Influence of the M3-M4 intracellular domain upon nicotinic acetylcholine receptor assembly, targeting and function.
ABSTRACT: The aim of this study was to investigate the influence of the intracellular domain of nicotinic acetylcholine receptor (nAChR) subunits upon receptor assembly, targeting and functional properties.Because most nAChR subunits form functional receptors only as heteromeric complexes, it can be difficult to examine the influence of individual subunits or subunit domains in isolation. A series of subunit chimaeras was constructed which contain the intracellular loop region (located between the M3 and M4 transmembrane domains) from nAChR subunits alpha1-alpha10 or beta1-beta4. All of these chimaeras contain common extracellular and transmembrane domains (from the nAChR alpha7 subunit and the 5-hydroxytryptamine receptor 5-HT(3A) subunit, respectively), thereby facilitating both homomeric receptor assembly and detection with radiolabelled or fluorescent alpha-bungarotoxin.The nAChR M3-M4 intracellular loop domain had no significant effect upon levels of total subunit protein detected in transfected cells but had a significant influence upon levels of both cell surface and intracellular assembled receptors. Comparisons of functional properties revealed a significant influence of the intracellular loop domain upon both single-channel conductance and receptor desensitization. In addition, studies conducted in polarized epithelial cells demonstrate that the nAChR loop can influence receptor targeting, resulting in either polarized (apical) or non-polarized distribution.Evidence has been obtained which demonstrates that the large intracellular loop domain of nAChR subunits can exert a profound influence upon receptor assembly, targeting and ion channel properties.
Project description:Nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT(3)Rs) are members of the superfamily of neurotransmitter-gated ion channels. Both contain five subunits which assemble to form either homomeric or heteromeric subunit complexes. With the aim of identifying the influence of subunit domains upon receptor assembly and function, a series of chimaeras have been constructed containing regions of the neuronal nAChR alpha 7 subunit and the 5-HT(3) receptor (3A) subunit.A series of subunit chimaeras containing alpha 7 and 5-HT(3A) subunit domains have been constructed and expressed in cultured mammalian cells. Properties of the expressed receptors have been examined by means of radioligand binding, agonist-induced changes in intracellular calcium and patch-clamp electrophysiology.Subunit domains which influence properties such as rectification, desensitization and conductance have been identified. In addition, the influence of subunit domains upon subunit folding, receptor assembly and cell-surface expression has been identified. Co-expression studies with the nAChR-associated protein RIC-3 revealed that, in contrast to the potentiating effect of RIC-3 on alpha 7 nAChRs, RIC-3 caused reduced levels of cell-surface expression of some alpha 7/5-HT(3A) chimaeras.Evidence has been obtained which demonstrates that subunit transmembrane domains are critical for efficient subunit folding and assembly. In addition, functional characterization of subunit chimaeras revealed that both extracellular and cytoplasmic domains exert a dramatic and significant influence upon single-channel conductance. These data support a role for regions other than hydrophobic transmembrane domains in determining ion channel properties.
Project description:Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR alpha7 subunit without compromising formation of functional receptors.A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplasmic loop between the third and fourth transmembrane domains of the mouse nAChR alpha7 subunit (alpha7Y). SH-EP1 cells were transfected with mouse nAChR wild type alpha7 subunits (alpha7) or with alpha7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with (125)I-labeled alpha-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.Whole-cell currents revealed that alpha7Y nAChRs and alpha7 nAChRs were functional with comparable EC(50) values for the alpha7 nAChR-selective agonist, choline, and IC(50) values for the alpha7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that alpha7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little alpha7Y localized to the plasma membrane, typical of alpha7 nAChRs.nAChRs composed as homooligomers of alpha7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of alpha7 nAChRs. alpha7Y nAChRs may be used to elucidate properties of alpha7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.
