GABA Rho Research Articles

Novel GABAA‐rho1 interactions with acid sensing ion channel ligands (1059.3)

γ‐ amino butyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain, and targets the ionotropic GABAA receptors. GABAC, or GABAA‐rho, is a subclass of GABAA receptors composed entirely of rho (ρ) subunits and are located on the axonal terminal of retinal bipolar cells, where it not only exhibits a tonic inhibitory current, but also regulates the GABA‐A and other GABAA‐rho synaptic currents (Jones et al 2011). GABAA‐rho exhibits unique properties, such as insensitivity to select antagonists of the heteromeric GABAA receptors (Korpi et al., 2002). A group of ligands, which possess a guanidine group, have been shown to influence GABAA receptors. These compounds, such as (S)‐2‐Guanidinopropionic acid and guanidine acetic acid were competitive antagonists for the GABAA‐rho1 receptor. Other guanidine compounds that are acid sensing ion channel (ASIC) ligands, might also exhibit unique effects on the GABAA‐rho1 receptor. We hypothesize that these ASIC ligands will exhibit unique intrinsic activities on the GABAA‐rho1 receptor, which is different from that of the heteromeric GABAA receptor. The human GABAA‐rho1 receptors were expressed in HEK‐293T cells, and activity was analyzed using whole cell patch‐clamp electrophysiology. When co‐applied with GABA and compared to the GABA concentration profile, one ligand was found to decrease the maximal response, with no change in the GABA EC50. This indicates characteristics of non‐competitive inhibition, while a different ligand shifted the GABA EC50 to lower GABA concentrations, suggesting this guanidine compound is a positive allosteric modulator. When applied alone, it failed to directly activate GABA rho1 receptors. These contrasting effects suggest that these ligands act at two binding sites within the GABAA‐rho architecture. Future experiments will focus on additional characterization of these novel effects on GABA rho receptors and offer a novel chemical structure to design novel GABAA‐rho therapeutics.Grant Funding Source: Supported by: Welch Foundation Grant No: BK‐1736, NIH training grant: NBA T32 AG020494

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

Truncated glycine receptors that have been found in human patients suffering from the neuromotor disorder hyperekplexia or in spontaneous mouse models resulted in non-functional ion channels. Rescue of function experiments with the lacking protein portion expressed as a separate independent domain demonstrated restoration of glycine receptor functionality in vitro. This construct harbored most of the TM3-4 loop, TM4, and the C terminus and was required for concomitant transport of the truncated α1 and the complementation domain from the endoplasmic reticulum toward the cell surface, thereby enabling complex formation of functional glycine receptors. Here, the complementation domain was stepwise truncated from its N terminus in the TM3-4 loop. Truncation of more than 49 amino acids led again to loss of functionality in the receptor complex expressed from two independent domain constructs. We identified residues 357-418 in the intracellular TM3-4 loop as being required for reconstitution of functional glycine-gated channels. All complementation constructs showed cell surface protein expression and correct orientation according to glycine receptor topology. Moreover, we demonstrated that the truncations did not result in a decreased protein-protein interaction between both glycine receptor domains. Rather, deletions of more than 49 amino acids abolished conformational changes necessary for ion channel opening. When the TM3-4 loop subdomain harboring residues 357-418 was expressed as a third independent construct together with the truncated N-terminal and C-terminal glycine receptor domains, functionality of the glycine receptor was again restored. Thus, residues 357-418 represent an important determinant in the process of conformational rearrangements following ligand binding resulting in channel opening.

