Subunit Subtypes Research Articles

Major Differences in Inhibitory Synaptic Transmission onto Two Neocortical Interneuron Subclasses

Locally projecting GABAergic interneurons are the major providers of inhibition in the neocortex and play a crucial role in several brain functions. Neocortical interneurons are connected via electrical and chemical synapses that may be crucial in modulating complex network oscillations. We investigated the properties of spontaneous and evoked IPSCs in two morphologically and physiologically identified interneuron subtypes, the fast-spiking (FS) and low threshold-spiking (LTS) cells in layer V of rodent sensorimotor cortex. We found that IPSCs recorded in FS cells were several orders of magnitude more frequent, larger in amplitude, and had faster kinetics than IPSCs recorded in LTS cells. GABA(A) receptor alpha- and beta-subunit selective modulators, zolpidem and loreclezole, had different effects on IPSCs in FS and LTS interneurons, suggesting differential expression of GABA(A) receptor subunit subtypes. These pharmacological data indicated that the alpha1 subunit subtype is poorly expressed by LTS cells but makes a large contribution to GABA(A) receptors on FS cells. This was confirmed by experiments performed in genetically modified mice in which the alpha1 subunit had been made insensitive to benzodiazepine-like agonists. These results suggest that differences in IPSC waveform are likely attributable to distinctive expression of GABA(A) receptor subunits in FS and LTS cells. The particular properties of GABAergic input on different interneuronal subtypes might have important consequences for generation and pacing of cortical rhythms underlying several brain functions. Moreover, selective pharmacological manipulation of distinct inhibitory circuits might allow regulation of pyramidal cell activities under specific physiological and pathophysiological conditions.

Specific sites in the Beta Interaction Domain of a schistosome Ca2+ channel beta subunit are key to its role in sensitivity to the anti-schistosomal drug praziquantel.

Praziquantel, the drug of choice against schistosomiasis, disrupts calcium (Ca2+) homeostasis in schistosomes via an unknown mechanism. Voltage-gated Ca2+ channels are heteromultimeric transmembrane protein complexes that contribute to impulse propagation and also regulate intracellular Ca2+ levels. Beta subunits modulate the properties of the pore-forming alpha1 subunit of high voltage-activated Ca2+ channels. Unlike other Ca2+ channel beta subunits, which have current stimulatory effects, a beta subunit subtype found in S. mansoni (SmbetaA) and S. japonicum (Sjbeta) dramatically reduces current levels when co-expressed with Ca2+ channel alpha1 subunits in Xenopus oocytes. It also confers praziquantel sensitivity to the mammalian Cav2.3 alpha1 subunit. The Beta Interaction Domains (BIDs) of SmbetaA and Sjbeta lack 2 conserved serines that each constitute a consensus site for protein kinase C (PKC) phosphorylation. Here, we use site-directed mutagenesis of schistosome beta subunits to show that these unique functional properties are correlated with the absence of these consensus PKC sites in the BID. Furthermore, a second schistosome beta subunit subtype contains both serines in the BID, enhances currents through alpha1 subunits, and does not confer praziquantel sensitivity. Thus, phosphorylation sites in the BID may play important roles in defining the modulatory properties and pharmacological sensitivities of schistosome Ca2+ channel beta subunits.

α1 and α6 subunits specify distinct desensitization, deactivation and neurosteroid modulation of GABA A receptors containing the δ subunit

GABA A receptor α subunit subtypes have distinct CNS distributions and confer different pharmacological and biophysical properties to αβγ receptor isoforms. However, the α subtype-dependent properties of αβδ receptor isoforms that may be targeted to extrasynaptic sites remain poorly understood. We investigated the properties of α1β3δ and α6β3δ receptor currents evoked by concentration jumps using a saturating GABA concentration (1 mM). α1β3δ receptor currents desensitized slowly, deactivated rapidly and displayed voltage-dependence only of peak amplitude. In contrast, α6β3δ receptor currents had voltage-dependent increased desensitization and slower deactivation, but did not show rectification. The neurosteroid THDOC (1 μM) enhanced α1β3δ more than α6β3δ currents, but increased the extent of desensitization and prolonged deactivation for both receptor isoforms. α1–α6 and α6–α1 chimeras (spliced in transmembrane domain 1) suggested that differences in deactivation rate and its voltage-dependence correlated with N-terminal domains, while the extent of desensitization and its voltage-dependence correlated with C-terminal domains. Both chimeras showed outward rectification. α1 subunit-like THDOC enhancement was observed with the α1–α6 chimera, but the α6–α1 chimera did not confer α6 subunit-like enhancement, suggesting that multiple α1 subunit domains contributed to neurosteroid efficacy. Thus, α subunit subtypes may regulate the kinetic and pharmacological properties of tonic neuronal inhibition.