Project description:Nicotinic acetylcholine receptors (nAChR) are members of the Cys-loop ligand-gated ion channel superfamily. Muscle nAChR are heteropentamers that assemble from two ?, and one each of ?, ?, and ? subunits. Each subunit is composed of three domains, extracellular, transmembrane and intracellular. The transmembrane domain consists of four ?-helical segments (M1-M4). Pioneering structural information was obtained using electronmicroscopy of Torpedo nAChR. The recently solved X-ray structure of the first eukaryotic Cys-loop receptor, a truncated (intracellular domain missing) glutamate-gated chloride channel ? (GluCl?) showed the same overall architecture. However, a significant difference with regard to the vertical alignment between the channel-lining segment M2 and segment M3 was observed. Here, we used functional studies utilizing disulfide trapping experiments in muscle nAChR to determine the spatial orientation between M2 and M3. Our results are in agreement with the vertical alignment as obtained when using the GluCl? structure as a template to homology model muscle nAChR, however, they cannot be reconciled with the current Torpedo nAChR model. The vertical M2-M3 alignments as observed in X-ray structures of prokaryotic Gloeobacter violaceus ligand-gated ion channel and GluCl? are in agreement. Our results further confirm that this alignment in Cys-loop receptors is conserved between prokaryotes and eukaryotes.
Project description:The nicotinic acetylcholine receptor (nAChR) ?3 subunit is thought to serve an accessory role in nAChR subtypes expressed in dopaminergic regions implicated in drug dependence and reward. When ?3 subunits are expressed in excess, they have a dominant-negative effect on function of selected nAChR subtypes. In this study, we show, in Xenopus oocytes expressing ?2, ?3 or ?4 plus either ?2 or ?4 subunits, that in the presumed presence of similar amounts of each nAChR subunit, co-expression with wild-type ?3 subunits generally (except for ?3*-nAChR) lowers amplitudes of agonist-evoked, inward peak currents by 20-50% without having dramatic effects (? 2-fold) on agonist potencies. By contrast, co-expression with mutant ?3(V9'S) subunits generally (except for ?4?2*-nAChR) increases agonist potencies, consistent with an expected gain-of-function effect. This most dramatically demonstrates formation of complexes containing three kinds of subunit. Moreover, for oocytes expressing nAChR containing any ? subunit plus ?4 and ?3(V9'S) subunits, there is spontaneous channel opening sensitive to blockade by the open channel blocker, atropine. Collectively, the results indicate that ?3 subunits integrate into all of the studied receptor assemblies and suggest that natural co-expression with ?3 subunits can influence levels of expression and agonist sensitivities of several nAChR subtypes.
Project description:A cytosine to thymidine (C ? T) missense mutation in the signal peptide (SP) sequence (rs2472553) of the nicotinic acetylcholine receptor (nAChR) ?2 subunit produces a threonine-to-isoleucine substitution (T22I) often associated with nicotine dependence (ND). We assessed effects on function of ?2*-nAChR ('*'indicates presence of additional subunits) of this mutation, which could alter SP cleavage, RNA/protein secondary structure, and/or efficiency of transcription, translation, subunit assembly, receptor trafficking or cell surface expression. Two-electrode voltage clamp analyses indicate peak current responses to ACh or nicotine are decreased 2.8-5.8-fold for putative low sensitivity (LS; 10:1 ratio of ?:? subunit cRNAs injected) ?2?2- or ?2?4-nAChR and increased for putative high sensitivity (HS; 1:10 ?:? subunit ratio) ?2?2- (5.7-15-fold) or ?2?4- (1.9-2.2-fold) nAChR as a result of the mutation. Agonist potencies are decreased 1.6-4-fold for putative LS or HS ?2(T22I)?2-nAChR or for either ?2*-nAChR subtype formed in the presence of equal amounts of subunit cRNA, slightly decreased for LS ?2(T22I)?4-nAChR, but increased 1.4-2.4-fold for HS ?2(T22I)?4-nAChR relative to receptors containing wild-type ?2 subunits. These effects suggest that the ?2 subunit SP mutation generally favors formation of LS receptor isoforms. We hypothesize that lower sensitivity of human ?2*-nAChR to nicotine could contribute to increased susceptibility to ND. To our knowledge this is the first report of a SP mutation having a functional effect in a member of cys-loop family of ligand-gated ion channels.
