Figure 3 Modulation of GABA-activated currents in cultured cerebellar granule cell neurones by H+ at late developmental times A and B, membrane currents were activated by 2.5 M GABA and recorded at -50 mV holding potential from granule neurones at 14 DIV. Cells were exposed to GABA at pH 5.4 (A) and pH 9.4 (B) followed by a recovery at pH 7.4. C, pH titration for the peak (left) and steady-state (right) GABA-activated currents at 12 DIV (), 14 DIV (), 16 DIV (), 17 DIV () and 20 DIV () (n = 15). The data for the steady-state GABA-induced responses were fitted with the pH model and pKa values determined (Table 1).

To assess whether modulation by H⁺ was affecting the GABA-activated current via a voltage-dependent mechanism, current-voltage (I-V) relationships were determined at early and late times for the peak and steady-state responses to GABA at pH 7.4 and 5.4. At 5 DIV, both the peak and steady-state I-V relationships displayed outward rectification (Fig. 4A) with low pH reducing the I-V slopes in a voltage-insensitive manner. In contrast, at 15 DIV, the peak I-V relationships displayed slight outward rectification (Fig. 4B) with low pH only slightly increasing the slope in a voltage-insensitive manner. However, the steady-state I-V relationships displayed significant outward rectification (Fig. 4B) with low pH increasing the chord conductance in a voltage-insensitive manner. The GABA response reversal potential was unaffected by low pH for neurones at either 5 or 15 DIV.

		View larger version [in this window] [in a new window]
Figure 4 Voltage dependence of GABA-activated current modulation by H+ A and B, current-voltage relationships for the peak (left) and steady-state (right) GABA-activated responses at 5 DIV (A; 10 M GABA) and 15 DIV (B; 2.5 M GABA) at pH 7.4 () and pH 5.4 (). The curves for peak and steady-state GABA-activated responses were generated using either 2nd or 3rd order polynomials. The GABA reversal potentials did not deviate significantly from 0 mV.

Effect of development and H⁺ on single GABA channel currents in outside-out patches

To ascertain the mechanism underlying the observed change in the proton sensitivity during development of GABA_A receptors, outside-out patches were formed from the cell bodies of granule cells over a similar time period and the single GABA channel currents and associated kinetics were studied. Outside-out patches, maintained at -70 mV and exposed to 1 M GABA, rapidly responded with brief single openings or bursts of single-channel currents (Fig. 5) which disappeared in control Krebs solution. At 7 DIV, single-channel GABA-activated currents exhibited a notable decrease in the number of channel openings at high H⁺ concentrations (pH 5.4; Fig. 5A and B) which was not observed at 14 DIV (Fig. 5C and D) and could underlie the changes noted in whole-cell GABA-activated currents at these external pH values.

		View larger version [in this window] [in a new window]
Figure 5 H+ modulation of single-channel GABA-activated currents from cerebellar granule neurones at differing developmental stages GABA (1 M)-activated single-channel currents recorded from 2 representative neurones at 7 DIV (A and B) and 14 DIV (C and D) at -70 mV holding potential. For the patch from a 7 DIV neurone, at pH 7.4, openings are composed of single events and bursts. A selected time period (continuous bar) has been expanded to show the different conductance levels indicated by dotted lines. In pH 5.4 Krebs solution (B), the frequency of channel opening was reduced. For the patch from a 14 DIV neurone, exposure to pH 5.4 (D) has little effect on channel frequency or conductance. These records have been filtered at 1.5 kHz for display.

Analysis of the GABA channel conductances at pH 7.4 revealed three states designated as low (12.9 ± 1.03 pS), medium (17.9 ± 0.45 pS) and high (22.9 ± 0.8 pS) (n = 23 patches). Numerous transitions between the conductance states and direct transitions between the high conductance state and the shut state indicated that these states might represent multiple conductances of the same channel; however, in other patches there was clear evidence of medium and low conductance states being accessed directly from the shut state without traversing the high conductance state in accordance with different GABA_A receptors with discrete conductance states. To determine whether the conductance states changed during development, outside-out patches were obtained from 4 to 16 DIV; however, the relationship between single-channel conductance and development appeared to show no clear correlation (Fig. 6).

These three conductance states were also analysed at different external pH values and were found not to differ in value from the ranges 12-14 pS and 16-17 pS to 22-26 pS (Fig. 6). Moreover, the conductance states were also present in similar proportions at each external pH (5.4 to 9.4) with the main conductance states (high and medium), accounting for most of the ion channel openings, registering over 70% of events (Fig. 6, inset). Thus the single-channel conductance states and their relative frequency demonstrated no correlation, either during development, or after changing external pH (Fig. 6; n = 97 patches).