A histidine residue in the extracellular N-terminal domain of the GABA A receptor α5 subunit regulates sensitivity to inhibition by zinc

The divalent cation zinc is abundant in the brain, particularly in the mossy fibers of the hippocampus. Recent evidence suggests that zinc is packaged into some synaptic vesicles in this region and can be co-released with neurotransmitter. Zinc inhibits the activity of GABA A receptors and the sensitivity of the receptor to zinc is influenced by its α subunit subtype composition. The α4, α5 and α6 subunits confer greater sensitivity to zinc than receptors containing other α subunits. The α4 and α5 subunits are highly expressed in hippocampal neurons, and likely mediate any effects of zinc on GABAergic neurotransmission in this area. The α5 subunit contains a unique histidine residue in the N-terminal extracellular domain while the other α subunits have an aspartate residue in this location. Point mutations were created to exchange the histidine and aspartate residues of the α1 and α5 subunits. Receptors containing the mutated α5 (H195D) subunit had reduced sensitivity to zinc, while α1 (D191H)β3γ2L receptors had increased sensitivity to zinc, similar to the α5β3γ2L wild type receptors. These findings indicate that histidine195 of the α5 subunit plays an important role in determining the sensitivity of recombinant GABA A receptors to zinc.

Calcium Channel β Subunits Differentially Regulate the Inhibition of N-type Channels by Individual Gβ Isoforms

The direct inhibition of N- and P/Q-type calcium channels by G protein betagamma subunits is considered a key mechanism for regulating presynaptic calcium levels. We have recently reported that a number of features associated with this G protein inhibition are dependent on the G protein beta subunit isoform (Arnot, M. I., Stotz, S. C., Jarvis, S. E., Zamponi, G. W. (2000) J. Physiol. (Lond.) 527, 203-212; Cooper, C. B., Arnot, M. I., Feng, Z.-P., Jarvis, S. E., Hamid, J., Zamponi, G. W. (2000) J. Biol. Chem. 275, 40777-40781). Here, we have examined the abilities of different types of ancillary calcium channel beta subunits to modulate the inhibition of alpha(1B) N-type calcium channels by the five known different Gbeta subunit subtypes. Our data reveal that the degree of inhibition by a particular Gbeta subunit is strongly dependent on the specific calcium channel beta subunit, with N-type channels containing the beta(4) subunit being less susceptible to Gbetagamma-induced inhibition. The calcium channel beta(2a) subunit uniquely slows the kinetics of recovery from G protein inhibition, in addition to mediating a dramatic enhancement of the G protein-induced kinetic slowing. For Gbeta(3)-mediated inhibition, the latter effect is reduced following site-directed mutagenesis of two palmitoylation sites in the beta(2a) N-terminal region, suggesting that the unique membrane tethering of this subunit serves to modulate G protein inhibition of N-type calcium channels. Taken together, our data suggest that the nature of the calcium channel beta subunit present is an important determinant of G protein inhibition of N-type channels, thereby providing a possible mechanism by which the cellular/subcellular expression pattern of the four calcium channel beta subunits may regulate the G protein sensitivity of N-type channels expressed at different loci throughout the brain and possibly within a neuron.

Astrocytes Regulate N-Methyl-d-aspartate Receptor Subunit Composition Increasing Neuronal Sensitivity to Excitotoxicity

We have examined the dependence of rat cerebellar granule neurons (CGNs) for protection against glutamate toxicity. Under co-culture conditions, rat CGNs require astrocytes to protect against glutamate. The CGNs become more sensitive to glutamate toxicity in co-culture than when grown in cultures with only low numbers of astrocytes. If the protection of the astrocytes was withdrawn or blocked, this sensitivity led to neuronal death. Differing changes in NMDA receptor subunit subtype composition were noted depending on the conditions in which the CGNs were grown. Suppression of individual NMDA subunit subtypes by oligonucleotide knockdown resulted in inhibition of toxicity. This result implies that astrocytes regulate the expression of NMDA receptor subunit subtypes which influence neuronal sensitivity to glutamate toxicity.

