Nicotine-induced up-regulation and desensitization of alpha4beta2 neuronal nicotinic receptors depend on subunit ratio

Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different alpha4beta2 subunit ratios (1alpha:4beta, 2alpha:3beta, and 4alpha:1beta) expressed in Xenopus oocytes. The presence of alpha4 subunit in the oocyte plasmatic membrane increased linearly with the amount of alpha4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the alpha4 subunit. The 2alpha4:3beta2 subunit ratio displayed the highest ACh sensitivity. Nicotine dose-response curves for the 1alpha4:4beta2 and 2alpha4:3beta2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 microm. A biphasic curve for 4alpha4:1beta2 was obtained at nicotine concentrations higher than 300 microm. The 1alpha4:4beta2 subunit ratio exhibited the lowest ACh- and nicotine-induced macroscopic current, whereas 4alpha4:1beta2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of beta2:alpha4 subunits increased. All three alpha4beta2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2alpha4: 3beta2 subunit ratio. Our data suggest that the subunit ratio of alpha4beta2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate alpha4beta2 receptor activation and desensitization.     

Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A

Nicotinic acetylcholine receptors (nAChRs) containing alpha3 and beta2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related alpha6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog ofalpha-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric alpha6/alpha3beta2beta3 nAChRs with an IC(50) of 1.2 nm; in contrast, its IC(50) on the closely related alpha3beta2 as well as non-alpha6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the alpha3 subunit inserted into the homologous positions of the alpha6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the alpha6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the alpha3 subunit increased the IC(50) of the peptide by >1000-fold. Conversely, when these key residues inalpha3 were replaced with those fromalpha6, the IC(50) decreased by almost 150-fold. Similar effects were seen with other alpha6-selective conotoxins, suggesting the general importance of thesealpha6 residues in conferring selective binding.     

Hydroxylated residues influence desensitization behaviour of recombinant alpha3 glycine receptor channels

The human glycine receptor subunit alpha3 exists in two splice variants (alpha3K/L), with alpha3L bearing an additional segment of 15 amino acids within the cytoplasmic TM3-4 loop. Homomeric alpha3K glycine receptors show faster desensitization than alpha3L receptors. Ion channel properties were compared of alpha3L, alpha3K, and of the triple mutant alpha3LDeltaOH = alpha3L(T358A/Y367F/S370A), where hydroxyl functions of the spliced insert had been removed by site-directed mutagenesis. Upon recombinant expression in HEK 293 cells, patch-clamp recording experiments revealed that removal of hydroxyl functions primarily affected receptor desensitization. The fraction of non-desensitizing current was 68 +/- 13% for alpha3L, 21 +/- 13% for alpha3K, and 48 +/- 16% for alpha3LDeltaOH. Desensitization time constants at saturating glycine concentration were 8.4 +/- 2.8 s, 1.9 +/- 2.3 s, and 2.8 +/- 0.4 s, for alpha3L, alpha3K, and the triple mutant alpha3LDeltaOH, respectively. In contrast, single-channel and whole-cell properties were similar for all three constructs. Thus, ion channel activation, desensitization, and conductance properties are independently controlled by distinct structural elements. Hydroxyl functions within the M3-4 loop of the glycine receptor alpha3 subunit are crucial, but not exclusive, determinants of receptor desensitization.     

The calcium sensor protein visinin-like protein-1 modulates the surface expression and agonist sensitivity of the alpha 4beta 2 nicotinic acetylcholine receptor

The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast two-hybrid library using the large cytoplasmic domain of the alpha4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The alpha4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant alpha4beta2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native alpha4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant alpha4beta2 AChRs up-regulated their surface expression levels approximately 2-fold and increased their agonist sensitivity to acetylcholine approximately 3-fold. The modulation of the recombinant alpha4beta2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of alpha4beta2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.     

Incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors

Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking alpha (alpha2-alpha4, alpha6) and beta (beta2, beta4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (beta4_alpha2, beta4_alpha3, beta4_alpha4, and beta2_alpha4) when coexpressed with the corresponding beta monomer subunit. All tandems expressed when injected alone, except for beta4_alpha3, which produced functional channels only together with beta4 monomer and was chosen for further characterization. These channels produced from beta4_alpha3 tandem constructs plus beta4 monomer were identical with receptors expressed from monomer alpha3 and beta4 constructs in acetylcholine sensitivity and in the number of alpha and beta subunits incorporated in the channel gate. However, separately mutating the beta subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of beta subunits from the tandem and one from the beta monomer construct, whereas the rest incorporated two or three beta monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily.     