Project description:Functional heterologous expression of naturally expressed mouse ?6*-nicotinic acetylcholine receptors (m?6*-nAChRs; where "*" indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid m?6m?4h?3- (? 5-8-fold) or WT m?6m?4m?3-nAChRs (? 2-fold) yielded higher function than m?6m?4-nAChRs. Function was not detected when m?6 and m?2 subunits were expressed together or in the additional presence of h?3 or m?3 subunits. However, function emerged upon expression of m?6m?2m?3(V9'S)-nAChRs containing ?3 subunits having gain-of-function V9'S (valine to serine at the 9'-position) mutations in transmembrane domain II and was further elevated 9-fold when h?3(V9'S) subunits were substituted for m?3(V9'S) subunits. Studies involving WT or gain-of-function chimeric mouse/human ?3 subunits narrowed the search for domains that influence functional expression of m?6*-nAChRs. Using h?3 subunits as templates for site-directed mutagenesis studies, substitution with m?3 subunit residues in extracellular N-terminal domain loops "C" (Glu(221) and Phe(223)), "E" (Ser(144) and Ser(148)), and "?2-?3" (Gln(94) and Glu(101)) increased function of m?6m?2*- (? 2-3-fold) or m?6m?4* (? 2-4-fold)-nAChRs. EC50 values for nicotine acting at m?6m?4*-nAChR were unaffected by ?3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops ?2-?3 and E) interfaces of ?3 subunits are some of the molecular impediments for functional expression of m?6m?2?3- or m?6m?4?3-nAChRs.
Project description:The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human alpha4beta2 and rat alpha4beta4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [(125)I]epibatidine and 5[(125)I]A-85380. Our results show that both agonists photoincorporated into the beta4 subunit with little or no labeling of the beta2 and alpha4 subunits respectively. [(125)I]epibatidine labeling in the beta4 subunit was mapped to two overlapping proteolytic fragments that begin at beta4V102 and contain Loop E (beta4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that beta4Q117 in Loop E is the principal site of [(125)I]epibatidine labeling. This was accomplished by substituting residues in the beta2 subunit with the beta4 homologs and finding [(125)I]epibatidine labeling in beta4 and beta2F119Q subunits with little, if any, labeling in alpha4, beta2, or beta2S113R subunits. Finally, functional studies established that the beta2F119/beta4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.
Project description:We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9' or 13' positions (L9'S or V13'S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation. However, coexpression with β3 subunits potentiates rather than suppresses function of all-human, all-mouse, or hybrid α6((L9'S or V13'S))β4*- or α6(N143D+M145V)(L9'S)β2*-nAChR. This contrasts with the lack of consistent function when α6((L9'S or V13'S)) and β2 subunits are expressed alone or in the presence of wild-type β3 subunits. These results provide evidence that gain-of-function hα6hβ2*-nAChR (i.e. hα6(N143D+M145V)(L9'S)hβ2hβ3 nAChR) could be produced in vitro. These studies also indicate that nAChR β3 subunits can be assembly partners in functional α6*-nAChR and that 9' or 13' mutations in the nAChR α6 subunit second transmembrane domain can act as gain-of-function and/or reporter mutations. Moreover, our findings suggest that β3 subunit coexpression promotes function of α6*-nAChR.
Project description:Despite the apparent function of naturally expressed mammalian ?6*-nicotinic acetylcholine receptors (?6*-nAChR; where * indicates the known or possible presence of additional subunits), their functional and heterologous expression has been difficult. Here, we report that coexpression with wild-type ?3 subunits abolishes the small amount of function typically seen for all-human or all-mouse ?6?4*-nAChR expressed in Xenopus oocytes. However, levels of function and agonist potencies are markedly increased, and there is atropine-sensitive blockade of spontaneous channel opening upon coexpression of ?6 and ?4 subunits with mutant ?3 subunits harboring valine-to-serine mutations at 9'- or 13'-positions. There is no function when ?6 and ?2 subunits are expressed alone or in the presence of wild-type or mutant ?3 subunits. Interestingly, hybrid nAChR containing mouse ?6 and human (h) ?4 subunits have function potentiated rather than suppressed by coexpression with wild-type h?3 subunits and potentiated further upon coexpression with h?3(V9'S) subunits. Studies using nAChR chimeric mouse/human ?6 subunits indicated that residues involved in effects seen with hybrid nAChR are located in the ?6 subunit N-terminal domain. More specifically, nAChR h?6 subunit residues Asn-143 and Met-145 are important for dominant-negative effects of nAChR h?3 subunits on h?6h?4-nAChR function. Asn-143 and additional residues in the N-terminal domain of nAChR h?6 subunits are involved in the gain-of-function effects of nAChR h?3(V9'S) subunits on ?6?2*-nAChR function. These studies illuminate the structural bases for effects of ?3 subunits on ?6*-nAChR function and suggest that unique subunit interfaces involving the complementary rather than the primary face of ?6 subunits are involved.
Project description:Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal ?4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated ?4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type ?4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the ?4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the ?4?2 nAChR.