Gamma-aminobutyric Acid Receptor Channels Research Articles

Structural model of ρ1 GABAC receptor based on evolutionary analysis: Testing of predicted protein–protein interactions involved in receptor assembly and function

The homopentameric rho1 GABA(C) receptor is a ligand-gated ion channel with a binding pocket for gamma-aminobutyric acid (GABA) at the interfaces of N-terminal extracellular domains. We combined evolutionary analysis, structural modeling, and experimental testing to study determinants of GABA(C) receptor assembly and channel gating. We estimated the posterior probability of selection pressure at amino acid residue sites measured as omega-values and built a comparative structural model, which identified several polar residues under strong selection pressure at the subunit interfaces that may form intersubunit hydrogen bonds or salt bridges. At three selected sites (R111, T151, and E55), mutations disrupting intersubunit interactions had strong effects on receptor folding, assembly, and function. We next examined the role of a predicted intersubunit salt bridge for residue pair R158-D204. The mutant R158D, where the positively charged residue is replaced by a negatively charged aspartate, yielded a partially degraded receptor and lacked membrane surface expression. The membrane surface expression was rescued by the double mutant R158D-D204R, where positive and negative charges are switched, although the mutant receptor was inactive. The single mutants R158A, D204R, and D204A exhibited diminished activities and altered kinetic profiles with fast recovery kinetics, suggesting that R158-D204 salt bridge perhaps stabilizes the open state of the GABA(C) receptor. Our results emphasize the functional importance of highly conserved polar residues at the protein-protein interfaces in GABA(C) rho1 receptors and demonstrate how the integration of computational and experimental approaches can aid discovery of functionally important interactions.

Molecular, pharmacological and functional properties of GABAA receptors in anterior pituitary cells

Anterior pituitary cells express gamma-aminobutyric acid (GABA)-A receptor-channels, but their structure, distribution within the secretory cell types, and nature of action have not been clarified. Here we addressed these questions using cultured anterior pituitary cells from postpubertal female rats and immortalized alphaT3-1 and GH(3) cells. Our results show that mRNAs for all GABA(A) receptor subunits are expressed in pituitary cells and that alpha1/beta1 subunit proteins are present in all secretory cells. In voltage-clamped gramicidin-perforated cells, GABA induced dose-dependent increases in current amplitude that were inhibited by bicuculline and picrotoxin and facilitated by diazepam and zolpidem in a concentration-dependent manner. In intact cells, GABA and the GABA(A) receptor agonist muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABA(B) receptor agonist baclofen was ineffective, suggesting that chloride-mediated depolarization activates voltage-gated calcium channels. Consistent with this finding, RT-PCR analysis indicated high expression of NKCC1, but not KCC2 cation/chloride transporter mRNAs in pituitary cells. Furthermore, the GABA(A) channel reversal potential for chloride ions was positive to the baseline membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells and modulation of spontaneous electrical activity. These results indicate that secretory pituitary cells express functional GABA(A) receptor-channels that are depolarizing.

Developmental changes in expression of GABAA receptor‐channels in rat intrinsic cardiac ganglion neurones

The effects of gamma-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at -60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABA(A) receptor agonists muscimol and taurine, and inhibited by the GABA(A) receptor antagonists, bicuculline and picrotoxin. The GABA(A0) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABA(A) receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at -100 mV was approximately 20 times higher for intracardiac neurones obtained from neonatal rats (P2-5) compared with adult rats (P45-49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system.

Nitrous oxide-induced enhancement of gamma-aminobutyric acidA-mediated chloride currents in acutely dissociated hippocampal neurons.