Voltage-jump relaxation kinetics for wild-type and chimeric beta subunits of neuronal nicotinic receptors

We have studied the voltage-jump relaxation currents for a series of neuronal nicotinic acetylcholine receptors resulting from the coexpression of wild-type and chimeric beta 4/beta 2 subunits with alpha 3 subunits in Xenopus oocytes. With acetylcholine as the agonist, the wild-type alpha 3 beta 4 receptors displayed five- to eightfold slower voltage-jump relaxations than did the wild-type alpha 3 beta 2 receptors. In both cases, the relaxations could best be described by two exponential components of approximately equal amplitudes over a wide range of [ACh]'s. Relaxation rate constants increased with [ACh] and saturated at 20- to 30-fold lower concentrations for the alpha 3 beta 2 receptor than for the alpha 3 beta 4 receptor, as observed previously for the peak steady state conductance. Furthermore, the chimeric beta 4/beta 2 subunits showed a transition in the concentration dependence of the rate constants in the region between residues 94 and 109, analogous to our previous observation with steady state conductances. However, our experiments with a series of beta- subunit chimeras did not localize residues that govern the absolute value of the kinetic parameters. Hill coefficients for the relaxations also differed from those previously measured for steady state responses. The data reinforce previous conclusions that the region between residues 94 and 109 on the beta subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.     

An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine

In smoker's brain, rodent brain, and in cultured cells expressing nicotinic receptors, chronic nicotine treatment induces an increase in the total number of high affinity receptors for acetylcholine and nicotine, a process referred to as up-regulation. Up-regulation induced by 1 mm nicotine reaches 6-fold for alpha3beta2 nicotinic receptors transiently expressed in HEK 293 cells, whereas it is much smaller for alpha3beta4 receptors, offering a rationale to investigate the molecular mechanism underlying up-regulation. In this expression system binding sites are mainly intracellular, as shown by [(3)H]epibatidine binding experiments and competition with the impermeant ligand carbamylcholine. Systematic analysis of beta2/beta4 chimeras demonstrates the following. (i) The extracellular domain critically contributes to up-regulation. (ii) Only residues belonging to two beta2 segments, 74-89 and 106-115, confer up-regulation to beta4, mainly by decreasing the amount of binding sites in the absence of nicotine; on an atomic three-dimensional model of the alpha3beta2 receptor these amino acids form a compact microdomain that mainly contributes to the subunit interface and also faces the acetylcholine binding site. (iii) The beta4 microdomain is sufficient to confer to beta2 a beta4-like up-regulation. (iv) This microdomain makes an equivalent contribution to the up-regulation differences between alpha4beta2 and alpha4beta4. We propose that nicotine, by binding to immature oligomers, elicits a conformational reorganization of the microdomain, strengthening the interaction between adjacent subunits and, thus, facilitating maturation processes toward high affinity receptors. This mechanism may be central to nicotine addiction, since alpha4beta2 is the subtype exhibiting the highest degree of up-regulation in the brain.     

Long-Term Opiate Exposure Leads to Reduction of the αi-1 Sufyunit of GTP-Binding Proteins

Desensitization or tolerance is a major consequence of long-term opiate exposure. The mechanism of opiate desensitization is only poorly understood. We report that exposure of rat spinal cord-dorsal root ganglion cocultured neurons to kappa-opiate agonist is accompanied by a 60-70% reduction in the level of the alpha i subunit of GTP-binding proteins. Using selective antibodies, which discriminate among the various alpha i subunit forms, it was found that the opiate treatment leads to a reduction in the amount of the alpha i-1 subunit. The levels of alpha s, alpha o, and beta subunits remain unchanged. This molecular event could underlie the development of tolerance and cross-tolerance to opiates.     

Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human alpha 4 beta 2 nicotinic acetylcholine receptors in Xenopus oocytes

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.     

The beta subunit determines the ligand binding properties of synaptic glycine receptors

Inhibitory glycine receptors (GlyRs) regulate motor coordination and sensory signal processing in spinal cord and other brain regions. GlyRs are pentameric proteins composed of membrane-spanning alpha and beta subunits. Here, site-directed mutagenesis combined with homology modeling based on the crystal structure of the acetylcholine binding protein identified key ligand binding residues of recombinant homooligomeric alpha1 and heterooligomeric alpha1beta GlyRs. This disclosed two highly conserved, oppositely charged residues located on adjacent subunit interfaces as being crucial for agonist binding. In addition, the beta subunit was found to determine the ligand binding properties of heterooligomeric GlyRs. Expression of an alpha1beta tandem construct and affinity purification of metabolically labeled GlyRs confirmed a subunit stoichiometry of 2alpha3beta. Because the beta subunit anchors GlyRs at synaptic sites, our results have important implications for the biosynthesis, clustering, and pharmacology of synaptic GlyRs.     