		View larger version [in this window] [in a new window]
Figure 6 Effect of development and H+ concentration on GABA single-channel conductance Analysis of the 3 GABA single-channel conductance states, 22-26 pS (high), 16-17 pS (medium) and 12-14 pS (low), at the stages of development 4-16 DIV, following exposure to external pH 5.4, 6.4, 7.4, 8.4 and 9.4. The percentage contribution of each conductance state is indicated in the inset. The data were collated from 98 outside-out patches of cerebellar granule neurones.

Effect of development and H⁺ on GABA channel open and closed times

Single-channel open and shut times were studied in order to determine whether these two parameters were altered with differing H⁺ concentrations and the stage of development in vitro. In particular, could changes in these parameters account for the reduction in channel activity at early times when exposing patches to pH 6.4 and 5.4 relative to pH 7.4? The mean open times were determined from 1 M GABA-activated single channels at 4-16 DIV at pH 5.4, 6.4, 7.4, 8.4 and 9.4. There was no clear correlation in the mean open times measured at any of the H⁺ concentrations studied during this developmental time period (Fig. 7A). In contrast, at pH values of 5.4 and 6.4, the mean shut time decreased during development (Fig. 7B), resulting in an increased probability of channel opening (P_o) (Fig. 7C). The increase in mean shut time and consequent reduction in P_o at early times for channels exposed to pH 5.4 and 6.4 could account for the reduced whole-cell peak and steady-state GABA-activated currents observed at low external pH. There was no significant change in the mean closed times or P_o between pH 7.4, 8.4 and 9.4.

		View larger version [in this window] [in a new window]
Figure 7 Effect of H+ on the mean open and shut times and Po during development Mean open (A) and shut (B) times in addition to open probability (Po; C) were collated from single GABA channels activated by 1 M GABA obtained from cerebellar neurones at 4-16 DIV at pH values 5.4, 6.4, 7.4, 8.4 and 9.4. Whilst mean open times are largely unaffected by pH, mean shut times, particularly at pH 5.4 and 6.4 are increased, an effect dependent upon the stage of neuronal development. The relationship of Po demonstrates a reduction at pH 5.4 and 6.4 at early development times. Data obtained from 95 outside-out patches held at -70 mV.

The distribution of all open times induced by GABA at early and late times at each H⁺ concentration was represented by open-time frequency histograms. Open-time frequency histograms were obtained at 7 and 13 DIV at pH 6.4, 7.4 and 8.4. In all open-time histograms two exponential functions were always required to fit the distribution with time constants representing the short (_O1) and long (_O2) open-time distributions. Open-time frequency histograms were constructed at each H⁺ concentration at 4-16 DIV and time constants, _O1 and _O2 (Fig. 8A), and relative areas, A_O1 and A_O2 (Fig. 8B), were ascertained. Most of the open durations (70-90%) appeared to be accounted for by _O2 (Fig. 8B). The open time constants were relatively unaffected by different extracellular H⁺ concentrations and there was no obvious correlation during the stages of development.

		View larger version [in this window] [in a new window]
Figure 8 Effect of H+ concentration on single-channel open times during GABAA receptor development in cerebellar neurones Single-channel open-time constants (O1 and O2; A) and the corresponding relative areas (AO1 and AO2; B) were measured at pH values 5.4, 6.4, 7.4, 8.4 and 9.4 at 4-16 DIV (n = 95). The time constants and relative areas were unaffected by varying extracellular H+ concentration with no obvious correlation over the developmental time period.

The distribution of all shut times was described by a frequency histogram fitted by the sum of three separate exponential functions, designated as _C1 for the short shut distribution, _C2 for the intermediate and _C3 for long shut periods. Shut-time frequency histograms were obtained at 5 and 14 DIV at pH 6.4, 7.4 and 8.4. Both _C1 and _C2 had very similar values throughout this period of development and at differing H⁺ concentrations, whereas _C3 was approximately 2.5 times longer at pH 5.4 and 6.4 at 4-9 DIV compared to all other determinations (Fig. 9). Time constants and their relative areas (A_C1-3) were ascertained from individual shut-time frequency histograms constructed at 4-16 DIV at pH 5.4, 6.4, 7.4, 8.4 and 9.4 (Figs 9 and 10A-C). During development, altering the extracellular pH had little effect on the short or intermediate shut time constants or the contribution of these components to the overall histogram. However, _C3 was significantly longer (Fig. 9) and also made a larger contribution to the shut time distribution (Fig. 10C) at early times at pH 5.4 and 6.4 compared to pH 7.4, 8.4 and 9.4.

		View larger version [in this window] [in a new window]
Figure 9 Effect of H+ concentration on all shut times for GABA-activated channels at different developmental stages The 3 shut time constants (short, C1; intermediate, C2; and long, C3) are plotted against the developmental stage of the cerebellar neurone and correlated with the pH of the Krebs solution. The short and intermediate shut time constants are largely unaffected by development or external pH; however, the long shut time constants are significantly greater at the lower pH values of 5.4 and 6.4 (n = 95). Patch holding potential, -70 mV.