Because the synaptic inhibition in the human brain is largely mediated by gamma-aminobutyric acid (GABA), the GABA receptor is of primary interest for the study of the working mechanism of general anesthetics. This article examines the interaction between this type of ion channel and nitrous oxide (N2O). Patch clamp recording techniques were applied to investigate the effects of N2O on GABA(A) receptor channels in a whole-cell configuration at room temperature. Acutely dissociated rat hippocampal cells from the CA1 region were used. Rapid application of the agonist muscimol and anesthetics (N2O, pentobarbital, and ethanol) was accomplished using a Y tube application system. Peak chloride (Cl-) currents were measured. Short-term application of muscimol (5-30 microM) with dissolved N2O (80%, approximately 22.5 mM) increased the Cl- current (approximately 140%) compared with muscimol alone. This effect is comparable with results the authors obtained with ethanol (800 mM) and pentobarbital (100 microM). Prolonged exposure (9 min) to N2O further increased Cl- currents by an additional 50%. Concentrations of N2O lower than 12 mM did not show an enhancement of this current, whereas application of N2O alone did not result in any Cl- conductance. These results indicate that N2O can enhance GABA(A) channel-mediated Cl- currents by modulating the effect of the specific GABA(A) agonist; it is not active by itself.

Distribution of GABA receptor rho subunit transcripts in the rat brain.

The gamma-aminobutyric acid (GABA) receptor rho subunits recently cloned from rat and human retina are thought to form GABA receptor channels belonging to a pharmacologically distinct receptor class, termed GABA(C). In this work we have examined the distribution of rho1, rho2 and rho3 subunits, and found expression of all three transcripts in several regions of the rat nervous system. In situ hybridization revealed expression of rho2 in the adult rat retina and some other parts of the visual pathways. A high local rho2 expression was seen in the superficial grey layer of the superior colliculus, and in the dorsal lateral geniculate nucleus. Expression was also detected in the 6th layer of visual cortex and in the CA1 pyramidal cell layer of hippocampus. With reverse transcriptase-polymerase chain reaction, expression of rho1 was mainly seen in the adult rat retina and dorsal root ganglia, as well as, at a significantly lower level, in the superior colliculus, hippocampus, brain stem, thalamus, postnatal day 8 (P8) superior colliculus and P8 hippocampus. Expression pattern of rho3 mRNA was clearly different from that of rho1 and rho2, being strongest in the hippocampus, and significantly lower in the retina, dorsal root ganglia and cortex. No rho3 expression was observed in adult or P8 superior colliculus or in P8 hippocampus. The present results clearly demonstrate that expression of GABA receptor rho subunits is not restricted to the retina, but significant expression can also be detected in many other brain regions, especially in those belonging to the visual pathways. The expression pattern of the rho subunits may be helpful in solving the functional significance of the receptors formed from these subunits.

A receptor with GABAC-like pharmacology in invertebrate neurones in culture.

We have characterized in crustacean neurones in culture a receptor for gamma-aminobutyric acid (GABA) which conforms to the pharmacological profile of the proposed type-C GABA receptor (GABAC) found in the vertebrate retina. It is associated with a chloride-selective ion channel and is blocked by picrotoxin. It is neither inhibited by bicuculline nor activated by baclofen, while diazepam and phenobarbital are without modulatory effect. Like the GABAC-like receptor of the vertebrate retina it is activated by the folded GABA analogue cis-4-aminocrotonic acid (CACA). Desensitization is moderate allowing for a more sustained action of GABA. Single channel recordings revealed a bicuculline-resistant GABA- and CACA-activated chloride channel with a conductance about eight times higher than that described for the bicuculline-resistant GABA receptor channel from the rat retina.

The molecular biology of chloride channels.

The molecular biology of chloride channels is a new field, having begun only 5 years ago with the cloning, on the basis of partial amino acid sequence information, of complementary DNAs for subunits of the neuronal glycine and gamma-aminobutyric acid-receptor channels. The sequences of these subunits that form heterooligomeric channels revealed a general similarity with the structural motif employed by the well-studied and previously cloned nicotinic acetylcholine-receptor subunits. The principal distinguishing feature was the rings of positively charged residues within the loops separating the putative transmembrane helices of the new chloride selective channels. A great diversity of subunit types with specific functional and pharmacologic properties as well as localizations within the central nervous system have been identified and cloned. Chloride channels are known to play an important role in both secretion and reabsorption of salt and fluid across wet epithelia, and the cystic fibrosis transmembrane conductance regulator, cloned by genetic means, has been shown to be centrally involved in both of these processes. It is regulated by a complex mechanism involving both ATP binding and phosphorylation at multiple sites. Expression cloning techniques have yielded a voltage-gated family of channels with a large number of potential membrane spanning segments; one member is responsible for the resting potential of skeletal muscle. An entirely different low molecular weight-voltage-gated channel, possibly regulated by osmolarity changes, is the most recent chloride channel to be cloned. It almost certainly will not be the last.