Loreclezole inhibition of recombinant α1β1γ2L GABA A receptor single channel currents

Loreclezole had two different effects on GABA A receptor (GABAR) currents. When applied to GABARs that contained a β2 or β3 subunit subtype, but not a β1 subtype, loreclezole potentiated the peak current evoked by sub-maximal concentrations of GABA. Loreclezole also increased the rate and degree of apparent desensitization of GABAR whole-cell currents, an effect that was independent of the β subunit subtype, suggesting that potentiation and inhibition of GABAR current by loreclezole occurred through separate sites. We used patch-clamp recording from outside-out and inside-out patches from L929 fibroblasts transiently transfected with rat GABAR subunits to examine the properties of inhibition of α1β1γ2L single channel currents by loreclezole. Loreclezole decreased the mean open time of the channel by decreasing the average durations of the open states. Loreclezole also increased the occurrence of a closed component with an average duration near 20 ms. Inhibition by loreclezole was not voltage-dependent. Loreclezole was equally effective when applied to the intracellular side of the receptor, suggesting that its binding site was readily accessible from both sides of the membrane. Pre-application of loreclezole effectively inhibited the GABAR current in macropatches, indicating that binding did not require an open channel. These findings were consistent with a mechanism of allosteric modulation at a site formed by the membrane spanning regions of the receptor.

Human Neuronal γ-Aminobutyric AcidAReceptors: Coordinated Subunit mRNA Expression and Functional Correlates in Individual Dentate Granule Cells

gamma-Aminobutyric acid(A) receptors (GABARs) are heteromeric proteins composed of multiple subunits. Numerous subunit subtypes are expressed in individual neurons, which assemble in specific preferred GABAR configurations. Little is known, however, about the coordination of subunit expression within individual neurons or the impact this may have on GABAR function. To investigate this, it is necessary to profile quantitatively the expression of multiple subunit mRNAs within individual cells. In this study, single-cell antisense RNA amplification was used to examine the expression of 14 different GABAR subunit mRNAs simultaneously in individual human dentate granule cells (DGCs) harvested during hippocampectomy for intractable epilepsy. alpha4, beta2, and delta-mRNA levels were tightly correlated within individual DGCs, indicating that these subunits are expressed coordinately. Levels of alpha3- and beta2-mRNAs, as well as epsilon- and beta1-mRNAs, also were strongly correlated. No other subunit correlations were identified. Coordinated expression could not be explained by the chromosomal clustering of GABAR genes and was observed in control and epileptic rats as well as in humans, suggesting that it was not species-specific or secondary to epileptogenesis. Benzodiazepine augmentation of GABA-evoked currents also was examined to determine whether levels of subunit mRNA expression correlated with receptor pharmacology. This analysis delineated two distinct cell populations that differed in clonazepam modulation and patterns of alpha-subunit expression. Clonazepam augmentation correlated positively with the relative expression of alpha1- and gamma2-mRNAs and negatively with alpha4- and delta-mRNAs. These data demonstrate that specific GABAR subunit mRNAs exhibit coordinated control of expression in individual DGCs, which has significant impact on inhibitory function.

GABAA Receptors Expressed in Undifferentiated Human Teratocarcinoma NT2 Cells Differ from Those Expressed by Differentiated NT2-N Cells