Biological Activity of GABA Neurotransmitter Conjugated with Gold Nanoparticles in Xenopus Laevis Oocytes Transfected with GABA Rho 1 Receptor

Gold nanoparticles are of great interest to the scientific community owing to their potential applications in biology and medicine, earned by their extraordinary optical and chemical properties; counting their excellent biocompatibility, colloidal stability and suitable surface properties for conjugation with biomaterials. In this study, GABA, a classic neurotransmitter was conjugated with gold nanoparticles (Au@GABA). The gold nanoconjugates were synthesized by forming a covalent bond between gold nanoparticles and GABA. Au@GABA nanoconjugates showed excellent optical features and superior stability under physiological conditions. Results from UV-Vis, FTIR and Raman spectroscopies proved the optimum optical properties and chemical composition of the nanoconjugates. The surface charge was characterized by gel electrophoresis and the morphological properties by scanning electron microscopy. Bare gold nanoparticles and nanoconjugates were tested by electrophysiological recording in oocytes of Xenopus laevis expressing GABA rho 1 receptor.

A Molecular Dynamics Study on the Effect of Disulfide Bonds in Cys-Loop Ligand-Gated GABAA Receptors

GABAA-receptors (GABARs) are chloride ion channels in the ‘cys-loop receptor’ superfamily that are major inhibitory neuroreceptors. Upon agonist binding, GABARs open and increase intraneuronal chloride ion concentration, hyperpolarize the cell and inhibit neural transmission. Reducing agents will break the disulfide bonds in the cys-loop of the receptor. Indeed, it has been speculated that sulfhydryl compounds modify proteins via thiol/disulfide redox reactions and serve as neuromodulators (1). Reducing reagents were found to decrease the GABA EC50 in the highly homologous GABAC-receptor, while oxidizing reagents increased the EC50 (2). Conversely, conflicting findings suggests the mechanism of action of redox modulation does not alter GABAR agonist-binding affinity (3). However, studies are complicated by the fact that mutagenesis of the cysteines commonly produces failure of subunit assembly (4). Thus, to investigate the effect of disulfide bond breakage on the receptor we generated various GABAR homology models with and without disulfide bonds. These systems were subjected to ligand docking and extensive molecular dynamics simulations to determine if the GABA affinity was modulated. This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.1. Allan, A. M. and L. D. Baier. 1992. Effect of thiol group modification on ion flux and ligand binding properties of the GABAA-benzodiazepine receptor chloride channel complex. Synapse 10:310-316.2. Calero, C. I. and D. J. Calvo. 2008. Redox modulation of homomeric rho1 GABA receptors. J Neurochem 105:2367-2374.3. Pan, Z. H., et al. 2000. Redox modulation of recombinant human GABA(A) receptors. Neuroscience 98:333-338.4. Amin, J., et al. 1994. The agonist binding site of the gamma-aminobutyric acid type A channel is not formed by the extracellular cysteine loop. Mol Pharmacol 45:317-323.LLNL-ABS-502871

Rho1 GABAC receptors are expressed in fibrous and cartilaginous layers of chick sclera and located on sclera fibroblasts and chondrocytes

rho(1) GABA(C) receptor antagonists inhibit myopia in chick but the site of this effect is not known. The sclera ultimately determines the shape and size of the globe and thus an untested possibility is that GABA agents have a scleral mechanism. Whether rho(1) GABA(C) receptors are expressed and located in chick sclera is unknown. Real-time PCR, western blot and immunohistochemistry were used to determine whether rho1 GABA(C) receptors are expressed and located in chick fibrous and cartilaginous sclera. Both layers of the chick sclera were positive for rho1 GABA(C) receptor mRNA (PCR) and protein (western blot) expression and labeling was observed in both fibroblasts and chondrocytes of the fibrous and cartilaginous layers (immunohistochemistry). These investigations clearly show that chick sclera possesses rho(1) GABA(C) receptors. The sclera is thus a potential previously unrecognized site for activity of rho(1) GABA(C) agents.