		View larger version [in this window] [in a new window]
Figure 10 Relative contributions of the 3 shut time constants at various stages of development and at different H+ concentrations The relative areas of the shut time constants corresponding to AC1 (C1), AC2 (C2) and AC3 (C3) were ascertained from individual shut time frequency histograms constructed at 4-16 DIV at pH 5.4, 6.4, 7.4, 8.4 and 9.4 (n = 95). Note the increased contribution from long shut times at low pH and early development times in C.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Developmental properties of GABA_A receptors in cultured granule cells

The cerebellar granule cell GABA_A receptor subunit repertoire has been investigated extensively both in vivo and in vitro (Korpi & Luddens, 1993; Mertens et al. 1993; Caruncho & Costa, 1994; Khan et al. 1994, 1996; Quirk et al. 1994; Caruncho et al. 1995; Korpi et al. 1995; Pollard et al. 1995; Jones et al. 1997). In adult rat brain, in situ hybridisation studies revealed the presence of mRNAs for the subunits 1, 6, 2, 3, 2 and (Laurie et al. 1992; Persohn et al. 1992). Gao & Fritschy (1995) demonstrated that granule cells taken from P7 rats expressed the same subunit complement after 9 DIV as adult granule cells in vivo, concluding that the expression of GABA_A receptor subunits is not critically dependent on the establishment of GABAergic input. Overall the expression of GABA_A receptor subunits by cerebellar granule cells in vitro is a dynamic process with individual subunits appearing to be regulated independently (Zheng et al. 1994).

The change in GABA potency observed in the present study at 11-12 DIV, together with the change in pH sensitivity could reflect a dynamic change in GABA_A receptor subunit composition from cultures derived from 4-day-old rats. This might reflect subunit exchanges within single receptor complexes or the appearance of a mixture of receptor populations. Moreover, the increased Hill coefficient at later development times would be in accordance with increased cooperativity for channel activation, although whether this is due to subunit composition changes is less clear. Using colloidal gold particles, the GABA_A receptor subunit density in the plasma membrane of cerebellar granule cells cultured from 8-day-old rats for 3-14 DIV increased asynchronously; the 1 subunit increased approximately 2-fold by 7 DIV and over 5-fold by 14 DIV; the 6 subunit remained very low by 7 DIV but increased by over 4-fold by 14 DIV; and the density of 2/3, 2 and subunits increased steadily from 3 to 14 DIV (Zheng et al. 1994).

Using recombinant GABA_A receptors comprising subunits expected to be found in cerebellar granule neurones, substitution of 1 for 6 subunits decreased the GABA EC₅₀ values for both 622L and 632L GABA_A receptors to 2 M (Saxena & Macdonald, 1996), comparable to the EC₅₀ of 1 M reported for rat 622S GABA_A receptors (Sincoff et al. 1996). Interestingly, swapping 2L with the subunit, forming 62 and 63 constructs, revealed GABA EC₅₀ values of 0.2 and 0.3 M, respectively (Saxena & Macdonald, 1996). These changes in the GABA EC₅₀ values observed with different combinations of subunits may underlie the different EC₅₀ values (10-2.5 M) obtained in the present study during development. This change could reflect increased 6 subunit expression during development (Zheng et al. 1994) and incorporation presumably into 2 subunit-containing receptors since 62/3 receptors displayed a 6-fold increase in GABA sensitivity compared to 62/32L constructs (Saxena & Macdonald, 1996).

Previous studies using cultured rat cerebellar granule cells estimated GABA EC₅₀ values to be from 2.3 M (Robello et al. 1993) to 20.7 M (Kilic et al.1993). Interestingly, 2.3 M is similar to the EC₅₀ of 2.5 M obtained at 'late times' in the present study and is explained by Robello et al. (1993) using cultures after 5 DIV obtained from 8-day-old cerebella. The study by Kilic et al. (1993) utilised cultures grown in a low K⁺ environment, which may prevent GABA_A receptor maturation (Beattie & Siegel, 1993; Zhu et al. 1995). This is supported by EC₅₀ values of 1.4 and 13.8 M obtained from cerebellar granule cells grown in high and low K⁺, respectively (Zhu et al. 1995), indicating the probable importance of K⁺-induced depolarisation in attaining maturation of GABA_A receptors in vitro. Recording from granule cells in cerebellar brain slices, a GABA EC₅₀ of 45.2 M was obtained (Kaneda et al. 1995). This higher value may reflect the presence of GABA uptake and a slower speed of agonist application.

Interaction of H⁺ ions at GABA_A receptors: dependence on receptor subunit composition?

Previous studies have demonstrated that native GABA_A receptor function can be differentially influenced by reducing external pH resulting in potentiated (Gallagher et al. 1983; Pasternack et al. 1996), reduced (Groul et al. 1980; Smart, 1992; Pasternack et al. 1996), or unaffected (Tang et al. 1990) GABA-activated responses. The differential effects of H⁺ are probably due, at least in part, to GABA_A receptor heterogeneity since the subunit composition of recombinant GABA_A receptors markedly affects their sensitivities to H⁺ (Krishek et al. 1996). For example, 112S GABA_A receptors were relatively insensitive to external H⁺, whereas the addition of a subunit to this receptor or exchanging 1 for a 2 subunit resulted in a reduction in GABA-activated responses at both low and high pH (Krishek et al. 1996).