During CNS development, changes occur in expression of GABA(A) receptor subunit subtypes and GABA(A) receptor pharmacological and biophysical properties. We used reverse transcription PCR and whole-cell-recording techniques to determine whether GABA(A) receptor expression and function also changed during retinoic acid-induced differentiation of human Ntera 2 (NT2) teratocarcinoma cells into neuron-like cells (NT2-N cells). In undifferentiated NT2 cells only alpha5, beta3, gamma3, and pi subtype mRNAs were detected. NT2 GABA(A) receptor currents had a maximal amplitude of 52 pA and an EC(50) of 4.0 microM, were relatively insensitive to enhancement by zolpidem and diazepam, and were enhanced by loreclezole and inhibited by lanthanum, zinc, and furosemide. In contrast, in NT2-N cells after 13 weeks of retinoic acid treatment, all GABA(A) receptor subtype mRNAs were detected. Maximal peak whole-cell currents were approximately 50-fold larger than NT2 cell currents, and the GABA EC(50) was higher (39.7 microM). In 13 week NT2-N cells, diazepam, zolpidem, loreclezole, and lanthanum had only small effects on GABA(A) receptor currents, and the zinc IC(50) for current inhibition was significantly higher than that for NT2 cells. In a previous study, we showed that NT2-N cells after 5 weeks of retinoic acid treatment had moderate peak currents, GABA EC(50,) and zinc IC(50) but that currents were robustly enhanced by diazepam, zolpidem, and loreclezole. During differentiation of NT2 cells to NT2-N cells, GABA(A) receptors underwent changes in subunit expression and pharmacology that were similar to many of the developmental changes in GABA(A) receptors that occur in CNS neurons.

Characterization of nonenterotoxigenic Escherichia coli strains producing F17 fimbriae isolated from diarrheic lambs and goat kids.

Forty-five ovine and caprine nonenterotoxigenic Escherichia coli strains producing F17-related fimbriae were characterized with respect to the fimbrial structural subunit and adhesin subtypes produced. In addition, several characteristics related to the virulence of strains producing F17 fimbriae were studied. Most of the strains (73%) possessed the f17cA structural subunit gene, whereas the f17aA and f17dA genes were detected only on three (6%) and two (4%) strains, respectively. The f17bA gene was not detected. All but one of these strains possessed the f17G genes of the adhesin subfamily II. The only strain having the f17G gene of subfamily I possessed the structural subunit gene f17dA. Sequencing of the f17A and f17G genes of four selected strains confirmed the association of f17cA and f17dA structural subunit genes with the f17G genes of the adhesin subfamily II. These results indicated that adhesins of the subfamily II are prominent among ovine and caprine isolates and that they are indistinctly associated with the F17 structural subunit subtypes on these field strains. CS31A- and CNF2-related genes were not detected. Most of the strains adhered in vitro to ovine intestinal brush borders (36 of 45) and agglutinated the erythrocytes of different species in the presence of D-mannose (39 of 45). F17-positive strains produced colicin V (57%) and were resistant to the bactericidal effect of serum (91%) in significantly higher percentages than F17-negative strains (34% produced colicin V, and 66% were serum resistant). Thus, most of the studied ovine and caprine strains showed phenotypic characteristics of septicemic strains.

A New β Subtype-specific Interaction in α1ASubunit Controls P/Q-type Ca2+ Channel Activation

The cytoplasmic beta subunit of voltage-dependent calcium channels modulates channel properties in a subtype-specific manner and is important in channel targeting. A high affinity interaction site between the alpha1 interaction domain (AID) in the I-II cytoplasmic loop of alpha1 and the beta interaction domain (BID) of the beta subunit is highly conserved among subunit subtypes. We describe a new subtype-specific interaction (Ss1) between the amino-terminal cytoplasmic domain of alpha1A (BI-2) and the carboxyl terminus of beta4. Like the interaction identified previously () between the carboxyl termini of alpha1A and beta4 (Ss2), the affinity of this interaction is lower than AID-BID, suggesting that these are secondary interactions. Ss1 and Ss2 involve overlapping sites on beta4 and are competitive, but neither inhibits the interaction with AID. The interaction with the amino terminus of alpha1 is isoform-dependent, suggesting a role in the specificity of alpha1-beta pairing. Coexpression of beta4 in Xenopus oocytes produces a reduced hyperpolarizing shift in the I-V curve of the alpha1A channel compared with beta3 (not exhibiting this interaction). Replacing the amino terminus of alpha1A with that of alpha1C abolishes this difference. Our data contribute to our understanding of the molecular organization of calcium channels, providing a functional basis for variation in subunit composition of native P/Q-type channels.