Retinal vascular leakage occurring in GABA Rho-1 subunit deficient mice

Recent studies demonstrate that GABAergic activity elicits relaxation of retinal arterioles leading to an increase in blood flow. It has also been found that GABAnergic activity in the retina of mice with diabetic retinopathy is suppressed. In this study, we provide further evidence that lack of GABAergic activity significantly alters vasculature development as well as the hypoxia-induced angiogenic response. Using GABA C receptor ρ 1 subunit-knockout mice ( rho-1 −/−), our results demonstrate that in hypoxia-induced retinas a severe vascular leakage occurred in 2 week-old rho-1 −/− mice compared with their wildtype counterparts. In addition, our results also showed that all of the rho-1 −/− mice developed significant retinal vascular leakages by 48 weeks-of-age. Microarray and real-time PCR experiments revealed a unique angiogenesis-related gene expression pattern. This suggests that retinal vascular disorders of rho-1 −/− mice results from significant up-regulation of angiogenic genes and concomitant down-regulation of anti-angiogenic genes. The study results are consistent with the pathological changes of the retinal vascular leakage seen in diabetic retinopathy. Our data indicate that the GABA C ρ 1 subunit plays a role in maintaining both homeostasis and balance of retinal neurotransmitter function. Knockout of the retinal GABA C ρ 1-subunit leads to changes in vascular permeability similar to the pathological changes induced by retinal hypoxic conditions.

Carisoprodol-Mediated Modulation of GABAA Receptors: In Vitro and in Vivo Studies

Carisoprodol is a frequently prescribed muscle relaxant. In recent years, this drug has been increasingly abused. The effects of carisoprodol have been attributed to its metabolite, meprobamate, a controlled substance that produces sedation via GABA(A) receptors (GABA(A)Rs). Given the structural similarities between carisoprodol and meprobamate, we used electrophysiological and behavioral approaches to investigate whether carisoprodol directly affects GABA(A)R function. In whole-cell patch-clamp studies, carisoprodol allosterically modulated and directly activated human alpha1beta2gamma2 GABA(A)R function in a barbiturate-like manner. At millimolar concentrations, inhibitory effects were apparent. Similar allosteric effects were not observed for homomeric rho1 GABA or glycine alpha1 receptors. In the absence of GABA, carisoprodol produced picrotoxin-sensitive, inward currents that were significantly larger than those produced by meprobamate, suggesting carisoprodol may directly produce GABAergic effects in vivo. When administered to mice via intraperitoneal or oral routes, carisoprodol elicited locomotor depression within 8 to 12 min after injection. Intraperitoneal administration of meprobamate depressed locomotor activity in the same time frame. In drug discrimination studies with carisoprodol-trained rats, the GABAergic ligands pentobarbital, chlordiazepoxide, and meprobamate each substituted for carisoprodol in a dose-dependent manner. In accordance with findings in vitro, the discriminative stimulus effects of carisoprodol were antagonized by a barbiturate antagonist, bemegride, but not by the benzodiazepine site antagonist, flumazenil. The results of our studies in vivo and in vitro collectively suggest the barbiturate-like effects of carisoprodol may not be due solely to its metabolite, meprobamate. Furthermore, the functional traits we have identified probably contribute to the abuse potential of carisoprodol.

A Molecular Basis for Agonist and Antagonist Actions at GABAC Receptors

We modelled the N-terminal ligand-binding domain of the rho1 GABA(C) receptor based on the Lymnaea stagnalis acetylcholine-binding protein (L-AChBP) crystal structure using comparative modelling and validated using flexible docking guided by known mutagenesis studies. A range of known rho1 GABA(C) receptor ligands comprising seven full agonists, 10 partial agonists, 43 antagonists and 12 inactive molecules were used to evaluate and validate the models. Of the 50 models identified, six models that allowed flexible ligand docking in accordance with the experimental data were selected and used to study detailed receptor-ligand interactions. The most refined model to accommodate all known active ligands featured a cavity comprising of a volume of 488 A(3). A detailed analysis of the interaction between the rho1 GABA(C) receptor model and the docked ligands revealed possible H-bonds and cation-pi interactions between the different ligands and binding site residues. Based on quantum mechanical/molecular mechanical (QM/MM) calculations, the model showed distinctive conformations of loop C that provided a molecular basis for agonist and antagonist actions. Agonists elicit loop C closure, while a more open loop C was observed upon antagonist binding. The model differentiates the role for key residues known to be involved in either binding and/or gating.