Characterization of the enantiomeric 3-substituted-1,4-benzodiazepin-2-ones binding to the GABA—BDZ—chloride ionophore receptor complex

Gamma aminobutyric acid (GABA) is an important and abundant inhibitory neurotransmitter in the mammalian central nervous system (CNS). Its interaction with the GABAA receptor subtypes opens anion channels in the membrane through which chloride ions pass down their electrochemical gradient, thus stabilizing the resting level of membrane potential. GABA* receptors, associated with the anion channel, are part of a protein complex containing distinct but interacting recognition sites for convulsants [such as picrotoxin and t-butylbicyclo-phosphorothionate (TBPS)] and depressants [such as benzodiazepines (BDZs) and barbiturates] [ 11. Therefore, the macromolecular make-up and interactions of the BDZ binding sites with the GABA receptor and chloride channel have to be accounted for in models of BDZ receptor function [2-41. Several papers report the structure activity relationships as well as the influence of the stereochemistry of BDZs on their in vitro binding to the CNS receptor [5-111. However, to the best of our knowledge, nothing is known about BDZ receptor function depending on the enantioselective binding of chiral BDZs to their receptor. In the present paper the interaction to the CNS receptor of a series of chiral3-substituted1,4-benzodiazepin-2-ones, resolved as pure enantiomers by preparative chiral HPLC, was studied. The study of the interaction of the BDZ receptor with the GABA receptor and chloride channel was utilized to profile the in vitro differences between enantiomeric BDZs as agonists, antagonists, and inverse agonists. Quantifiable differences in the coupling of the BDZ receptors to GABA receptors can be determined by measuring the GABA-shift in the affinities of BDZs to displace 3H-flunitrazepam [2, 11-131. Thus, the influence of the stereochemistry on the pharmacological profile of the BDZ receptor ligands has been investigated as GABA-ratio determination. Further, the study of possible interaction between BDZ receptor and the chloride channel has been performed by the use of 35S-TBPS (a selective label of chloride anion recognition site). Thus the effect exerted by chiral BDZs on the chloride channel regulation was evaluated by the modulation of 35S-TBPS binding.

General anesthetics modulate GABA receptor channel complex in rat dorsal root ganglion neurons

The effects of halothane, isoflurane, and enflurane on ionic currents induced by bath application of gamma-amino-butyric acid (GABA) were studied with the rat dorsal root ganglion neurons maintained in primary culture. The whole-cell patch clamp technique was used to record the current. In normal neurons before exposure to anesthetics, GABA at low concentrations (1-3 x 10(-6) M) induced a small sustained inward current. At higher concentrations (3 x 10(-5) M-1 x 10(-3) M), GABA induced a large inward current, which decayed to a steady-state level (desensitization). Halothane (0.86 mM), isoflurane (0.96 mM), and enflurane (1.89 mM), each equivalent to the respective 2 minimum alveolar concentration (MAC) units, augmented the sustained current evoked by 3 x 10(-6) M GABA to 330-350% of control and the peak current evoked by 3 x 10(-5) M of GABA to 136-145% of control. The decay phase of the current was accelerated by the anesthetics, the time for the current to decline to 70% of the peak being reduced to 23-39% of control. In contrast, the densitized steady-state current evoked by high concentrations of GABA was decreased by anesthetics. In conclusion, general anesthetics exert a dual effect on the GABA receptor channel complex: to potentiate the nondesensitized (both peak and sustained) current and to suppress the desensitized steady-state current. The potentiation of the GABA receptor channel response may be a primary action of anesthetics leading to surgical anesthesia.

