Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating

The nicotinic receptor (AChR) is a pentamer of homologous subunits with an alpha(2)betaepsilondelta composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15' of the M1 domain is phenylalanine in alpha subunits while it is isoleucine in non-alpha subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant alpha subunit (alphaF15'I) as well as those containing the reverse mutations in the non-alpha subunits (betaI15'F, deltaI15'F, and epsilonI15'F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the beta subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of betaI15'F of AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant in the diliganded receptor, and a significant increase opening in the absence of agonist. Mutations at betaI15' showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an approximately 70% closed-state-like environment for the beta15' position at the transition state of gating. The overall results identify position 15' as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.     

Thermal denaturation of the Na,K-ATPase provides evidence for alpha-alpha oligomeric interaction and gamma subunit association with the C-terminal domain

Thermal denaturation can help elucidate protein domain substructure. We previously showed that the Na,K-ATPase partially unfolded when heated to 55 degrees C (Arystarkhova, E., Gibbons, D. L., and Sweadner, K. J. (1995) J. Biol. Chem. 270, 8785-8796). The beta subunit unfolded without leaving the membrane, but three transmembrane spans (M8-M10) and the C terminus of the alpha subunit were extruded, while the rest of alpha retained its normal topology with respect to the lipid bilayer. Here we investigated thermal denaturation further, with several salient results. First, trypsin sensitivity at both surfaces of alpha was increased, but not sensitivity to V8 protease, suggesting that the cytoplasmic domains and extruded domain were less tightly packed but still retained secondary structure. Second, thermal denaturation was accompanied by SDS-resistant aggregation of alpha subunits as dimers, trimers, and tetramers without beta or gamma subunits. This implies specific alpha-alpha contact. Third, the gamma subunit, like the C-terminal spans of alpha, was selectively lost from the membrane. This suggests its association with M8-M10 rather than the more firmly anchored transmembrane spans. The picture that emerges is of a Na,K-ATPase complex of alpha, beta, and gamma subunits in which alpha can associate in assemblies as large as tetramers via its cytoplasmic domain, while beta and gamma subunits associate with alpha primarily in its C-terminal portion, which has a unique structure and thermal instability.     

Membrane tethering enables an extracellular domain of the acetylcholine receptor alpha subunit to form a heterodimeric ligand-binding site

The first step of assembly of the nicotinic acetylcholine receptor (AChR) of adult skeletal muscle is the specific association of the alpha subunit with either delta or epsilon subunits to form a heterodimer with a ligand-binding site. Previous experiments have suggested that het erodimer formation in the ER arises from interaction between the luminal, NH2-terminal domains of the subunits. When expressed in COS cells with the delta subunit, however, the truncated NH2-terminal domain of the subunit folded correctly but did not form a heterodimer. Association with the delta subunit occurred only when the NH2-terminal domain was retained in the ER and was tethered to the membrane by its own M1 transmembrane domain, by the transmembrane domain of another protein, or by a glycolipid link. In each case, the ligand-binding sites of the resulting heterodimers were indistinguishable from that formed when the full-length alpha subunit was used. Attachment to the membrane may promote interaction by concentrating or orienting the subunit; alternatively, a membrane-bound factor may facilitate subunit association.     

The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly.

Ligand-gated ion channels are oligomeric membrane proteins in which homologous subunits specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we used a dominant-negative assay in which truncated subunits of the mouse muscle acetylcholine receptor (AChR) were coexpressed with the four wild-type subunits in transfected COS cells. Fragments of the alpha, delta, and gamma subunits consisting solely of the extracellular N-terminal domain blocked surface expression of the AChR and the formation of alpha delta heterodimers, an early step in the assembly pathway of the AChR. Immunoprecipitation and sucrose gradient sedimentation experiments showed that an N-terminal fragment of the alpha subunit forms a specific complex with the intact delta subunit. Thus the extracellular N-terminal domain of the alpha, delta, and gamma subunits contains the information necessary for specific subunit association.     

Rapsyn-mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high-density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the alpha subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn-induced clustering of the AChR beta, gamma, and delta subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into alphadelta dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild-type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn-mediated clustering of an assembly intermediate, the alphadelta dimer.     