As the GABA_A receptor subunit composition changes with development in cerebellar granule neurones it appears likely that their pH sensitivity profile may also alter. Accordingly, low pH reduced peak and steady-state GABA-activated responses at early times and had little influence on peak but enhanced steady-state responses at late times.

At alkaline pH, there was an enhanced peak with little change in the steady-state GABA response at early times, whereas at late times, peak currents were uninfluenced and steady-state GABA-activated currents were reduced. The results of Robello et al. (1994) certainly accord with our data at late times, i.e. increased and decreased steady-state GABA-activated currents at low and high pH, respectively.

The likelihood of different GABA_A receptor combinations co-existing during the development of single granule cells (Mathews et al. 1994; Tia et al. 1996) suggests that it is impossible to predict the exact in vivo composition of any one receptor construct from the pH sensitivity of whole-cell currents. However, by increasing the density of 6 compared to 1 subunits with development in granule neurones (and assuming they assemble into discrete receptors, see Wisden et al. 1996 for review) then 122S and 622S GABA_A receptors could form prevalent species. Notably, GABA-activated responses recorded from 122S constructs are reduced at low pH (Krishek et al. 1996) whereas responses transduced by 622S receptors are potentiated (B. J. Krishek & T. G. Smart, unpublished observations); however, the pH sensitivities of other possible subunit assembly permutations, 132S, 12/32s, 62/32S, 12/32s, 162/32S, 162/32s, have yet to be established. Expressed 122 and 322 receptor combinations when exposed to acidic pH reduced GABA-activated currents to a comparable extent suggesting that 1 and 3 subunits do not differentially affect receptor sensitivity to H⁺ (Huang & Dillon, 1999). For other anion-permeable members of the ligand-gated ion channel family, such as the rat GABA_C receptor 1 subunit, low pH reduced GABA-activated currents (Wegelius et al. 1996) and the human 1 isoform was also only sensitive at low pH (Rivera et al. 2000). Similarly, the human glycine receptor 1 subunit was inhibited in a competitive manner by low pH (Harvey et al. 1999).

The pH titration analyses of cerebellar granule cell GABA_A receptors reveal remarkably similar pK_a values whether or not these relationships are determined at early or late times for peak or steady-state GABA-activated currents. The values of 6.7, 5.6 and 6.8 (Table 1) are all close to that predicted for H⁺ interacting with histidine residues (Mahler & Cordes, 1971), possibly suggesting a role for these amino acid residues in modulating GABA-activated currents. However, the precise pK_a values for such candidate residues are likely to be influenced by the nature of their immediate neighbours. Analysis of the pK_a values for the steady-state currents at early and late times (5.6 and 6.8, respectively) revealed that they are significantly different. It might be postulated that this is due to a change in receptor subunit composition affecting the charge distribution around a proton binding site; however, it is equally plausible that subunit exchanges may subtly affect receptor structure/conformation, which can then easily cause minor changes (shifts in dipoles, charged side chains, etc.) in pK_a values for a large protein such as the GABA_A receptor.

Mechanism of proton interaction at granule cell GABA_A receptors

Recording GABA-activated single-channel currents in outside-out cerebellar granule cell patches suggested that H⁺ inhibition of the whole-cell peak and steady-state currents at early times involves primarily an increase in the long shut times causing a reduced probability of GABA channel opening. A similar effect has been attributed to H⁺ inhibition of GABA-activated currents in hypothalamic neurones (Huang & Dhillon, 1999). In addition, at early times, the lack of sensitivity to alkaline pH during whole-cell steady-state current recordings can be entirely accounted for by the lack of a significant change in single-channel conductance, open and closed times or probability of opening. However, at late times, with external pH 5.4 and 6.4, there was a decrease in the long shut time with an increased open probability from 7 to 13 DIV which probably underlies the small but perceptible H⁺-induced enhancement in the whole-cell GABA-activated steady-state currents. Thus, unusually in a single cell type in the nervous system, H⁺ modulation of GABA-activated currents can be differential at selected stages of development, possibly based on the GABA_A receptor subunit composition (Krishek et al. 1996).

The mechanism of H⁺ inhibition of GABA-activated currents at early times could occur by H⁺ stabilising the receptor in one or more shut or desensitised states. It is unlikely to be due to an acceleration of the closure of channels through the unbinding of agonist as this would be expected to affect the short and intermediate shut times and not solely the long shut times as seen. At late times, H⁺ enhancement of GABA-induced currents could result from promotion of open state formation from either the closed or desensitised states for GABA_A receptors which may well possess a different subunit composition from those present at early times.