Enantioselective actions of 4-amino-3-hydroxybutanoic acid and (3-amino-2-hydroxypropyl)methylphosphinic acid at recombinant GABAC receptors

The R- and S-enantiomers of 4-amino-3-hydroxybutanoic acid (GABOB) were full agonists at human recombinant rho1 GABA(C) receptors. Their enantioselectivity (R>S) matched that reported for their agonist actions at GABA(B) receptors, but was the opposite to that reported at GABA(A) receptors (S>R). The corresponding methylphosphinic acid analogues proved to be rho1 GABA(C) receptor antagonists with R(+)-CGP44533 being more potent than S(-)-CGP44532, thus showing the opposite enantioselectivity to the agonists R(-)- and S(+)-GABOB. These studies highlight the different stereochemical requirements for the hydroxy group in these analogues at GABA(A), GABA(B) and GABA(C) receptors.

Random assembly of GABA rho1 and rho2 subunits in the formation of heteromeric GABA(C) receptors.

1. Various combinations of the rho subunits (rho(1A), rho(1B), rho(2A), rho(2B)) of GABA(C) receptors cloned from white perch retina were expressed in Xenopus oocytes, and electrophysiological and pharmacological methods were used to test their ability to co-assemble into heteromeric receptors. Simultaneous injection of the two subunits, irrespective of their relative proportions, led invariably to the formation of a preponderance of heteromeric receptors. 2. The GABA deactivation responses elicited from these cells could be described by a single exponential decay, and their pharmacological responses deviated significantly from those expected of a simple mixture of two homomeric rho(1) and rho(2) receptors. In contrast, a double exponential function comprising fast and slow components was required to fit the GABA deactivation responses elicited from oocytes sequentially expressing rho(1) and rho(2) subunits, a condition that favors the formation of a mixture of homomeric rho(1) and rho(2) receptors. 3. Both the GABA-response kinetics and the sensitivity to picrotoxin of the heteromeric perch rho(1B)rho(2A) receptor varied with the proportion of the subunit RNA injected, indicating there is no fixed stoichiometry for their co-assembly into heteromeric rho(1)rho(2) receptors. 4. If native GABA(C) receptors in retinal neurons behave in a similar manner as in the oocyte expression system, these finding suggest that the properties of their GABA(C) receptors are likely to be influenced by the transcription/translation efficiency of GABA rho subunit genes.

A Cation-π Binding Interaction with a Tyrosine in the Binding Site of the GABA C Receptor

GABA(C) (rho) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HT(3), and glycine receptors. As in other members of this family, the agonist binding site of GABA(C) receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-pi interaction to a tryptophan, the GABA(C) binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-pi binding ability of the side chain and EC(50) for receptor activation, thus demonstrating a cation-pi interaction between a tyrosine side chain and a neurotransmitter. Comparisons among four homologous receptors show variations in cation-pi binding energies that reflect the nature of the cationic center of the agonist.

Studies on the mechanisms of action of picrotoxin, quercetin and pregnanolone at the GABA rho 1 receptor.