Epitope mapping of antibodies to acetylcholine receptor alpha subunits using peptides synthesized on polypropylene pegs.

Concurrent synthesis of overlapping octameric peptides corresponding to the sequence of the Torpedo acetylcholine receptor (AChR) alpha subunit has been carried out on polypropylene supports functionalized with primary amino groups according to a method developed by M. Geysen [(1987) J. Immunol. Methods 102, 259-274]. The peptides on the solid supports have been used in an enzyme-linked immunosorbent assay. Interactions of the synthetic peptides with antibodies are then detected without removing them from the solid support. By this procedure, epitopes of both antisera and monoclonal antibodies to the Torpedo acetylcholine receptor, its subunits, and synthetic peptide fragments have been mapped. Both rat and rabbit antisera to the alpha subunit show major epitopes spanning the residues 150-165, 338-345, and 355-366 on the Torpedo AChR alpha subunit. Epitopes of monoclonal antibodies to these major epitopes and to others have been rather precisely mapped by using this technique with peptides of varying lengths. The specificity of several of these mAbs are of interest because they have been used in mapping the transmembrane orientation of the AChR alpha-subunit polypeptide chain.     

The beta subunit determines the ion selectivity of the GABAA receptor

The gamma-aminobutyric acid, type A (GABA(A)) receptor is a chloride-conducting receptor composed of alpha, beta, and gamma subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1-M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1-M2 loop of the beta(3) subunit to the corresponding amino acids of the alpha(7) nicotinic acetylcholine subunit rendered the GABA(A) receptor cation-selective upon co-expression with wild type alpha(2) and gamma(2) subunits. Similar mutations in the alpha(2) or gamma(2) subunits did not lead to such a change in ion selectivity. This suggests a unique role for the beta(3) subunit in determining the ion selectivity of the GABA(A) receptor. The pharmacology of the mutated GABA(A) receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn(2+) and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC(50) = 1.3 microm) compared with the wild type receptor (EC(50) = 6.4 microm) and a loss of desensitization to GABA of the mutant receptor.     

Membrane topology of the Alg14 endoplasmic reticulum UDP-GlcNAc transferase subunit

N-linked glycosylation begins in the endoplasmic reticulum with the synthesis of a highly conserved dolichol-linked oligosaccharide precursor. The UDP-GlcNAc glycosyltransferase catalyzing the second sugar addition of this precursor consists in most eukaryotes of at least two subunits, Alg14 and Alg13. Alg14 is a membrane protein that recruits the soluble Alg13 catalytic subunit from the cytosol to the face of the endoplasmic reticulum (ER) membrane where this reaction occurs. Here, we investigated the membrane topology of Saccharomyces cerevisiae Alg14 and its requirements for ER membrane association. Alg14 is predicted by most algorithms to contain one or more transmembrane spanning helices (transmembrane domains (TMDs)). We provide evidence that Alg14 contains a C-terminal cytosolic tail and an N terminus that resides within the ER lumen. However, we also demonstrate that Alg14 lacking this TMD is functional and remains peripherally associated with ER membranes, suggesting that additional domains can mediate ER association. These conclusions are based on the functional analysis of Alg13/Alg14 chimeras containing Alg13 fused at either end of Alg14 or truncated Alg14 variants lacking the predicted TMD; protease protection assays of Alg14 in intact ER membranes; and extraction of Alg14-containing ER membranes with high pH. These yeast Alg13-Alg14 chimeras recapitulate the phylogenetic diversity of Alg13-Alg14 domain arrangements that evolved in some protozoa. They encode single polypeptides containing an Alg13 domain fused to Alg14 domain in either orientation, including those lacking the Alg14 TMD. Thus, this Alg13-Alg14 UDP-GlcNAc transferase represents an unprecedented example of a bipartite glycosyltransferase that evolved by both fission and fusion.     