Single-channel properties: comparison of recombinant with native GABA_A receptors

The single-channel properties of only a few of the many possible combinations of the most abundant granule cell GABA_A receptor subunits (1, 6, 2, 3, 2 and ) have been examined in heterologous expression systems. Interestingly, the main conductance state revealed for 112S and 122S GABA_A receptors was 29 and 32 pS, respectively (Verdoorn et al. 1990; Angelotti & Macdonald, 1993). These studies indicate that, in the presence of 1 and 2 subunits, substitution of the 1 for the 2 subunit did not significantly alter the single-channel conductance, although there was a subconductance level of 21 pS seen for 112S GABA_A receptors (Angelotti & Macdonald, 1993). In contrast, removal of the 2 subunit to form 12 and 11 GABA_A receptors produced channels with lower conductances of 11 and 15-19 pS (Verdoorn et al. 1990; Moss et al. 1990; Angelotti & Macdonald, 1993). Addition of the subunit to 112L and 11 GABA_A receptors indicated that the single-channel conductances were comparable (30 pS (112L), 33 pS (112L) and 19 pS (11), 22 pS (11); Saxena & Macdonald, 1994). Interestingly, substitution of the 1 for the 6 subunit to form 612S GABA_A receptors revealed a similar single-channel conductance to that of its counterpart 112S receptor of 34 pS with a subconductance level of 18-23 pS (Angelotti et al. 1992). In the present study, there was no correlation between the single-channel conductance observed during development for each pH studied. These data are in accordance with the recombinant studies where substitution of 1 for 6, or 1 for 2 subunits, in the presence of the 2 subunit revealed little change in the channel conductance. The main difference between the present study and recombinant GABA_A receptor channels is the existence of multiple conductance states in granule neurones which may be derived from more than one receptor isoform.

It is therefore probable that the recombinant subunit species examined do not exactly match those constituting native GABA_A receptors in granule neurones. Multiple conductance states have been seen previously for neuronal GABA_A receptors. Analysis of GABA-activated single-channel currents in granule cells from thin cerebellar slices revealed the presence of two (16 and 28 pS; Kaneda et al. 1995) or three conductance states (28, 17 and 12 pS; Brickley et al. 1999). Moreover, for cultured cerebellar granule cells conductance states of 19 and 31 pS with a subconductance state of 22 pS were revealed (Kilic et al. 1993). In the present study the main conductance state at pH 7.4 was 23 pS with a further two conductance levels discernible at 17 and 13 pS, which encompasses the previous studies on granule cells.

Physiological implication

Transient changes in extracellular pH occur under physiological conditions, including during synaptic transmission where acidic contents of transmitter vesicles cause an extracellular acid shift within the synaptic cleft (Krishtal et al. 1987). Furthermore, activation of GABA_A receptors induces changes in the external pH due to bicarbonate efflux through the anion-selective channels (Kaila & Voipio, 1987; Kaila, 1994). Fluctuations in external pH are also known to occur as a consequence of pathological disease processes such as ischaemia, hypoxic insult to neural tissues and epileptiform activity (Chesler, 1990; Chesler & Kaila, 1992). The effect of changing the H⁺ concentration on GABA_A receptors of the cerebellar granule cells has been shown to be dependent on the stage of development in vitro, which presumably reflects the pH sensitivity of these receptors in vivo. Therefore, acidification of the extracellular space would reduce inhibitory neurotransmission in immature neurones, whilst the GABA response would be unaffected or enhanced in mature neurones depending on the rate of acidification and recovery. Thus modulation by H⁺ of granule cell GABA-activated currents could have physiological implications for cerebellar neuronal activity and ultimately motor coordination.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

