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

The effect of GABAA 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. 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 uM. For granule cells before 11 DIV, the peak GABA-activated current was inhibited at low external pH and enhanced at high pH with a pKa of 6.6. For the steady-state response, low pH was inhibitory with a pKa of 5.56. After 11 DIV, the peak GABA-activated current was largely pH insensitive; however, the steady-state current was potentiated at low pH with a pKa of 6.84. 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. 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. 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. 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 GABAA receptors. The mode of interaction of H+ at the single-channel level and implications of such interactions at cerebellar granule cell GABAA receptors are discussed.

Interaction of H+ and Zn2+ on recombinant and native rat neuronal GABAA receptors.

1. The interaction of Zn2+ and H+ ions with GABAA receptors was examined using Xenopus laevis oocytes expressing recombinant GABAA receptors composed of subunits selected from alpha1, beta1, gamma2S and delta types, and by using cultured rat cerebellar granule neurones. 2. The potency of Zn2+ as a non-competitive antagonist of GABA-activated responses on alpha1beta1 receptors was reduced by lowering the external pH from 7.4 to 5.4, increasing the Zn2+ IC50 value from 1.2 to 58.3 microM. Zinc-induced inhibition was largely unaffected by alkaline pH up to pH 9.4. 3. For alpha1beta1delta subunits, concentration-response curves for GABA were displaced laterally by Zn2+ in accordance with a novel mixed/competitive-type inhibition. The Zn2+ IC50 at pH 7.4 was 16.3 microM. Acidification of Ringer solution resulted in a reduced antagonism by Zn2+ (IC50, 49.0 microM) without affecting the type of inhibition. At pH 9.4, Zn2+ inhibition remained unaffected. 4. The addition of the gamma2S subunit to the alpha1beta1delta construct caused a marked reduction in the potency of Zn2+ (IC50, 615 microM), comparable to that observed with alpha1beta1gamma2S receptors (IC50 639 microM). GABA concentration-response curves were depressed in a mixed/non-competitive fashion. 5. In cultured cerebellar granule neurones, Zn2+ inhibited responses to GABA in a concentration-dependent manner. Lowering external pH from 7.4 to 6.4 increased the IC50 from 139 to 253 microM. 6. The type of inhibition exhibited by Zn2+ on cerebellar granule neurones, previously grown in high K+-containing culture media, was complex, with the GABA concentration-response curves shifting laterally with reduced slopes and similar maxima. The Zn2+-induced shift in the GABA EC50 values was reduced by lowering the external pH from 7.4 to 6.4. 7. The interaction of H+ and Zn2+ ions on GABAA receptors suggests that they share either a common regulatory pathway or coincident binding sites on the receptor protein. The apparent competitive mode of block induced by Zn2+ on alpha1beta1delta receptors is shared by GABAA receptors on cerebellar granule neurones, which are known to express delta-subunit-containing receptors. This novel mechanism is masked when a gamma2 subunit is incorporated into the receptor complex, revealing further diversity in the response of native GABAA receptors to endogenous cations.

Regulation of neuronal and recombinant GABA(A) receptor ion channels by xenovulene A, a natural product isolated from Acremonium strictum.

Xenovulene A (XR368) is a natural product exhibiting little structural resemblance with classical benzodiazepines yet is able to displace high-affinity ligand binding to the benzodiazepine site of the gamma-aminobutyric acid (GABA)A receptor. We have characterized this compound and an associated congener (XR7009) by use of radioligand binding and electrophysiological methodologies with native neurons and the Xenopus oocyte expression system. Xenovulene A, and the more potent XR7009, inhibited [3H]flunitrazepam binding to rat forebrain with Ki values of 7 and 192 nM, and 1.7 and 42 nM, respectively, each site accounting for approximately 50% of the total specific binding. In cerebellar and spinal cord membranes, these ligands identified only single binding sites. These ligands demonstrated no intrinsic agonist activity at recombinant GABA(A) receptors comprising alpha1beta1gamma2S subunits expressed in Xenopus oocytes, yet at 1 microM both significantly potentiated the GABA-induced response and reduced the GABA EC50 from 10.9 (control) to 5.1 (Xenovulene A) or 2.7 microM (XR7009). The rank potency order for enhancement of the 10 microM GABA response is: XR7009 (EC50, 0.02 microM) > diazepam (0.03) > Xenovulene A (0.05) > flurazepam (0.17). The activity of XR368 and XR7009 was reduced by the benzodiazepine antagonist, flumazenil, and absent in receptors devoid of the gamma2 subunit. These agents exhibited receptor subtype selectivity because alpha3beta1gamma2S receptors were less sensitive to these compounds relative to alpha1 subunit-containing receptors, whereas alpha6beta1gamma2S receptors were completely insensitive. Potentiation of the response to GABA on native GABA(A) receptors in cortical neurons substantiates the profile of the novel structures of Xenovulene A and XR7009 as specific benzodiazepine agonists.