Subunit dependent modulation of GABA A receptor function by neuroactive steroids

Neurosteroids are potent, endogenous modulators of GABA A receptor function in the central nervous system. The endogenous progesterone metabolite allopregnanolone (ALP) and the synthetic steroid compound alphaxalone (AFX) have been shown to both directly activate and potentiate GABA A receptor-activated membrane current ( I GABA). The role of different α and γ subunit subtypes in modulation of I GABA by ALP and AFX was investigated using recombinant GABA A receptor isoforms expressed in Xenopus oocytes. Changing or removal of the α subunit subtype altered the efficacy of both ALP and AFX (α2β1γ2L>α1β1γ2L≫β1γ2L) to potentiate I GABA, but did not alter the potency of the neuroactive steroids at these receptor isoforms. The efficacy of ALP to enhance I GABA was also dependent on the γ subunit subtype (α1β1γ3>α1β1γ2L=α1β1γ1). AFX also had higher efficacy in the α1β1γ3 receptor isoform compared to α1β1γ1. In contrast to ALP, the potency of AFX was greater in the α1β1γ3 and α1β1γ1 receptor isoforms compared to α1β1γ2L. This study provides evidence that the α subunit subtype determines the efficacy, but not the potency, of these neuroactive steroids to potentiate I GABA. The γ3 subunit subtype increases the maximal efficacy of neuroactive steroids compared to other γ subunit subtypes. These results suggest that the heteromeric assembly of different GABA A receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABA A receptors that in turn produce distinctly different modulatory interactions between neuroactive steroids acting at the GABA A receptor.

Spontaneous and gamma-aminobutyric acid (GABA)-activated GABA(A) receptor channels formed by epsilon subunit-containing isoforms.

A new gamma-aminobutyric acid (GABA)A receptor (GABAR) subunit class, epsilon, has recently been cloned and shown to form functional channels when coexpressed with both alpha and beta subunits. We report that the combination of alpha1beta3epsilon subunit subtypes expressed in L929 cells produced functional chloride ion channels that were both spontaneously active and gated by the application of extracellular GABA. When cells were voltage-clamped at -75 mV in the whole-cell configuration, holding currents of 50 to 300 pA associated with increased noise were consistently recorded. The application of pentobarbital and loreclezole, which increase GABAR currents, increased the holding current, whereas the application of zinc and picrotoxin, which reduce GABAR currents, reduced the holding current in a concentration-dependent manner. Coexpression of alpha1beta3gamma2L, alpha1beta3delta, alpha1epsilon, beta3epsilon, alpha1beta3, or epsilon subtypes did not produce holding currents that were sensitive to picrotoxin (30 microM). Cells expressing alpha1 beta3epsilon subtypes had concentration-dependent GABAR currents that were potentiated by pentobarbital, loreclezole, and lanthanum and inhibited by zinc and furosemide. Spontaneous and GABAR single-channel currents from alpha1beta3epsilon receptors had single-channel conductances of approximately 24 pS. The biophysical properties and the effects of allosteric modulators were similar for spontaneous and evoked GABAR currents, suggesting that a single GABAR isoform was responsible for both currents. These data extend the pharmacological characterization of epsilon-containing GABARs and demonstrate that incorporation of the epsilon subunit permits spontaneous channel gating while preserving the structural information necessary for GABA sensitivity.

GABAA receptor subunit gamma2 and delta subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts.

1. To determine their contributions to the rapid kinetic properties of GABAA receptor (GABAR) currents, alpha1 and beta3 subunit subtypes without or with delta or gamma2L subtypes were transiently coexpressed in mouse L929 fibroblasts to produce alpha1beta3, alpha1beta3delta, or alpha1beta3gamma2L GABAR isoforms. 2. Brief (2-3 ms) applications of 1 mM GABA to outside-out membrane patches containing alpha1beta3, alpha1beta3delta, or alpha1beta3gamma2L isoforms elicited currents that activated rapidly with monophasic time courses and deactivated rapidly with biphasic time courses. alpha1beta3gamma2L currents exhibited a slower mean deactivation rate (76.1 ms) than alpha1beta3 (34.1 ms) or alpha1beta3delta currents (42.8 ms). 3. During 1 mM GABA applications, alpha1beta3gamma2L currents activated more rapidly (0.46 ms) than alpha1beta3 currents (1.7 ms) or alpha1beta3delta currents (2.4 ms). During 4000 ms GABA applications, alpha1beta3 and alpha1beta3gamma2L currents desensitized with triphasic time courses to similar extents (alpha1beta3, 94.6 %; alpha1beta3gamma2L, 92.4 %) and with similar mean rates (alpha1beta3, 352 ms; alpha1beta3gamma2L, 462 ms). In contrast, alpha1beta3delta currents desensitized only 55.6 % with a biphasic time course and slower mean rate (1260 ms). 4. These experiments demonstrated that the alpha1beta3 heterodimer formed a GABAR channel with rapid deactivation and rapid and nearly complete desensitization. Addition of the delta subunit did not alter the activation rate, but produced a receptor with slower and less complete desensitization. Addition of the gamma2L subtype increased activation rate, prolonged deactivation and changed the pattern of rapid desensitization. 5. Rapid kinetic and steady-state single-channel data were used to construct kinetic models that predicted the behaviour of the alpha1beta3gamma2L and alpha1beta3delta currents. These models represent a reconciliation of macroscopic and steady-state single-channel data for GABARs and provide a framework for systematically assessing the functional significance of different GABAR isoforms.