ANGELOTTI T. P. & MACDONALD, R. L. (1993). Assembly of GABAA receptor subunits: 11 and 112S subunits produce simple unique ion channels with dissimilar single-channel properties. Journal of Neuroscience 13, 1429-1440	[Abstract]
ANGELOTTI T. P., TAN, F., CHAHINE, K. G. & MACDONALD, R. L. (1992). Molecular and electrophysiological characterization of an allelic variant of the rat 6 GABAA receptor subunit. Molecular Brain Research 16, 173-178	[Medline]
BEATTIE C. E. & SIEGEL, R. E. (1993). Developmental cues modulate GABAA receptor subunit mRNA expression in cultured cerebellar granule neurons. Journal of Neuroscience 13, 1784-1792	[Abstract]
BONNERT T. P., MCKERNAN, R. M., FARRAR, S., LE BOURDELLES, B., HEAVENS, R. P., SMITH, D. W., HEWSON, L., RIGBY, M. R., SIRINATHSINGHJI, D. J., BROWN, N., WAFFORD, K. A. & WHITING, P. J. (1999). T, a novel -aminobutyric acid type A receptor subunit. Proceedings of the National Academy of Sciences of the USA 96, 9891-9896.	[Abstract/Full Text]
BOVOLIN P., SANTI, M. R., PUIA, G., COSTA, E. & GRAYSON, D. (1992). Expression pattern of -aminobutyric acid type A receptor subunit mRNAs in primary cultures of granule neurons and astrocytes from neonatal rat cerebella. Proceedings of the National Academy of Sciences of the USA 89, 9344-9348	[Abstract]
BRICKLEY S. G., CULL-CANDY, S. G. & FARRANT, M. (1999). Single-channel properties of synaptic and extrasynaptic GABAA receptors suggest differential targeting of receptor subtypes. Journal of Neuroscience 19, 2960-2973	[Abstract/Full Text]
CARUNCHO H. J. & COSTA, E. (1994). Double-immunolabelling analysis of GABAA subunits in label-fracture replicas of cultured rat cerebellar granule cells. Receptor Channels 2, 143-153
CARUNCHO H. J., PUIA, G., MOHLER, H. & COSTA, E. (1995). The density and distribution of six GABAA receptor subunits in primary cultures of rat cerebellar granule cells. Neuroscience 67, 583-593	[Medline]
CHESLER M. (1990). The regulation and modulation of pH in the nervous system. Progress in Neurobiology 34, 401-427	[Medline]
CHESLER M. & KAILA, K. (1992). Modulation of pH by neuronal activity. Trends in Neurosciences 15, 396-402	[Medline]
DAVIES P. A., HANNA, M. C., HALES, T. G. & KIRKNESS, E. F. (1997). Insensitivity to anaesthetic agents conferred by a class of GABAA receptor subunit. Nature 385, 820-823	[Medline]
GALLAGHER J. P., NAKAMURA, J. & SHINNOCK-GALLAGHER, P. (1983). The effects of temperature, pH and Cl- pump inhibitors on GABA responses recorded from cat dorsal root ganglia. Brain Research 267, 249-259	[Medline]
GAO B. & FRITSCHY, J.-M. (1995). Cerebellar granule cells in vitro recapitulate the in vivo pattern of GABAA-receptor subunit expression. Developmental Brain Research 88, 1-16	[Medline]
GROUL D. L., BARKER, J. L., HUANG, L.-Y., MACDONALD, J. F. & SMITH, T. G. (1980). Hydrogen ions have multiple effects on the excitability of cultured mammalian neurons. Brain Research 183, 247-252	[Medline]
HARVEY R. J., THOMAS, P., JAMES, C. H., WILDERSPIN, A. & SMART, T. G. (1999). Identification of an inhibitory Zn2+ binding site on the human glycine receptor 1 subunit. Journal of Physiology 520, 53-64	[Abstract/Full Text]
HUANG R. Q. & DILLON, G. H. (1999). Effect of extracellular pH on GABA-activated current in rat recombinant receptors and thin hypothalamic slices. Journal of Neurophysiology 82, 1233-1243	[Abstract/Full Text]
JONES A., KORPI, E. R., MCKERNAN, R. M., PELZ, R., NUSSER, Z., MAKELA, R., MELLOR, J. R., POLLARD, S., BAHN, S., STEPHENSON, F. A., RANDALL, A. D., SIEGHART, W., SOMOGYI, P., SMITH, A. J. H. & WISDEN, W. (1997). Ligand-gated ion channel subunit partnerships: GABAA receptor 6 subunit gene inactivation inhibits subunit expression. Journal of Neuroscience 17, 1350-1362	[Abstract/Full Text]
KAILA K. (1994). Ionic basis of GABAA receptor channel function in the nervous system. Progress in Neurobiology 42, 489-537	[Medline]
KAILA K. & VOIPIO, J. (1987). Postsynaptic fall in intracellular pH induced by GABA-activated bicarbonate conductance. Nature 330, 163-165	[Medline]
KAILA K., VOIPIO, J., PAALASMAA, P., PASTERNACK, M. & DEISZ, R. A. (1993). The role of bicarbonate in GABAA receptor-mediated IPSPs of rat neocortical neurones. Journal of Physiology 464, 273-289	[Abstract]
KANEDA M., FARRANT, M. & CULL-CANDY, S. G. (1995). Whole-cell and single channel currents activated by GABA and glycine in granule cells of the rat cerebellum. Journal of Physiology 485, 419-435	[Abstract]
KHAN Z. U., GUTIERREZ, A. & DE BLAS, A. L. (1994). The subunit composition of a GABAA/benzodiazepine receptor from rat cerebellum. Journal of Neurochemistry 63, 371-374	[Abstract]
KHAN Z. U., GUTIERREZ, A. & DE BLAS, A. L. (1996). The 1 and 6 subunits can coexist in the same cerebellar GABAA receptor maintaining their individual benzodiazepine-binding specificities. Journal of Neurochemistry 66, 685-691.	[Abstract]
KILIC G., MORAN, O. & CHERUBINI, E. (1993). Currents activated by GABA and their modulation by Zn2+ in cerebellar granule cells in culture. European Journal of Neuroscience 5, 65-72	[Medline]
KORPI E. R., KUNER, T., SEEBURG, P. H. & LUDDENS, H. (1995). Selective antagonist for the cerebellar granule cell-specific -aminobutyric acid type A receptor. Molecular Pharmacology 47, 283-289	[Abstract]
KORPI E. R. & LUDDENS, H. (1993). Regional -aminobutyric acid sensitivity of t-butylbicyclophosphorol[35S]thionate binding depends on -aminobutyric acidA receptor subunit. Molecular Pharmacology 44, 87-92	[Abstract]
KRISHEK B. J., AMATO, A., CONNOLLY, C. N., MOSS, S. J. & SMART, T. G. (1996). Proton sensitivity of the GABAA receptor is associated with the receptor subunit composition. Journal of Physiology 492, 431-443	[Abstract]