Subunit-selective contribution to channel gating of the M4 domain of the nicotinic receptor

The muscle nicotinic receptor (AChR) is a pentamer of four different subunits, each of which contains four transmembrane domains (M1-M4). We recently showed that channel opening and closing rates of the AChR depend on a hydrogen bond involving a threonine at position 14' of the M4 domain in the alpha-subunit. To determine whether residues in equivalent positions in non-alpha-subunits contribute to channel gating, we mutated deltaT14', betaT14', and epsilonS14' and evaluated changes in the kinetics of acetylcholine-activated currents. The mutation epsilonS14'A profoundly slows the rate of channel closing, an effect opposite to that produced by mutation of alphaT14'. Unlike mutations of alphaT14', epsilonS14'A does not affect the rate of channel opening. Mutations in deltaT14' and betaT14' do not affect channel opening or closing kinetics, showing that conserved residues are not functionally equivalent in all subunits. Whereas alphaT14'A and epsilonS14'A subunits contribute additively to the closing rate, they contribute nonadditively to the opening rate. Substitution of residues preserving the hydrogen bonding ability at position 14' produce nearly normal gating kinetics. Thus, we identify subunit-specific contributions to channel gating of equivalent residues in M4 and elucidate the underlying mechanistic and structural bases.     

Local constraints in either the GluN1 or GluN2 subunit equally impair NMDA receptor pore opening

The defining functional feature of N-methyl-d-aspartate (NMDA) receptors is activation gating, the energetic coupling of ligand binding into opening of the associated ion channel pore. NMDA receptors are obligate heterotetramers typically composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits that gate in a concerted fashion, requiring all four ligands to bind for subsequent opening of the channel pore. In an individual subunit, the extracellular ligand-binding domain, composed of discontinuous polypeptide segments S1 and S2, and the transmembrane channel-forming domain, composed of M1-M4 segments, are connected by three linkers: S1-M1, M3-S2, and S2-M4. To study subunit-specific events during pore opening in NMDA receptors, we impaired activation gating via intrasubunit disulfide bonds connecting the M3-S2 and S2-M4 in either the GluN1 or GluN2A subunit, thereby interfering with the movement of the M3 segment, the major pore-lining and channel-gating element. NMDA receptors with gating impairments in either the GluN1 or GluN2A subunit were dramatically resistant to channel opening, but when they did open, they showed only a single-conductance level indistinguishable from wild type. Importantly, the late gating steps comprising pore opening to its main long-duration open state were equivalently affected regardless of which subunit was constrained. Thus, the NMDA receptor ion channel undergoes a pore-opening mechanism in which the intrasubunit conformational dynamics at the level of the ligand-binding/transmembrane domain (TMD) linkers are tightly coupled across the four subunits. Our results further indicate that conformational freedom of the linkers between the ligand-binding and TMDs is critical to the activation gating process.     

Dislocation of membrane proteins in FtsH-mediated proteolysis.

Escherichia coli FtsH degrades several integral membrane proteins, including YccA, having seven transmembrane segments, a cytosolic N-terminus and a periplasmic C-terminus. Evidence indicates that FtsH initiates proteolysis at the N-terminal cytosolic domain. SecY, having 10 transmembrane segments, is also a substrate of FtsH. We studied whether and how the FtsH-catalyzed proteolysis on the cytosolic side continues into the transmembrane and periplasmic regions using chimeric proteins, YccA-(P3)-PhoA-His6-Myc and SecY-(P5)-PhoA, with the alkaline phosphatase (PhoA) mature sequence in a periplasmic domain. The PhoA domain that was present within the fusion protein was rapidly degraded by FtsH when it lacked the DsbA-dependent folding. In contrast, both PhoA itself and the TM9-PhoA region of SecY-(P5)-PhoA were stable when expressed as independent polypeptides. In the presence of DsbA, the FtsH-dependent degradation stopped at a site near to the N-terminus of the PhoA moiety, leaving the PhoA domain (and its C-terminal region) undigested. The efficiency of this degradation stop correlated well with the rapidity of the folding of the PhoA domain. Thus, both transmembrane and periplasmic domains are degraded by the processive proteolysis by FtsH, provided they are not tightly folded. We propose that FtsH dislocates the extracytoplasmic domain of a substrate, probably using its ATPase activity.     

Subunit folding and alpha delta heterodimer formation in the assembly of the nicotinic acetylcholine receptor. Comparison of the mouse and human alpha subunits.