Modulation of GABAA receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABAA receptor isoforms

Neuroactive steroids are potent, selective allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In order to study the influence of subunit subtypes of the GABAA receptor on modulation of receptor function by neuroactive steroids, we expressed human recombinant GABAA receptors in Xenopus oocytes. GABA-activated membrane current, and the modulatory effects of the endogenous neurosteroid 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) and the synthetic steroid anesthetic 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) were measured using two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha1beta1gamma2L and alpha1beta2gamma2L receptor isoforms. In contrast, alphaxalone was much more effective as a positive allosteric modulator on the alpha1beta1gamma2L receptor isoform. In the absence of the gamma2L subunit subtype, allopregnanolone had much greater efficacy, but its potency was decreased. Allopregnanolone was much more effective on the alpha1beta1 receptor isoform compared with the alpha1beta2 receptor isoform. The potency for alphaxalone to potentiate the GABA response was not altered in the absence of the gamma2L subunit subtype, although its efficacy was greatly enhanced. Both allopregnanolone and alphaxalone produced nonparallel leftward shifts in the GABA concentration-response relationship in the absence of the gamma2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma2L subunit subtype was present. The 3beta-pregnane isomers epipregnanolone and isopregnanolone both inhibited the ability of allopregnanolone and alphaxalone to potentiate GABAA receptor function. However, the degree of block produced by the 3beta-pregnane steroid isomers was dependent on the type of receptor isoform studied and the neuroactive steroid tested. Isopregnanolone, the 3beta-isomer of allopregnanolone, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha1beta2gamma2L receptor isoform compared with the alpha1beta1gamma2L receptor isoform, and also produced a greater degree of block of potentiation caused by allopregnanolone compared with alphaxalone. Our results support the hypothesis that the heteromeric assembly of different GABAA receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABAA receptors, which in turn produces distinctly different modulatory interactions between neuroactive steroids acting at the GABAA receptor. The alpha and gamma subunit subtypes may have the greatest influence on allopregnanolone modulation of GABAA receptor function, whereas the beta and gamma subunit subtypes appear to be most important for the modulatory effects of alphaxalone.Key words: GABAA receptor, neurosteroid, allopregnanolone, alphaxalone, Xenopus oocyte.

The Role of an α Subtype M2-M3His in Regulating Inhibition of GABAAReceptor Current by Zinc and Other Divalent Cations