Proton sensitivity of the GABA(A) receptor is associated with the receptor subunit composition.

1. Modulation of GABA(A) receptors by external H(+) was examined in cultured rat sympathetic neurones, and in Xenopus laevis oocytes and human embryonic kidney (HEK) cells expressing recombinant GABA(A) receptors composed of combinations of alpha 1, beta 1, beta 2, gamma 2S and delta subunits. 2. Changing the external pH from 7.4 reduced GABA-activated currents in sympathetic neurones. pH titration of the GABA-induced current was fitted with a pH model which predicted that H(+) interact with two sites (PK(a) values of 6.4 and 7.2). 3. For alpha 1 beta 1 GABA(A) receptors, low external pH (< 7.4) enhanced responses to GABA. pH titration predicted the existence of two sites with PK(a) values of 6.6 and 7.5. The GABA concentration-response curve was shifted to the left by low pH and non-competitively inhibited at high pH (> 7.4). 4. alpha 1 beta 1 gamma 2S receptor constructs were not affected by external pH, whereas exchanging the beta 1 subunit for beta 2 conferred a sensitivity to pH, with predicted PK(a) values of 5.16 and 9.44. 5. Low pH enhanced the responses to GABA on alpha 1 beta 1 delta subunits, whilst high pH caused an inhibition (PK(a) values of 6.6 and 9.9). The GABA concentration-response curves were enhanced (pH 5.4) or reduced (pH 9.4) with no changes in the GABA EC(50). 6. Immunoprecipitation with subunit and epitope-specific antisera to alpha 1, beta 1 and delta subunits demonstrated that these subunits could co-assemble in cell membranes. 7. Expression of alpha 1 beta 1 gamma 2S delta constructs resulted in a 'bell-shaped' pH titration relationship. Increasing or decreasing external pH inhibited the responses to GABA. 8. The pH sensitivity of recombinant GABA(A) receptors expressed in HEK cells was generally in accordance with data accrued from Xenopus oocytes. However, rapid application of GABA to alpha 1 beta 1 constructs at high pH (> 7.4) caused an increased peak and reduced steady-state current, with a correspondingly increased rate of desensitization. 9. Modulation of GABA(A) receptor function was apparently unaffected by the internal pH. Moreover, pH values between 5 and 9.5 did not significantly affect the charge distribution on the zwitterionic GABA molecules. 10. In conclusion, this study demonstrates that external pH can either enhance, have little effect, or reduce GABA-activated responses, and this is apparently dependent on the receptor subunit composition. The potential importance of H(+) sensitivity of GABA(A) receptors is discussed.

Homomeric beta 1 gamma-aminobutyric acid A receptor-ion channels: evaluation of pharmacological and physiological properties.

The ubiquitous distribution of gamma-aminobutyric acid A (GABAA) receptor beta subunits throughout the central nervous system is in accord with a vital role in receptor structure and function. Homomeric beta subunits have been reported to be either GABA-gated or capable of forming anion-selective channels that lacked GABA-gating properties. With electrophysiological recording techniques, we examined the properties of the murine Beta 1 subunit, addressed whether the homomeric receptor is expressed independently from the host cell's genome, and investigated whether these channels can open spontaneously. Murine beta 1 subunits, expressed in Xenopus oocytes or A293 cells, were unaffected by GABA or bicuculline; however, the resting membrane conductances were reduced by picrotoxin, zinc, or penicillin-G. In comparison, the expression of bovine beta1 subunits formed GABA-gated C1- channels. For murine beta 1 subunits, both pentobarbitone and propofol increased the membrane conductance, although the benzodiazepine ligands flurazepam, flumazenil, and methyl-6,7-dimethoxy-4 ethyl-beta-carboline-3-carboxylate were inactive. Oocytes injected with murine beta 1 cRNA in the presence of actinomycin D (to block host cell DNA transcription) expressed beta1 channels that were indistinguishable from those derived from previous cDNA injections in cells capable of normal transcription. Single-channel recording from murin beta 1 cDNA-injected oocytes revealed spontaneously opening channels with a main state conductance of 18 pS. Picrotoxin inhibited the channel openings by reducing the probability of opening. We concluded that murine beta 1 subunits can form functional ion channels that are not gated by GABA but can be closed by some noncompetitive GABA antagonists. Interestingly, previous observations of spontaneously opening ion channels with properties similar to those found for the murine beta 1 receptor suggest that a limited expression of homomeric beta subunit-ion channels may exist in vivo.

Assembly and cell surface expression of heteromeric and homomeric gamma-aminobutyric acid type A receptors.