Antiepileptic Drug Actions

Antiepileptic drugs (AEDs) vary in their efficacy against generalized tonic-clonic, myoclonic, and absence seizures, suggesting different mechanisms of action. Phenytoin (PHT), carbamazepine (CBZ), and valproate (VPA) reduced the ability of mouse central neurons to sustain high-frequency repetitive firing of action potentials (SRF) at therapeutic free serum concentrations. Phenobarbital (PB) and the benzodiazepines (BZDs), diazepam (DZP), clonazepam (CZP), and lorazepam (LZP), also reduced SRF, but only at supratherapeutic free serum concentrations achieved in treatment of generalized tonic-clonic status epilepticus. These AEDs interact with sodium channels to slow the rate of recovery of the channels from inactivation. The BZDs and PB enhanced gamma-aminobutyric acid (GABA) responses evoked on mouse central neurons by binding to two different sites on the GABAA receptor channel. BZDs increased the frequency of GABA receptor channel openings. In contrast, barbiturates increased the open duration of these channels. VPA enhanced brain GABA concentration and may enhance release of GABA from nerve terminals. Ethosuximide (ESM) reduced a small transient calcium current which has been shown to be involved in slow rhythmic firing of certain neurons. Reduction of SRF, enhancement of GABA-ergic inhibition, and reduction of calcium current may be, in part, the bases for AED action against generalized tonic-clonic, myoclonic, and absence seizures, respectively.

Patch-clamp study of gamma-aminobutyric acid receptor Cl- channels in cultured astrocytes.

The membrane channels operated by gamma-aminobutyric acid (GABA) were studied in cultured astrocytes from rat cerebral hemispheres by using patch-clamp techniques. The channel properties appeared to be very similar, in many respects, to those present in neuronal cell membranes. The Cl- -selective channels were activated after the sequential binding of two GABA molecules to the receptor, as deduced from the slope of the dose-response curve. Single-channel currents displayed multiple conductance states of 12 pS, 21 pS, 29 pS, and 43 pS, with the main-state conductance being 29 pS. The gating properties could be described by a sequential reaction scheme for agonist-activated channels. GABA-induced whole-cell currents were potentiated by the benzodiazepine receptor agonist diazepam and also, to a lesser extent, by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate an inverse agonist. In neurons and chromaffin cells, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate reduces the sensitivity of the GABA receptor, indicating that neuronal and glial GABA/benzodiazepine receptor--Cl- channel complexes are different. Glial GABA receptor channels could be of functional importance in buffering extracellular Cl- in the cleft of the GABAergic synapse.

Ankyrin and spectrin associate with voltage-dependent sodium channels in brain

The segregation of voltage-dependent sodium channels to specialized regions of the neuron is crucial for propagation of an action potential. Studies of their lateral mobility indicate that sodium channels are freely mobile on the neuronal cell body but are immobile at the axon hillock, presynaptic terminal and at focal points along the axon. To elucidate the mechanisms that regulate sodium channel topography and mobility, we searched for specific proteins from the brain that associate with sodium channels. Here we show that sodium channels labelled with 3H-saxitoxin (STX) are precipitated in the presence of exogenous brain ankyrin by anti-ankyrin antibodies and that 125I-labelled ankyrin binds with high affinity to sodium channels reconstituted into lipid vesicles. The cytoplasmic domain of the erythrocyte anion transporter competes for the latter interaction. Neither the neuronal GABA (gamma-aminobutyric acid) receptor channel complex nor the dihydropyridine (DHP) receptor bind brain ankyrin. The results indicate that brain ankyrin links the voltage-dependent sodium channel to the underlying cytoskeleton and may help to maintain axolemmal membrane heterogeneity and control sodium channel mobility.

Mechanism of anion permeation through channels gated by glycine and gamma‐aminobutyric acid in mouse cultured spinal neurones.