KRISHTAL O. A., OSIPCHUK, Y. U., SHELEST, T. N. & SMIRNOFF, S. V. (1987). Rapid extracellular pH transients related to synaptic transmission in rat hippocampal slices. Brain Research 436, 352-356	[Medline]
LAURIE D. J., WISDEN, W. & SEEBURG, P. H. (1992). The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III Embryonic and postnatal development. Journal of Neuroscience 12, 4151-4172	[Abstract]
MAHLER H. R. & CORDES, E. H. (1971). Biological Chemistry, 2nd edn, p. 44. Harper & Row, New York
MATHEWS G. C., BOLOS-SY, A. M., HOLLAND, K. D., ISENBERG, K. E., COVEY, D. F., FERRENDELLI, J. A. & ROTHMAN, S. M. (1994). Developmental alteration in GABAA receptor structure and physiological properties in cultured cerebellar granule neurons. Neuron 13, 149-158	[Medline]
MERTENS S., BENKE, D. & MOHLER, H. (1993). GABAA receptor populations with novel subunit combinations and drug binding profiles identified in brain by 5- and -subunit-specific immunopurification. Journal of Biological Chemistry 268, 5965-5973	[Abstract]
MOSS S. J., SMART, T. G., PORTER, N. M., NAYEEM, N., DEVINE, J., STEPHENSON, F. A., MACDONALD, R. L. & BARNARD, E. A. (1990). Cloned GABA receptors are maintained in a stable cell line: allosteric and channel properties. European Journal of Pharmacology 189, 77-88	[Medline]
PASTERNACK M., SMIRNOV, S. & KAILA, K. (1996). Proton modulation of functionally distinct GABAA receptors in acutely isolated pyramidal neurons of rat hippocampus. Neuropharmacology 35, 1279-1288	[Medline]
PERSOHN E., MALHERBE, P. & RICHARDS, J. G. (1992). Comparative molecular neuroanatomy of cloned GABAA receptor subunits in rat CNS. Journal of Comparative Neurology 326, 193-216	[Medline]
POLLARD S., THOMPSON, C. L. & STEPHENSON, F. A. (1995). Quantitative characterization of 6 and 16 subunit-containing native -aminobutyric acidA receptors of adult rat cerebellum demonstrates two subunits per receptor oligomer. Journal of Biological Chemistry 270, 21285-21290	[Abstract/Full Text]
QUIRK K., GILLARD, N. P., RAGAN, C. I. WHITING, P. J. & MCKERNAN, R. M. (1994). Model of subunit composition of -aminobutyric acid A receptor subtypes expressed in rat cerebellum with respect to their and / subunits. Journal of Biological Chemistry 269, 16020-16028	[Abstract]
RIVERA C., WEGELIUS, K., REEBEN, M., KAILA, K. & PASTERNACK, M. (2000). Different sensitivities of human and rat p1 GABA receptors to extracellular pH. Neuropharmacology 39, 977-989	[Medline]
ROBELLO M., AMICO C. & CUPELLO, A. (1993). Regulation of GABAA receptor in cerebellar granule cells in culture: differential involvement of kinase activities. Neuroscience 53, 131-138	[Medline]
ROBELLO M., BALDELLI, P. & CUPELLO, A. (1994). Modulation by extracellular pH of the activty of GABAA receptors on rat cerebellum granule cells. Neuroscience 61, 833-837	[Medline]
SAXENA N. C. & MACDONALD, R. L. (1994). Assembly of GABAA receptor subunits: Role of the subunit. Journal of Neuroscience 14, 7077-7086	[Abstract]
SAXENA N. C. & MACDONALD, R. L. (1996). Properties of putative cerebellar -aminobutyric acidA receptor isoforms. Molecular Pharmacology 49, 567-579	[Abstract]
SIEGHART W. (1995). Structure and pharmacology of -aminobutyric acidA receptor subtypes. Pharmacological Reviews 47, 182-234
SIGEL E., BAUR, R., TRUDE, G., MOHLER, H. & MALHERBE, P. (1990). The effect of subunit composition of rat brain GABAA receptors on channel function. Neuron 5, 703-711	[Medline]
SINCOFF R., TANGUY, J., HAMILTON, B., CARTER, D., BRUNNER, E. A. & YEH, J. Z. (1996). Halothane acts as a partial agonist of the 622S GABAA receptor. FASEB Journal 10, 1539-1545	[Abstract]
SMART T. G. (1992). A novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones. Journal of Physiology 447, 587-625	[Abstract]
SMART T. G. & CONSTANTI, A. (1982). A novel effect of zinc on the lobster muscle GABA receptor. Proceedings of the Royal Society B 215, 327-341
TAKEUCHI A. & TAKEUCHI, N. (1967). Anion permeability of the inhibitory postsynaptic membrane of the crayfish neuromuscular junction. Journal of Physiology 191, 575-590	[Medline]
TANG C.-M., DICHTER, M. & MORAD, M. (1990). Modulation of N-methyl-D-aspartate channels by extracellular H+. Proceedings of the National Academy of Sciences of the USA 87, 6445-6559	[Abstract]
TIA S., WANG, J. F., KOTCHABHAKDI, N. & VICINI, S. (1996). Developmental changes of inhibitory synaptic currents in cerebellar granule neurons: role of GABAA receptor 6 subunit. Journal of Neuroscience 16, 3630-3640	[Abstract/Full Text]
VERDOORN T. A., DRAGUHN, A., YMER, S., SEEBURG, P. H. & SAKMANN, B. (1990). Functional properties of recombinant rat GABAA receptors depend upon subunit composition. Neuron 4, 919-928	[Medline]
WEGELIUS K., REEBEN, M., RIVERA, C., KAILA, K., SAARMA, M. & PASTERNACK, M. (1996). The p1 GABA receptor cloned from rat retina is down-modulated by protons. NeuroReport 7, 2005-2009	[Medline]
WISDEN W., KORPI, E. R. & BAHN, S. (1996). The cerebellum: a model system for studying GABAA receptor diversity. Neuropharmacology 35, 1139-1160	[Medline]
ZHENG T. M., SANTI, M. R., BOVOLIN, P., MARLIER, L. M. & GRAYSON, D. R. (1993). Developmental expression of the 6 GABA-A receptor subunit mRNA occurs only after cerebellar granule cell migration. Developmental Brain Research 75, 91-103	[Medline]
ZHENG T. M., ZHU, W. J., PUIA, G., VICINI, S., GRAYSON, D. R., COSTA, E. & CARUNCHO, H. J. (1994). Changes in -aminobutyrate type A receptor subunit mRNAs, translation product expression, and receptor function during neuronal maturation in vitro. Proceedings of the National Academy of Sciences of the USA 91, 10952-10956	[Medline]
ZHU W. J., VICINI, S., HARRIS, B. T. & GRAYSON, D. R. (1995). NMDA-mediated modulation of -aminobutyric acid type A receptor function in cerebellar granule neurons. Journal of Neuroscience 15, 7692-7701.	[Abstract]