We have used the mouse alpha (alpha M) and human alpha (alpha H) subunits to investigate the molecular mechanisms of assembly of the mammalian acetylcholine receptor (AChR) transiently expressed in COS cells. COS cells expressing hybrid receptors incorporating alpha H along with other mouse subunits exhibited a 2-fold higher level of surface alpha-bungarotoxin (BuTx) binding than cells expressing the wild-type mouse AChR. When expressed either alone or with the delta subunit in COS cells, alpha H acquired the BuTx binding conformation (alpha Tx) more efficiently than did alpha M. By oligonucleotide-directed mutagenesis we showed that 2 residues in the amino-terminal domain were responsible for the differences between alpha M and alpha H. Alpha MST, the modified mouse alpha subunit, both folded more efficiently to form alpha Tx and was more effective in forming a stable alpha delta heterodimer than was alpha M. The kinetics of alpha Tx and alpha delta heterodimer formation revealed that the delta subunit increased the conversion of immature forms of the alpha subunit into the BuTx binding form and therefore provides evidence for interaction between the delta subunit and the immature form of the alpha subunit. These results provide evidence of the importance of the amino-terminal domains of the AChR subunits in the assembly process.     

Structural dynamics of the M4 transmembrane segment during acetylcholine receptor gating

The transition state structures that link the stable end states of allosteric proteins are largely unresolved. We used single-molecule kinetic analysis to probe the dynamics of the M4 transmembrane segments during the closed<==>open isomerization of the neuromuscular acetylcholine receptor ion channel (AChR). We measured the slopes (phi) of the free energy relationships for 87 mutants, which reveal the open- versus closed-like characters of the mutated residues at the transition state and hence the sequence and organization of gating molecular motions. phi was constant throughout the length of the alpha subunit M4 segment with an average value of 0.54, suggesting that this domain moves as a unit, approximately midway through the reaction. Analysis of a hybrid construct indicates that the two alpha subunits move synchronously. Between subunits, the sequence of M4 motions is alpha-epsilon-beta. The AChR ion channel emerges as a dynamic nanomachine with many moving parts.     

Cholesterol interacts with transmembrane alpha-helices M1, M3, and M4 of the Torpedo nicotinic acetylcholine receptor: photolabeling studies using [3H]Azicholesterol

The photoactivatable sterol probe [3alpha-(3)H]6-Azi-5alpha-cholestan-3beta-ol ([3H]Azicholesterol) was used to identify domains in the Torpedo californica nicotinic acetylcholine receptor (nAChR) that interact with cholesterol. [3H]Azicholesterol partitioned into nAChR-enriched membranes very efficiently (>98%), photoincorporated into nAChR subunits on an equal molar basis, and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine, consistent with photoincorporation at the nAChR lipid-protein interface. Sites of [3H]Azicholesterol incorporation in each nAChR subunit were initially mapped by Staphylococcus aureus V8 protease digestion to two relatively large homologous fragments that contain either the transmembrane segments M1-M2-M3 (e.g., alphaV8-20) or M4 (e.g., alphaV8-10). The distribution of [3H]Azicholesterol labeling between these two fragments (e.g., alphaV8-20, 29%; alphaV8-10, 71%), suggests that the M4 segment has the greatest interaction with membrane cholesterol. Photolabeled amino acid residues in each M4 segment were identified by Edman degradation of isolated tryptic fragments and generally correspond to acidic residues located at either end of each transmembrane helix (e.g., alphaAsp-407). [3H]Azicholesterol labeling was also mapped to peptides that contain either the M3 or M1 segment of each nAChR subunit. These results establish that cholesterol likely interacts with the M4, M3, and M1 segments of each subunit, and therefore, the cholesterol binding domain fully overlaps the lipid-protein interface of the nAChR.     

Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles

In our preceding paper [Ratnam, M., Sargent, P. B., Sarin, V., Fox, J. L., Le Nguyen, D., Rivier, J., Criado, M., & Lindstrom, J. (1986) Biochemistry (preceding paper in this issue)], we presented results from peptide mapping studies of purified subunits of the Torpedo acetylcholine receptor which suggested that the sequence beta 429-441 is on the cytoplasmic surface of the receptor. Since this finding contradicts earlier theoretical models of the transmembrane structure of the receptor, which placed this sequence of the beta subunit on the extracellular surface, we investigated the location of the corresponding sequence (389-408) and adjacent sequences of the alpha subunit by a more direct approach. We synthesized peptides including the sequences alpha 330-346, alpha 349-364, alpha 360-378, alpha 379-385, and alpha 389-408 and shorter parts of these peptides. These peptides corresponded to a highly immunogenic region, and by using 125I-labeled peptides as antigens, we were able to detect in our library of monoclonal antibodies to alpha subunits between two and six which bound specifically to each of these peptides, except alpha 389-408. We obtained antibodies specific for alpha 389-408 both from antisera against the denatured alpha subunit and from antisera made against the peptide. These antibodies were specific to alpha 389-396. In binding assays, antibodies specific for all of these five peptides bound to receptor-rich membrane vesicles only after permeabilization of the vesicles to permit access of the antibodies to the cytoplasmic surface of the receptors, suggesting that the receptor sequences which bound these antibodies were located on the intracellular side of the membrane. Electron microscopy using colloidal gold to visualize the bound antibodies was used to conclusively demonstrate that all of these sequences are exposed on the cytoplasmic surface of the receptor. These results, along with our previous demonstration that the C-terminal 10 amino acids of each subunit are exposed on the cytoplasmic surface, show that the hydrophobic domain M4 (alpha 409-426), previously predicted from hydropathy profiles to be transmembranous, does not, in fact, cross the membrane. Further, these results show that the putative amphipathic transmembrane domain M5 (alpha 364-399) also does not cross the membrane. Our results thus indicate that the transmembrane topology of a membrane protein cannot be deduced strictly from the hydropathy profile of its primary amino acid sequence. We present a model for the transmembrane orientation of receptor subunit polypeptide chains which is consistent with current data.     

The role of the carboxyl terminus in ClC chloride channel function

The human muscle chloride channel ClC-1 has a 398-amino acid carboxyl-terminal domain that resides in the cytoplasm and contains two CBS (cystathionine-beta-synthase) domains. To examine the role of this region, we studied various carboxyl-terminal truncations by heterologous expression in mammalian cells, whole-cell patch clamp recording, and confocal imaging. Channel constructs lacking parts of the distal CBS domain, CBS2, did not produce functional channels, whereas deletion of CBS1 was tolerated. ClC channels are dimeric proteins with two ion conduction pathways (protopores). In heterodimeric channels consisting of one wild type subunit and one subunit in which the carboxyl terminus was completely deleted, only the wild type protopore was functional, indicating that the carboxyl terminus supports the function of the protopore. All carboxyl-terminal-truncated mutant channels fused to yellow fluorescent protein were translated and the majority inserted into the plasma membrane as revealed by confocal microscopy. Fusion proteins of cyan fluorescent protein linked to various fragments of the carboxyl terminus formed soluble proteins that could be redistributed to the surface membrane through binding to certain truncated channel subunits. Stable binding only occurs between carboxyl-terminal fragments of a single subunit, not between carboxyl termini of different subunits and not between carboxyl-terminal and transmembrane domains. However, an interaction with transmembrane domains can modify the binding properties of particular carboxyl-terminal proteins. Our results demonstrate that the carboxyl terminus of ClC-1 is not necessary for intracellular trafficking but is critical for channel function. Carboxyl termini fold independently and modify individual protopores of the double-barreled channel.     