1. The ion-selective and ion transport properties of glycine receptor (GlyR) and gamma-aminobutyric acid receptor (GABAR) channels in the soma membrane of mouse spinal cord neurones were investigated using the whole-cell, cell-attached and outside-out patch versions of the patch-clamp technique. 2. Current-voltage (I-V) relations of transmitter-activated currents obtained from whole-cell measurements with 145 mM-Cl- intracellularly and extracellularly, showed outward rectification. In voltage-jump experiments, the instantaneous I-V relations were linear, and the steady-state I-V relations were rectifying outwardly indicating that the gating of GlyR and GABAR channels is voltage sensitive. 3. The reversal potential of whole-cell currents shifted 56 mV per tenfold change in internal Cl- activity indicating activation of Cl(-)-selective channels. The permeability ratio of K+ to Cl- (PK/PCl) was smaller than 0.05 for both channels. 4. The permeability sequence for large polyatomic anions was formate greater than bicarbonate greater than acetate greater than phosphate greater than propionate for GABAR channels; phosphate and propionate were not measurably permeant in GlyR channels. This indicates that open GlyR and GABAR channels have effective pore diameters of 5.2 and 5.6 A, respectively. The sequence of relative permeabilities for small anions was SCN- greater than I- greater than Br- greater than Cl- greater than F- for both channels. 5. GlyR and GABAR channels are multi-conductance-state channels. In cell-attached patches the single-channel slope conductances close to 0 mV membrane potential were 29, 18 and 10 pS for glycine, and 28, 17 and 10 pS for GABA-activated channels. The most frequently observed (main) conductance states were 29 and 17 pS for the GlyR and GABAR channel, respectively. 6. In outside-out patches with equal extracellular and intracellular concentrations of 145 mM-Cl-, the conductance states were 46, 30, 20 and 12 pS for GlyR channels and 44, 30, 19 and 12 pS for GABAR channels. The most frequently occurring main state was 46 pS for the GlyR and 30 pS for the GABAR channel. 7. Single-channel conductances measured in equal 140 mM concentrations of small anions on both membrane faces revealed a conductance sequence of Cl- greater than Br- greater than I- greater than SCN- greater than F- for both channels. This is nearly the inverse sequence of that found for the permeability of these ions indicating the presence of binding sites for ions in the channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Gamma-Aminobutyric acid receptor channels in adrenal chromaffin cells: a patch-clamp study.

We have studied membrane channels activated by gamma-aminobutyric acid (GABA) in adrenal medullary chromaffin cells by using patch-clamp techniques. These channels share many properties with GABA-receptor channels in the central nervous system. They are chloride-selective, blocked by the GABA antagonist bicuculline, and reversibly desensitized at high GABA concentrations. The dose-response curve has a slope of 2 in the Hill plot, indicating a bimolecular binding reaction of GABA to the receptor. Single-channel currents display multiple conductance states as do glycine-activated chloride channels in mouse spinal neurons. Gating properties of GABA-activated channels, as described by a sequential model for agonist-activated channels, are similar to gating properties in central neurons. GABA-induced currents are potentiated by diazepam, indicating that anxiolytic drugs like the benzodiazepines might be involved in the regulation of anxiety states in the peripheral nervous system.

Activation of multiple-conductance state chloride channels in spinal neurones by glycine and GABA.

In the mammalian central nervous system, glycine and gamma-aminobutyric acid (GABA) bind to specific and distinct receptors and cause an increase in membrane conductance to CI- (refs 5-7). Neurones in various regions of the nervous system show differential sensitivity to glycine and GABA; thus GABA and glycine receptors are spatially distinct from one another. However, on the basis of desensitization experiments on spinal cord neurones, it was suggested that the receptors for glycine and GABA may share the same CI- channel. We now report that in small membrane patches, isolated from the soma of spinal neurones, both receptor channels display several (multiple) conductance states. Two of the states are common to both receptor channels. However, the most frequently observed 'main conductance states' of the GABA and glycine receptor channels are different. Both channels display the same anion selectivity. We propose that one class of multistate CI- channel is coupled to either GABA or glycine receptors. The main conductance state adopted by this channel is determined by the receptor to which it is coupled.