Role of negatively charged amino acids in beta 4 F-loop in activation and desensitization of alpha 3 beta 4 rat neuronal nicotinic receptors

The role of negatively charged amino acids in the F-loop of the beta 4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the alpha 3 beta 4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at -40 mV produced inward currents which displayed biphasic desensitization. The EC50 of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC50 increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.     

Common determinants of single channel conductance within the large cytoplasmic loop of 5-hydroxytryptamine type 3 and alpha4beta2 nicotinic acetylcholine receptors

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (gamma) below the resolution of single channel recording (966 +/- 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range gamma values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4'), 436 (0'), and 440 (4') residues by 5-HT3B (-4'Gln, 0'Asp, and 4'Ala) residues increases gamma to 36.5 +/- 1.0 pS. The 0' residue makes the most substantial contribution to gamma of the 5-HT3AR. Replacement of 0'Arg by aspartate, glutamate (alpha7 nAChR subunit MA 0'), or glutamine (beta2 subunit MA 0') increases gamma to the resolvable range (>6 pS). By contrast, replacement of 0'Arg by phenylalanine (alpha4 subunit MA 0') reduced gamma to 416 +/- 107 fS. In reciprocal experiments with alpha4beta2 nAChRs (gamma = 31.3 +/- 0.8 pS), replacement of MA 0' residues by arginine in alpha4beta2(Q443R) and alpha4(F588R)beta2 reduced gamma slightly. By contrast, the gamma of double mutant alpha4(F588R)beta2(Q443R) was halved. The MA -4' and 4' residues also influenced gamma of 5-HT3ARs. Replacement of nAChR alpha4 or beta2 MA 4' residues by arginine made current density negligible. By contrast, replacement of both -4' residues by arginine produced functional nAChRs with substantially reduced gamma (11.4 +/- 0.5 pS). Homology models of the 5-HT3A and alpha4beta2 nAChRs against Torpedo nAChR revealed MA -4', 0', and 4' residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.     

Primary structure and expression of beta 2: a novel subunit of neuronal nicotinic acetylcholine receptors

A new subunit, beta 2, of the neuronal nicotinic receptor family has been identified. This subunit has the structural features of a non-agonist-binding subunit. We provide evidence that beta 2 can substitute for the muscle beta 1 subunit to form a functional nicotinic receptor in Xenopus oocytes. Expression studies performed in oocytes have demonstrated that three different neuronal nicotinic acetylcholine receptors can be formed by the pairwise injection of beta 2 mRNA and each of the neuronal alpha subunit mRNAs. The beta 2 gene is expressed in PC12 cells and in areas of the central nervous system where the alpha 2, alpha 3, and alpha 4 genes are expressed. These results lead us to propose that the nervous system expresses diverse forms of neuronal nicotinic acetylcholine receptors by combining beta 2 subunits with different agonist-binding alpha subunits.     

Point mutation in the first transmembrane region of the beta 2 subunit of the gamma--aminobutyric acid type A receptor alters desensitization kinetics of gamma--aminobutyric acid- and anesthetic-induced channel gating

A conserved glycine residue in the first transmembrane (TM1) domain of the beta2 subunit has been identified to be involved with desensitization induced by gamma-aminobutyric acid (GABA) and anesthetics. Recombinant GABA(A) receptors expressed in Sf9 cells were recorded using semi-fast agonist application. Upon direct activation by GABA or anesthetics, the main effect of the TM1 point mutation on the beta2 subunit (G219F) was to slow the time constant (tau) of desensitization. At GABA concentrations eliciting maximum currents, the corresponding median tau values were 0.87 s (25-75% interval (0.76; 1.04 s)), 0.93 s (0.76; 1.23 s), and 1.36 s (1.17; 1.57 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than alpha1beta2gamma2 (p < 0.01) and alpha1(G223F)beta2gamma2 (p < 0.05). For pentobarbital-induced currents (500 microm), the corresponding median tau values were 1.36 s (0.81; 1.41 s), 1.47 s (1.31; 2.38 s), and 2.82 s (2.21; 5.56 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than that for alpha1beta2gamma2 (p < 0.01). The present findings suggest that this TM1 glycine residue is critical for the rate at which desensitization occurs and that both GABA and intravenous anesthetics implement an analogous pathway for generating desensitization.     