Acknowledgements

This work is supported by the MRC. B.J.K. was in receipt of a University of London Maplethorpe Fellowship.

Corresponding author

T. G. Smart: Department of Pharmacology, The School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK.
Email: tsmart{at}ulsop.ac.uk

This article has been cited by other articles:

				M. E. Wilkins, A. M. Hosie, and T. G. Smart Proton modulation of recombinant GABAA receptors: influence of GABA concentration and the {beta} subunit TM2-TM3 domain J. Physiol., September 1, 2005; 567(2): 365 - 377. [Abstract] [Full Text] [PDF]

				K. Schnizler, B. Saeger, C. Pfeffer, A. Gerbaulet, U. Ebbinghaus-Kintscher, C. Methfessel, E.-M. Franken, K. Raming, C. H. Wetzel, A. Saras, et al. A Novel Chloride Channel in Drosophila melanogaster Is Inhibited by Protons J. Biol. Chem., April 22, 2005; 280(16): 16254 - 16262. [Abstract] [Full Text] [PDF]

				H.-J. Feng and R. L. Macdonald Proton Modulation of {alpha}1{beta}3{delta} GABAA Receptor Channel Gating and Desensitization J Neurophysiol, September 1, 2004; 92(3): 1577 - 1585. [Abstract] [Full Text] [PDF]

				R.-Q. Huang, Z. Chen, and G. H. Dillon Molecular Basis for Modulation of Recombinant {alpha}1{beta}2{gamma}2 GABAA Receptors by Protons J Neurophysiol, August 1, 2004; 92(2): 883 - 894. [Abstract] [Full Text] [PDF]

J. W. Mozrzymas, E. D. Zarmowska, M. Pytel, and K. Mercik Modulation of GABAA Receptors by Hydrogen Ions Reveals Synaptic GABA Transient and a Crucial Role of the Desensitization Process J. Neurosci., September 3, 2003; 23(22): 7981 - 7992. [Abstract] [Full Text]