Sensitivity of GABAA receptors (GABARs) to inhibition by zinc and other divalent cations is influenced by the alpha subunit subtype composition of the receptor. For example, alpha6beta3gamma2L receptors are more sensitive to inhibition by zinc than alpha1beta3gamma2L receptors. We examined the role of a His residue located in the M2-M3 extracellular domain (rat alpha6 H273) in the enhanced zinc sensitivity conferred by the alpha6 subtype. The alpha1 subtype contains an Asn (N274) residue in the equivalent location. GABA-activated whole-cell currents were obtained from L929 fibroblasts after transient transfection with expression vectors containing GABAA receptor cDNAs. Mutation of alpha1 (alpha1(N274H)) or alpha6 (alpha6(H273N)) subtypes did not alter the GABA EC50 of alphabeta3gamma2L receptors. alpha1(N274H)beta3gamma2L receptor currents were as sensitive to zinc as alpha6beta3gamma2L receptor currents, although alpha6(H273N)beta3gamma2L receptor currents had the reduced zinc sensitivity of alpha1beta3gamma2L receptor currents. We also examined the activity of other inhibitory divalent cations with varying alpha subtype dependence: nickel, cadmium, and copper. alpha6beta3gamma2L receptor currents were more sensitive to nickel, equally sensitive to cadmium, and less sensitive to copper than alpha1beta3gamma2L receptor currents. Studies with alpha1 and alpha6 chimeric subunits indicated that the structural dependencies of the activity of some of these cations were different from zinc. Compared with alpha6beta3gamma2L receptor currents, alpha6(H273N)beta3gamma2L receptor currents had reduced sensitivity to cadmium and nickel, but the sensitivity to copper was unchanged. Compared with alpha1beta3gamma2L receptor currents, alpha1(N274H)beta3gamma2L receptor currents had increased sensitivity to nickel, but the sensitivity to cadmium and copper was unchanged. These findings indicate that H273 of the alpha6 subtype plays an important role in determining the sensitivity of recombinant GABARs to the divalent cations zinc, cadmium, and nickel, but not to copper. Our results also suggest that the extracellular N-terminal domain of the alpha1 subunit contributes to a regulatory site(s) for divalent cations, conferring high sensitivity to inhibition by copper and cadmium.

Modulatory and direct effects of propofol on recombinant GABA A receptors expressed in Xenopus oocytes: Influence of α- and γ2-subunits

Propofol (2,6-diisopropylphenol) is an intravenous general anaesthetic which can directly activate and positively modulate the GABA A receptor. The effects of propofol on human recombinant GABA A receptors were studied in Xenopus oocytes expressing either α 1 β 2, α 1 β 2 γ 2L, or α 2 β 2 γ 2L receptor isoforms. In all receptor isoforms tested, propofol was able to potentiate the GABA-activated currents in a concentration-dependent manner. Although propofol potentiated both α 1 β 2 and α 1 β 2 γ 2L receptor isoforms with equal affinity, the efficacy of propofol potentiation was markedly greater in the α 1 β 2 receptor isoform. In contrast, potentiation of the α 2 β 2 γ 2L receptor isoform by propofol occurred with higher affinity and lower efficacy than in the α 1 β 2 γ 2L receptor isoform. Propofol directly activated all three receptor isoforms in a concentration dependent manner. Addition of the γ 2L subunit subtype to the α 1 β 2 receptor isoform decreased receptor sensitivity to direct activation by propofol. Replacement of the α 1-subunit subtype with the α 2-subunit subtype increased receptor sensitivity to propofol's direct effects. These results suggest that the α-and γ 2L-subunit subtypes each have the ability to influence both the direct and modulatory actions of propofol on GABA A receptor function.

Modulation of GABA(A) receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABA(A) receptor isoforms.

Neuroactive steroids are potent, selective allosteric modulators of gamma-aminobutyric acid type A (GABA(A)) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In order to study the influence of subunit subtypes of the GABA(A) receptor on modulation of receptor function by neuroactive steroids, we expressed human recombinant GABA(A) receptors in Xenopus oocytes. GABA-activated membrane current, and the modulatory effects of the endogenous neurosteroid 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) and the synthetic steroid anesthetic 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) were measured using two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha1beta1gamma2L and alpha1beta2gamma2L receptor isoforms. In contrast, alphaxalone was much more effective as a positive allosteric modulator on the alpha1beta1gamma2L receptor isoform. In the absence of the gamma2L subunit subtype, allopregnanolone had much greater efficacy, but its potency was decreased. Allopregnanolone was much more effective on the alpha1beta1 receptor isoform compared with the alpha1beta2 receptor isoform. The potency for alphaxalone to potentiate the GABA response was not altered in the absence of the gamma2L subunit subtype, although its efficacy was greatly enhanced. Both allopregnanolone and alphaxalone produced nonparallel leftward shifts in the GABA concentration-response relationship in the absence of the gamma2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma2L subunit subtype was present. The 3beta-pregnane isomers epipregnanolone and isopregnanolone both inhibited the ability of allopregnanolone and alphaxalone to potentiate GABA(A) receptor function. However, the degree of block produced by the 3beta-pregnane steroid isomers was dependent on the type of receptor isoform studied and the neuroactive steroid tested. Isopregnanolone, the 3beta-isomer of allopregnanolone, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha1beta2gamma2L receptor isoform compared with the alpha1beta1gamma2L receptor isoform, and also produced a greater degree of block of potentiation caused by allopregnanolone compared with alphaxalone. Our results support the hypothesis that the heteromeric assembly of different GABA(A) receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABA(A) receptors, which in turn produces distinctly different modulatory interactions between neuroactive steroids acting at the GABA(A) receptor. The alpha and gamma subunit subtypes may have the greatest influence on allopregnanolone modulation of GABA(A) receptor function, whereas the beta and gamma subunit subtypes appear to be most important for the modulatory effects of alphaxalone.