1. The mechanisms of action of antagonists of the gamma-aminobutyric acid C (GABA(C)) receptor picrotoxin, quercetin and pregnanolone were studied. 2. Ionic currents (chloride), mediated through human homomeric GABA rho(1) receptors expressed in Xenopus oocytes, were recorded by two-electrode voltage clamp. 3. Dose-response (D-R) curves and kinetic measurements of GABA rho(1) currents were carried out in the presence or absence of antagonists. Use-dependent actions were also evaluated. 4. Picrotoxin, quercetin and pregnanolone exerted noncompetitive actions. 5. IC(50) values measured at the EC(50) for GABA (1 microM) were as follows: picrotoxin 0.6+/-0.1 microM (Hill coefficient n=1.0+/-0.2); quercetin 4.4+/-0.4 microM (n=1.5+/-0.2); pregnanolone 2.1+/-0.5 microM (n=0.8+/-0.1). 6. These antagonists produced changes only in the slope of the linear current-voltage relationships, which was indicative of voltage-independent effects. 7. The effect of picrotoxin on GABA rho(1) currents was use-dependent, strongly relied on agonist concentration and showed a slow onset and offset. The mechanism was compatible with an allosteric inhibition and receptor activation was a prerequisite for antagonism. 8. The effect of quercetin was use-independent, showed relatively fast onset and offset, and resulted in a slowed time course of the GABA-evoked currents. 9. The effect of pregnanolone was use-independent, presented fast onset and a very slow washout, and did not affect current activation. 10. All the antagonists accelerated the time course of deactivation of the GABA rho(1) currents.

Mutations at the GABA receptor selectivity filter: a possible role for effective charges.

An important feature of ligand-gated ion channels is their exquisite ability to discriminate between ions. Still, little is known about the mechanisms underlying, or structural determinates of, this ability. We examined the structural elements underlying the ionic selectivity of rho1 GABA receptors expressed in Xenopus oocytes and human embryonic kidney cells using site-directed mutagenesis and two-electrode voltage-clamp or patch-clamp techniques. The wild-type GABA receptor was chloride selective, with a small but significant permeability to potassium (PNa+ : PK+ : PCl- = 0 : 0.03 :1). Mutation of an alanine to glutamate at position 291 (thought to be located at the intracellular end of the second transmembrane domain), formed a channel that exhibited little discrimination among ions (0.70:0.87:1), while deletion of a neighbouring proline (290) was chloride selective, but had elevated cation permeabilities compared to the wild-type channel (0.12 : 0.14 : 1). Together, the two mutations (DeltaP290/A291E) caused a reversal of selectivity (2.72 : 3.59 : 1). We also examined the effects of neutralizing and reversing the charge of the adjacent, and highly conserved, arginine. Mutation of the neighbouring arginine to glutamate (R292E) increased the cation permeability similar to the DeltaP290/A291E double mutant (2.4 : 3.0 : 1), whereas neutral mutations at this position (R292M or R292C) retained chloride selectivity (0 : 0.11 : 1.0 and 0 : 0.14 : 1.0, respectively). Our experiments suggest that the effective charge near the presumed intracellular mouth of the pore is critical for ionic selectivity.

Medicinal Chemistry and Molecular Pharmacology of GABA-C Receptors

GABA(C) receptors belong to the nicotinicoid superfamily of ionotropic receptors that include nicotinic acetylcholine receptors, bicuculline-sensitive GABA(A) receptors, strychnine-sensitive glycine receptors and 5HT3 serotonin receptors. The GABA(C) receptor concept arose from medicinal chemical studies of a conformationally restricted analog of GABA. Receptors matching the predicted properties of GABA(C) receptors were cloned from the retina. Post cloning studies revealed the unique physiology and pharmacology of these relatively simple homomeric receptors. Three subtypes of GABA(C) receptors have been cloned from mammalian sources and pharmacological differences between the rho1, rho2 and rho3 GABA(C) receptors have been described. There is evidence for functional GABA(C) receptors in the retina, spinal cord, superior colliculus, pituitary and the gut and their involvement in vision, aspects of memory and sleep-waking behaviour. This review concentrates on the medicinal chemistry and molecular pharmacology of GABA(C) receptor subtypes emphasising possible new investigational tools with which to investigate further GABA(C) receptor function. The most useful currently available ligands that show some GABA(C) receptor subtype selectivity are TPMPA, P4PMA, imidazole-4-acetic acid, 2-methyl-TACA and (+/-)-TAMP.