Functional consequences of the loss of high affinity agonist binding to gamma-aminobutyric acid type A receptors. Implications for receptor desensitization

We reported previously that tyrosine 62 of the beta2 subunit of the gamma-aminobutyric acid, type A (GABA(A)) receptor is an important determinant of high affinity agonist binding and that recombinant alpha1beta2gamma2(L) receptors carrying the Y62S mutation lack measurable high affinity sites for [3H]muscimol. We have now examined the effects of disrupting these sites on the macroscopic desensitization properties of receptors expressed in Xenopus oocytes. Desensitization was measured by the ability of low concentrations of bath-perfused agonist to reduce the current responses elicited by subsequent challenges with saturating concentrations of GABA. Wild-type receptors were desensitized by pre-perfused muscimol with an IC50 approximately 0.7 microm, which correlates well with the lower affinity sites for this agonist that are measured in direct binding studies. Receptors carrying the beta2 Y62S and Y62F mutations desensitized at slightly higher (2-7-fold) agonist concentrations. However, at low perfusate concentrations, the Y62S-containing receptor recovered from the desensitized state even in the continued presence of agonist. The characteristics of desensitization in the wild-type and mutant receptors lead us to suggest that the major role of the high affinity agonist-binding site(s) of the GABA(A) receptor is not to induce desensitization but rather to stabilize the desensitized state once it has been formed.     

Extracellular domain nicotinic acetylcholine receptors formed by alpha4 and beta2 subunits

Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of alpha7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of alpha4 and beta2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with alpha4beta2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha4beta2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha4beta2 nAChRs when measured with [3H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of alpha4beta2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain alpha4 and beta2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha4 and beta2 play an important role for efficient expression of extracellular domain alpha4beta2 nAChRs that are high fidelity structural models of full-length alpha4beta2 nAChRs.     

Desensitization of the nicotinic acetylcholine receptor: Molecular mechanisms and effect of modulators

1. Loss of response after prolonged or repeated application of stimulus is generally termed desensitization. A wide variety of phenomena occurring in living organisms falls under this general definition of desensitization. There are two main types of desensitization processes: specific and non-specific. 2. Desensitization of the nicotinic acetylcholine receptor is triggered by prolonged or repeated exposure to agonists and results in inactivation of its ion channel. It is a case of specific desensitization and is an intrinsic molecular property of the receptor. 3. Desensitization of the nicotinic acetylcholine receptor at the neuromuscular junction was first reported by Katz and Thesleff in 1957. Desensitization of the receptor has been demonstrated by rapid kinetic techniques and also by the characteristic "burst kinetics" obtained from single-channel recordings of receptor activity in native as well as in reconstituted membranes. In spite of a number of studies, the detailed molecular mechanism of the nicotinic acetylcholine receptor desensitization is not known with certainty. The progress of desensitization is accompanied by an increase in affinity of the receptor for its agonist. This change in affinity is attributed to a conformational change of the receptor, as detected by spectroscopic and kinetic studies. A four-state general model is consistent with the major experimental observations. 4. Desensitization of the nicotinic acetylcholine receptor can be potentially modulated by exogenous and endogenous substances and by covalent modifications of the receptor structure. Modulators include the noncompetitive blockers, calcium, the thymic hormone peptides (thymopoietin and thymopentin), substance P, the calcitonin gene-related peptide, and receptor phosphorylation. Phosphorylation is an important posttranslational covalent modification that is correlated with the regulation and desensitization of the receptor through various protein kinases. 5. Although the physiological significance of desensitization of the nicotinic receptor is not yet fully understood, desensitization of receptors probably plays a significant role in the operation of the neuronal networks associated in memory and learning processes. Desensitization of the nicotinic receptor could also possibly be related to the neuromuscular disease, myasthenia gravis.     

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed in combination with beta2 or beta2gamma2. Surprisingly, the (alpha1/gamma2)(gamma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitization. Moreover, desensitization was observed for the (alpha1/gamma2)beta2gamma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.     

Unique assignment of inter-subunit association in GABA(A) alpha 1 beta 3 gamma 2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA(A) receptor (GABA(A)R alpha 1 beta 3 gamma 2). There is strong evidence that the heteropentameric receptor contains two alpha 1, two beta 3, and one gamma 2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either gamma 2 beta 3 alpha 1 beta 3 alpha 1 or gamma 2 alpha 1 beta 3 alpha 1 beta 3 configurations. Here we use molecular modeling to thread the relevant GABA(A)R subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA(A) sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA(A) sequences were threaded onto the AChBP template in the gamma 2 beta 3 alpha 1 beta 3 alpha 1 or gamma 2 alpha 1 beta 3 alpha 1 beta 3 arrangements. Only the gamma 2 alpha 1 beta 3 alpha 1 beta 3 arrangement satisfied three known criteria: (1) alpha 1 His(102) binds at the gamma 2 subunit interface in proximity to gamma 2 residues Thr(142), Phe(77), and Met(130); (2) alpha 1 residues 80-100 bind near gamma 2 residues 91-104; and (3) alpha 1 residues 58-67 bind near the beta 3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.