Phosphorylation of the amino-terminal head domain of the middle molecular mass 145-kDa subunit of neurofilaments. Evidence for regulation by second messenger-dependent protein kinases

To begin to understand the regulation and roles of neurofilament phosphorylation, we localized the phosphorylated domains on the 140-145-kDa neurofilament subunit (NF-M) and identified the protein kinases that may specifically phosphorylate the sites within these domains in vivo. Mouse retinal ganglion cells were labeled in vivo by injecting mice intravitreally with [32P]orthophosphate, and neurofilament-enriched fractions were obtained from the optic axons. Two-dimensional phosphopeptide map analysis of NF-M after digestion with alpha-chymotrypsin and trypsin revealed seven major (M8-M14) and at least eight minor (M1-M7 and M15) phosphopeptides. Two-dimensional phosphopeptide map analyses of NF-M phosphorylated in vitro by individual purified or endogenous axonal cytoskeleton-associated protein kinases showed that five peptides (M9-M13) were substrates for the heparin-sensitive second messenger-independent protein kinase(s). Protein kinase A and/or protein kinase C phosphorylated eight other peptides (M1-M8). Two alpha-chymotryptic peptides (C1 and C2) that were phosphorylated by protein kinase A but not by the endogenous independent kinase(s) were isolated by high performance liquid chromatography on a reverse-phase C8 column. Partial sequence analysis of peptides C1 (S R V S G P S ...) and C2 (S R G S P S T V S ...) showed that the peptides were localized on the head domain of NF-M at 25 and 41 residues from the amino terminus, respectively. Tryptic digest of peptide C1 (less than 12 kDa) generated the phosphopeptides M1-M6. Peptide C2 was a breakdown product of peptide C1. Since the polypeptide sites targeted by second messenger-independent kinase(s) associated with neurofilaments are localized on the carboxyl-terminal domain, separate aspects of NF-M function appear to be regulated by separate kinase systems that selectively phosphorylate head or tail domains of the polypeptide.     

Rapsyn‐mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high‐density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the α subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn‐induced clustering of the AChR β, γ, and δ subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into αδ dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild‐type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn‐mediated clustering of an assembly intermediate, the αδ dimer. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 486–501, 2003     

Assembly and clustering of acetylcholine receptors containing GFP-tagged epsilon or gamma subunits: selective targeting to the neuromuscular junction in vivo.

Acetylcholine receptor (AChR) gamma and epsilon subunits were tagged by green fluorescent protein (GFP) to analyse assembly and targeting in live muscle fibers at the neuromuscular junction. N- or C-terminal fusion polypeptides showed no fluorescence upon transfection of HEK cells. When GFP was inserted into the cytoplasmic loop connecting putative transmembrane regions M3 and M4, the gamma/GFP and epsilon/GFP subunits were fluorescent and formed together with the alpha, beta, and delta subunits GFP-tagged AChR complexes that were integrated into the plasma membrane. As the AChR were also clustered by rapsyn, the results indicate that the cytoplasmatic domains of the gamma and epsilon subunits may not be required for assembly and rapsyn-dependent clustering. The gamma/GFP and epsilon/GFP subunit-containing receptors were expressed in X. laevis oocytes and have affinities for acetylcholine similar to that of the wild-type receptors. Direct gene transfer into single muscle fibers reveals that gamma/GFP or epsilon/GFP polypeptides are expressed at the site of injection and are transported within the endoplasmatic reticulum. When reaching subsynaptic regions, both gamma/GFP or epsilon/GFP subunits compete with endogenous epsilon subunits to assemble GFP-tagged receptors, which are selectively targeted to the postsynaptic membrane.     

A basic cluster determines topology of the cytoplasmic M3-M4 loop of the glycine receptor alpha1 subunit

The inhibitory glycine receptor is a member of the ligand-gated ion channel superfamily of neurotransmitter receptors, which are composed of homologous subunits with four transmembrane segments (M1-M4), each. Here, we demonstrate that the correct topology of the glycine receptor alpha1 subunit depends critically on six positively charged residues within a basic cluster, RFRRKRR, located in the large cytoplasmic loop (designated M3-M4 loop) following the C-terminal end of M3. Neutralization of one or more charges of this cluster, but not of other charged residues in the M3-M4 loop, led to an aberrant translocation into the endoplasmic reticulum lumen of the M3-M4 loop. However, when two of the three basic charges located in the ectodomain linking M2 and M3 were neutralized, in addition to two charges of the basic cluster, endoplasmic reticulum disposition of the M3-M4 loop was prevented. We conclude that a high density of basic residues C-terminal to M3 is required to compensate for the presence of positively charged residues in the M2-M3 ectodomain, which otherwise impair correct membrane integration of the M3 segment.