Positive allosteric modulation by ultraviolet irradiation on GABA(A), but not GABA(C), receptors expressed in Xenopus oocytes.

1. Recombinant rat GABA(A) (alpha1beta2, alpha1beta2gamma2, beta2gamma2) and human GABA(C) (rho1) receptors were expressed in Xenopus oocytes to examine the effect of ultraviolet (UV) light on receptor function. 2. GABA-induced currents in individual oocytes expressing GABA receptors were tested by two-electrode voltage clamp before, and immediately after, 312 nm UV irradiation. 3. UV irradiation significantly potentiated 10 microM GABA-induced currents in alpha1beta2gamma2 GABA receptors. The modulation was irradiation dose dependent, with a maximum potentiation of more than 3-fold. 4. The potentiation was partially reversible and decayed exponentially with a time constant of 8.2 +/- 1.2 min toward a steady-state level which was still significantly elevated (2.7 +/- 0.3-fold) compared to the control level. 5. The effect of UV irradiation on GABA(A) receptors varied with receptor subunit composition. UV irradiation decreased the EC50 of the alpha1beta2, alpha1beta2gamma2 and beta2gamma2 GABA(A) receptors, but exhibited no significant effect on the rho1 GABA(C) receptor. 6. UV irradiation also significantly increased the maximum current 2-fold in alpha1beta2 GABA(A) receptors with little effect on the maximum of alpha1beta2gamma2 (1.1-fold) or beta2gamma2 (1.1-fold) GABA(A) receptors. 7. The effect of UV irradiation on GABA(A) receptors did not overlap the effect of the GABA receptor- allosteric modulator, diazepam. 8. The UV effect on GABA(A) receptors was not prevented by the treatment of the oocytes before and during UV irradiation with one of the following free-radical scavengers: 40 mM D-mannitol, 40 mM imidazole or 40 mM sodium azide. In addition, the effect was not mimicked by the free-radical generator, H2O2. 9. Potential significance and mechanism(s) of the UV effect on GABA receptors are discussed.

Recombinant GABA(C) receptors expressed in rat hippocampal neurons after infection with an adenovirus containing the human rho1 subunit.

1. A recombinant adenovirus was generated with the human rho1 GABA(C) receptor subunit (adeno-rho). Patch-clamp and antibody staining were employed to confirm functional expression of recombinant rho1 receptors after infection of human embryonic kidney cells (HEK293 cell line), human embryonic retinal cells (911 cell line), dissociated rat hippocampal neurons and cultured rat hippocampal slices. 2. Standard whole-cell recording and Western blot analysis using rho1 GABA(C) receptor antibodies revealed that recombinant rho1 receptors were expressed in HEK293 and 911 cells after adeno-rho infection and exhibited properties similar to those of rho1 receptors after standard transfection. 3. Cultured rat hippocampal neurons (postnatal day (P)3-P5) did not show a native GABA(C)-like current. After adeno-rho infection, however, a GABA(C)-like current appeared in 70-90 % of the neurons. 4. Five days after infection, expression of GABA(C) receptors in hippocampal neurons significantly decreased native GABA(A) receptor currents from 1200 +/- 300 to 150 +/- 70 pA (n = 10). The native glutamate-activated current was unchanged. 5. Hippocampal slices (P8) did not show a native GABA(C)-like current, although recombinant rho1 receptors could be expressed in cultured hippocampal slices after adeno-rho infection. 6. These data indicate that an adenovirus can be used to express recombinant GABA(C) receptors in hippocampal neurons. This finding could represent an important step towards the gene therapy of CNS receptor-related diseases.

Evidence for phosphorylation-dependent internalization of recombinant human rho1 GABAC receptors.