The ability of differing subunit combinations of gamma-aminobutyric acid type A (GABAA) receptors produced from murine alpha 1, beta 2, and gamma 2L subunits to form functional cell surface receptors was analyzed in both A293 cells and Xenopus oocytes using a combination of molecular, electrophysiological, biochemical, and morphological approaches. The results revealed that GABAA receptor assembly occurred within the endoplasmic reticulum and involved the interaction with the chaperone molecules immunoglobulin heavy chain binding protein and calnexin. Despite all three subunits possessing the ability to oligomerize with each other, only alpha 1 beta 2 and alpha 1 beta 2 gamma 2L subunit combinations could produce functional surface expression in a process that was not dependent on N-linked glycosylation. Single subunits and the alpha 1 gamma 2L and beta 2 gamma 2L combinations were retained within the endoplasmic reticulum. These results suggest that receptor assembly occurs by defined pathways, which may serve to limit the diversity of GABAA receptors that exist on the surface of neurons.

A functional comparison of the antagonists bicuculline and picrotoxin at recombinant GABAA receptors.

Allosteric modulation of GABAA receptor function by a number of ligands has been shown to be dependent on the subunit composition of the receptor complex. In this respect, modulation of GABAA receptors by the antagonists bicuculline and picrotoxin was examined in Xenopus laevis oocytes expressing recombinant GABAA receptors composed of combinations of murine alpha 1, beta 1, gamma 2S and gamma 2L subunits. Bicuculline and picrotoxin reduced GABA-activated responses mediated by GABAA receptors composed of alpha 1 beta 1, alpha 1 beta 1 gamma 2S and alpha 1 beta 1 gamma 2L subunits in a dose-dependent manner. GABA equilibrium concentration-response curves for each receptor construct were shifted to the right by increasing concentrations of bicuculline in a competitive manner, whereas picrotoxin induced a slight lateral shift as well as a depression of the maximum response consistent with a mixed/non-competitive inhibitory mechanism. GABA concentration-response curves in the absence and presence of bicuculline were subjected to Schild analysis, which revealed similar pKB values of approximately 5.9 for alpha 1 beta 1, alpha 1 beta 1 gamma 2S and alpha 1 beta 1 gamma 2L receptor constructs. Concentration inhibition curves were used to estimate IC50 for picrotoxin were relatively unaffected by the GABAA receptor isoforms used in this study, and in particular, by the absence of the gamma 2 subunit in the alpha 1 beta 1 GABAA receptor complex. The similarity of the pKBs reported in this study to those previously reported using native neuronal preparations, which are likely to represent heterogeneous GABAA receptor populations, further indicates the lack of dependence on receptor subunit composition for the inhibitory action of bicuculline.

m-sulphonate benzene diazonium chloride: a novel GABAA receptor antagonist.

A previously identified irreversible affinity label for the gamma-aminobutyric acid (GABA) binding site in rat brain membranes, m-sulphonate benzene diazonium chloride (MSBD), was characterized in functional studies using patch clamp and two-electrode voltage clamp recording techniques. MSBD did not exhibit any agonist activity on native GABAA receptors in cultured sympathetic ganglionic neurones but acted as an antagonist of GABA-induced membrane currents. Recombinant GABAA receptors composed of alpha 1, beta 1 and gamma 2S subunits were expressed in Xenopus oocytes following microinjection with cDNAs. Equilibrium dose-response curve analyses established that MSBD was a partially reversible, apparently non-competitive GABAA receptor antagonist. The IC50 for MSBD was estimated from an inhibition curve as 87 +/- 3 microM. In addition, the onset and recovery from MSBD-induced inhibition was independent of GABAA receptor activation. The relatively simple structure of this novel GABAA receptor antagonist, MSBD, is compared with known agonists and antagonists at the GABAA receptor. MSBD may be a useful pharmacological tool which could be used to deduce further information about the structure and function of agonist and antagonist binding sites on the GABAA receptor.

Regulation of GABAA receptor function by protein kinase C phosphorylation.

GABAA receptors possess consensus sequences for phosphorylation by PKC that are located on the presumed intracellular domains of beta and gamma 2 subunits. PKC phosphorylation sites were analyzed using purified receptor subunits and were located on up to 3 serine residues in beta 1 and gamma 2 subunits. The role of phosphorylation in receptor function was studied using recombinant receptors expressed in kidney cells and Xenopus oocytes and was compared with native neuronal GABAA receptors. For recombinant and native GABAA receptors, PKC phosphorylation caused a reduction in the amplitudes of GABA-activated currents without affecting the time constants for current decay. Selective site-directed mutagenesis of the serine residues reduced the effects of phorbol esters and revealed that serine 343 in the gamma 2 subunit exerted the largest effect on the GABA-activated response. These results indicate that PKC phosphorylation can differentially modulate GABAA receptor function.

Cloning and functional expression of a brain G-protein-coupled ATP receptor.

A cDNA encoding a novel member of the G-protein-coupled receptor (GCR) superfamily, an ATP receptor, has been isolated from an embryonic chick whole brain cDNA library by hybridization screening. The encoded protein has a sequence of 362 amino acids (41 kDa) and shares no more than 27% amino acid identity with any known GCR. When expressed as a complementary RNA (cRNA) in Xenopus oocytes a slowly-developing inward current was observed in response to application of ATP. The pharmacology of this expressed protein defines it as a P2Y purinoceptor.