Functional properties of recombinant GABA A receptors composed of single or multiple β subunit subtypes

GABA A receptor (GABAR) isoforms in the central nervous system are composed of combinations of α(1–6), β(1–4), γ(1–4), δ(1) and ε(1) subunit subtypes arranged in a pentamer. Many regions of the brain express high levels of mRNA encoding several different subunits and even multiple subunit subtypes. The stoichiometry of GABAR isoforms is unclear, and the number and identity of individual subunit subtypes that are coassembled remain uncertain. To examine the role of β subunit subtypes in the functional properties of GABARS and to determine whether multiple β subtypes can be coassembled in functional GABARs, plasmids containing cDNAs encoding rat β1 and/or β3, α5 and γ2L subtypes were cotransfected into L929 fibroblasts. The properties of the expressed receptor populations were determined using whole-cell and single-channel recording techniques. The α5β1γ2L isoform was less sensitive to GABA than the α5β3γ2L isoform. α5β1γ2L isoform currents were also insensitive to the allosteric modulator loreclezole, while α5β3γ2L isoform currents were strongly potentiated by loreclezole. Fibroblasts transfected with plasmids containing cDNAs for both β1 and β3 subtypes along with α5 and γ2L subtypes produced a receptor population with an intermediate sensitivity to GABA which was insensitive to loreclezole. These results suggest that functional GABARs can be formed that contain two different β subunit subtypes with properties different from receptors that contain only a single β subtype and that the β subunit subtypes influence the response of GABARs to GABA and to the allosteric modulator loreclezole.

The role of alpha1 and alpha6 subtype amino-terminal domains in allosteric regulation of gamma-aminobutyric acida receptors.

The gamma-aminobutyric acidA (GABA) receptor in the mammalian central nervous system is composed of pentameric combinations of alpha1-6, beta1-4, gamma1-3, delta1, and/or epsilon1 subunit subtypes. Although each of the different subunits influences the functional properties of gamma-aminobutyric acidA receptors (GABARs), the alpha subunit subtypes have been shown to be important for activation of the receptor by GABA and pentobarbital and the regulation of GABARs by numerous allosteric regulators, including benzodiazepines, furosemide, zinc, and lanthanum. However, with the exception of the benzodiazepines, the alpha subtype domain that is responsible for the action of these allosteric compounds is unknown. The alpha1 and alpha6 subtypes are among the most diverse of the alpha subunit family and confer a different responsiveness of GABARs to GABA and many of the allosteric modulators. These regulatory compounds act after extracellular application and therefore likely act on extracellular GABAR sites, the largest of which is the amino-terminal extracellular domain. To determine the role of this domain in the action of these allosteric regulatory agents, we constructed chimeras of the rat alpha1 and alpha6 subtypes with a splice site within the first putative transmembrane domain (TM). This separated the large extracellular amino-terminal domain from the transmembrane, intracellular, and TM2-3 and carboxyl-terminal extracellular domains of the subunit. The chimeric subtypes were expressed in L929 fibroblasts along with beta3 and gamma2L subtypes, and their pharmacological properties were determined with whole-cell electrophysiological recording. The alpha subtype amino-terminal extracellular domain was the primary determinant of GABA sensitivity and was responsible for the functional properties of activation by pentobarbital, sensitivity to diazepam, potentiation by lanthanum, and high affinity inhibition by furosemide. The remaining carboxyl-terminal domains influenced the GABA sensitivity and determined zinc sensitivity and low affinity inhibition by furosemide. Both domains were apparently required for inhibition by lanthanum.