1. Recombinant wild-type or mutant human rho1 GABA receptors were expressed in human embryonic kidney (HEK) 293 or monkey COS-7 cells and studied using the patch clamp technique. 2. Standard whole-cell recordings with 4 mM Mg-ATP in the patch pipette induced a time-dependent decrease in the GABA-activated current (IGABA) amplitude that was not the result of a decrease in GABA sensitivity. In contrast, IGABA remained stable when recordings were obtained using the perforated patch configuration or with standard whole-cell recording and no Mg-ATP in the patch pipette. 3. The inhibitors of serine/threonine protein kinases KN-62 (20 microM) or staurosporine (20 nM) prevented the time-dependent decrease in the amplitude of IGABA seen in the presence of ATP. Alkaline phosphatase (220 U ml-1), when added to the patch pipette in the absence of ATP, induced a transient potentiation of IGABA. Although the protein kinase C (PKC) activator 4beta-phorbol 12-myristate, 13-acetate (PMA) did not reduce the amplitude of IGABA, inclusion of the catalytic domain of PKC in the recording pipette accelerated the time-dependent decrease in current amplitude. These data suggest that phosphorylation is involved in the regulation of the amplitude of IGABA. 4. Mutation of the three PKC consensus sequences of the rho1 receptor had no significant effect on the decline in IGABA, indicating that direct phosphorylation of these putative sites on the rho1 receptor does not underlie the time-dependent decrease in amplitude. 5. In COS-7 cells transfected with wild-type rho1 receptors, the amplitude of IGABA had completely recovered to the original value when the same cells were repatched after 30-40 min, indicating that the decline in IGABA was a reversible process. 6. The inhibitor of actin filament formation cytochalasin B, when added to the patch pipette in the absence of ATP, induced a time-dependent inactivation suggesting that the actin cytoskeleton may play a role in the regulation of the amplitude. 7. Coincident with the decrease in the amplitude of IGABA, the cell capacitance significantly decreased in the presence of ATP in the patch pipette. This decrease in capacitance was not observed in the absence of Mg-ATP. The decrease in the membrane surface area suggests that receptor internalization could be a potential mechanism for the observed inactivation. 8. At 32 C, compared with 22 C, the rate and magnitude of the decline was increased dramatically. In contrast, at 16 C, no significant change in IGABA was observed over the 20 min recording time. This marked temperature sensitivity is consistent with receptor internalization as a mechanism for the time-dependent decline in IGABA. 9. The specificity of the decrease in IGABA was assessed by coexpressing the voltage-dependent potassium channel Kv1.4 along with the rho1 receptor in HEK293 cells. The amplitude of the potassium current (IKv1.4) exhibited very little decrement in comparison to IGABA suggesting that the putative GABA receptor internalization was not the consequence of a non-specific membrane retrieval.

SITES FOR ISOFLURANE ACTION ON INHIBITORY AMINO ACID RECEPTORS REVEALED BY MUTAGENESIS?

S456 Neuronal membrane proteins such as the GABA-A receptor (GABA-A R), glycine receptor (Gly-R), and neuronal nicotinic acetylcholine receptors may play an important role in mediating the pharmacologic responses to inhaled anesthetics such as isoflurane (Forane). Isoflurane potentiates the actions of submaximal agonist concentrations at Gly-R and GABA-A R, as do the alcohols. We have identified specific amino acid residues within the Gly-R and GABA-A R which are crucial for the allosteric regulation of these receptors by isoflurane. Methods: Point mutants were created using unique site elimination (USE) mutagenesis. All constructs were verified by sequencing. Electrophysiological analysis was carried out in Xenopus oocytes and HEK 293 cells. GABA or glycine was applied in known concentrations to determine the EC50 and EC10 for agonist, and alcohol or anesthetic effects were then determined against EC10 concentrations of agonist. Results: Receptor chimeras based on the Gly-R alpha 1 subunit and GABA rho 1 subunit were used to identify a critical domain of 45 amino acids within the Gly-R. A series of 14 point mutants of the Gly-R alpha 1 subunit was then expressed in Xenopus oocytes. Of this group, only the TM2 mutant Gly-R alpha 1 (Ser 267 Ile) is insensitive to ethanol. Mutation of Ser 267 to Tyr also abolished Gly-R sensitivity to enflurane. An inverse relationship was found between the molecular volume of the residue at 267 and the potentiation of receptor function by enflurane. The presence of small residues such as Gly and Ala at 267 was associated with strong potentiation of function, whereas when larger residues such as Phe, His and Trp were present, potentiation was small or undetectable. Mutation at the analogous position in the GABA-A R, alpha 1(Ser 270 Ile) beta 1 produced a receptor which is insensitive to ethanol and to isoflurane. Another mutant GABA-A R alpha 1(Ala 291 Trp) beta 1 is also insensitive to ethanol, isoflurane and enflurane. All of these mutants showed near normal gating by GABA and normal sensitivity to propofol and methohexital. Conclusions: These data suggest a crucial role of specific amino acid residues in the allosteric regulation of both Gly-R and GABA-A R by isoflurane. The structures of these receptor proteins are presently unknown, but available evidence suggests that Ser 270 in GABA-A R alpha 1 is oriented towards the hydrophobic interior of the protein. [2] One possibility is that Ser 270 and Ala 291 in the alpha subunit of the GABA-A R form part of a binding pocket for anesthetic molecules between TM2 and TM3. The highly specific and selective effects of GABA-A R and Gly-R mutagenesis reported here will allow for testing of specific hypotheses of anesthetic mechanism in the future. Supported by GM45129, GM 00623.

The N-terminal Domain of Human GABA Receptor ρ1 Subunits Contains Signals for Homooligomeric and Heterooligomeric Interaction

gamma-Aminobutyric acid type C (GABAC) receptors identified in retina appear to be composed of GABA rho subunits. The purpose of this study was to localize signals for homooligomeric assembly of rho1 subunits and to investigate whether the same region contained signals for heterooligomeric interaction with rho2 subunits. In vitro translated human rho1 was shown to be membrane-associated, and proteinase K susceptibility studies indicated that the N terminus was oriented in the lumen of ER-derived microsomal vesicles. This orientation suggested the involvement of the N terminus of rho1 in the initial steps of subunit assembly. To test this hypothesis, mutants were created containing only N-terminal sequences (N-rho1) or C-terminal sequences (C-rho1) of rho1. Co-immunoprecipitation studies revealed that N-rho1, but not C-rho1, interacted with rho1 in vitro. When coexpressed in Xenopus oocytes, N-rho1 interfered with rho1 receptor formation. Together, these data suggested that signals for rho1 homooligomeric assembly reside in the N-terminal half of the subunit. Sequential immunoprecipitations were then performed upon cotranslated rho1 and rho2 subunits which demonstrated that rho1 and rho2 interacted in vitro. Co-immunoprecipitation indicated that N-rho1 specifically associated with rho2. Therefore, the N-terminal regions of rho subunits contain the initial signals for both homooligomeric and heterooligomeric assembly into receptors with GABAC properties.

Insights into the activation mechanism of rho1 GABA receptors obtained by coexpression of wild type and activation-impaired subunits.

To gain insight into the activation mechanism of homomeric ligand-gated receptor-channels, we examined human homomeric rho1 GABA receptors with fewer than the normal number of agonist binding sites. This was accomplished by coexpressing different ratios of wild type and activation-impaired rho1 subunits. Dose-response relations from oocytes coexpressing wild type and mutant subunits were comprised of two components in terms of GABA sensitivity; one 'wild type'-like and the other 'mutant'-like. Applying the binomial hypothesis to subunit coassembly enabled use to correlate these two components of the GABA dose-response relations to the underlying chimaeric receptor subtypes. We demonstrate that the receptors activate near normal provided that they are comprised of at least three wild type subunits. Our data are consistent with five equivalent and independent GABA binding sites of which only three need bind GABA to open the pore. The two additional binding sites may increase the GABA sensitivity of the rho1 receptor and, when bound by agonist, stabilize the open state.