Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating

The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of α and non-α subunits, or homo-pentameric, composed of α7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between α1 and ? or α7 subunits. The replacement of M3 in α1 by ?M3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse ? chimeric subunit. The duration of the open state decreases with the increase in the number of α1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each α1M3 segment decreases the energy barrier of the closing process by ∼ 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of α1 sequence by α7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.     

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.     

A distinct contribution of the delta subunit to acetylcholine receptor channel activation revealed by mutations of the M2 segment.

Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12' position of the M2 segment) were examined at the single-channel level. For all subunits (alpha, beta, epsilon, and delta), a 12'P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12'P mutations were co-expressed with alpha subunits having a binding site mutation that slows channel opening (alphaD200N). In these AChRs, a 12'P mutation in epsilon or beta slowed the closing rate constant approximately 6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh (Kd). In contrast, a 12'P mutation in delta slowed the channel closing rate constant only approximately 2-fold and significantly increased both the channel opening rate constant and the Kd. Pairwise expression of 12'P subunits indicates that mutations in epsilon and beta act nearly independently, but one in delta reduces the effect of a homologous mutation in epsilon or beta. The results suggest that a 12'P mutation in epsilon and beta has mainly local effects, whereas one in delta has both local and distributed effects that influence both agonist binding and channel gating.     

The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels.

Purified preparations of the inhibitory glycine receptor (GlyR) contain alpha and beta subunits, which share homologous primary structures and a common transmembrane topology with other members of the ligand-gated ion channel superfamily. Here, a beta subunit-specific antiserum was shown to precipitate the [3H]strychnine binding sites localized on alpha subunits from membrane extracts of both rat spinal cord and mammalian cells co-transfected with alpha and beta cDNAs. Further, inhibition of alpha homo-oligomeric GlyRs by picrotoxinin, a non-competitive blocker of ion flow, was reduced 50- to 200-fold for alpha/beta hetero-oligomeric receptors generated by cotransfection. Site-directed mutagenesis identified residues within the second predicted transmembrane segment (M2) of the beta subunit as major determinants of picrotoxinin resistance. These data implicate the M2 segment in blocker binding to and lining of the GlyR chloride channel.     

The extracellular linker of muscle acetylcholine receptor channels is a gating control element

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2-M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the alpha subunit (alphaS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the alphaS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an approximately 92-fold increased gating equilibrium constant, which is consistent with an approximately 10-fold decreased EC(50) in the presence of ACh. With choline, this mutation accelerates channel opening approximately 28-fold, slows channel closing approximately 3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, alphaS269I acetylcholine receptors open at a rate of approximately 1.4 x 10(6) s(-1) and close at a rate of approximately 760 s(-1). These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of approximately 140 s(-1). Ile mutations at positions flanking alphaS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the alpha subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the alphaS269I mutation, Ile mutations at equivalent positions of the beta, straightepsilon, and delta subunits do not affect apparent open-channel lifetimes. However, in beta and straightepsilon, shifting the mutation one residue to the NH(2)-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2-M3L of the alpha subunit moves before the corresponding linkers of the beta and straightepsilon subunits.     

Identification of a domain in the delta subunit (S238-V264) of the alpha4beta3delta GABAA receptor that confers high agonist sensitivity

We have expressed the alpha4beta3delta and alpha4beta3gamma2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the alpha4beta3delta receptor (EC50 approximately 1.4 micromol/L) than of the alpha4beta3gamma2L subtype (EC50 approximately 27.6 micromol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, imidazole-4-amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co-expressing chimeric delta/gamma2L subunits with alpha4 and beta3 subunits. A stretch of amino acids in the delta subunit, S238-V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the delta and gamma2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.     

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.     

Multiple roles of the conserved key residue arginine 209 in neuronal nicotinic receptors.

We have examined the role of a highly conserved arginine (R209), which flanks the M1 transmembrane segment of nAChRs, in the biogenesis and function of neuronal nAChRs. Point mutations revealed that, in alphaBgtx-sensitive neuronal alpha7 nAChRs, the conserved arginine is required for the transport of assembled receptors to the cell surface. By contrast, R209 does not play any role in the transport of assembled alpha-Bgtx-insensitive neuronal alpha3beta4 nAChRs to the cell surface. However, a basic residue at this position of alpha3 and beta4 subunits is necessary for either synthesis, folding, or assembly of alpha3beta4 receptors. Moreover, electrophysiological experiments revealed that in alpha3beta4 receptors the conserved arginine of the alpha3 subunit is involved in either coupling agonist binding to the channel or regulating single channel kinetics.     

Molecular basis of the differential sensitivity of nematode and mammalian muscle to the anthelmintic agent levamisole

Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 microm at -70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the alpha subunit that differ between vertebrates and nematodes. The replacement of the conserved alphaGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that alphaGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs.     

Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated rat muscle

We used specific antibodies to gamma, delta, and epsilon subunits to characterize acetylcholine receptor (AChR) in extracts and at endplates of developing, adult, and denervated rat muscle. The AChRs in normal adult muscle were immunoprecipitated by anti-epsilon and anti-delta, but not by anti-gamma antibodies, whereas AChRs in denervated and embryonic muscles were precipitated by anti-gamma and anti-delta, but showed little or no reactivity to anti-epsilon antibodies. In immunofluorescence experiments, AChRs at neonatal endplates bound antibodies to gamma or delta, but not epsilon, subunit, whereas those in adult muscles bound antibodies to epsilon or delta, but not gamma, subunit. AChRs at denervated endplates and at developing endplates between postnatal days 9 and 16 bound all three antibodies. We conclude that the distribution of gamma and epsilon subunits of the AChR parallels the distribution of AChRs with embryonic and adult channel properties, respectively.     

Stable expression of the mouse nicotinic acetylcholine receptor in mouse fibroblasts. Comparison of receptors in native and transfected cells.

A stable cell line expressing mouse acetylcholine receptors (AChRs), named AM4, was established by cotransfecting into NIH 3T3 fibroblasts, alpha-, beta-, gamma-, and delta-subunit cDNAs plus the neor gene by calcium phosphate precipitation. Surface AChRs on AM4 cells contain all four subunits, sediment as a single approximately 9 S peak on sucrose gradients, and have the same ratio of alpha- to beta-subunits as surface AChRs from mouse BC3H-1 cells. The surface AChRs exhibit pharmacological properties identical to those obtained for BC3H-1 cells, including the association and dissociation rates of alpha-bungarotoxin, a low affinity and cooperative instantaneous dose-response curve, cooperative steady state agonist binding and desensitization, cooperative enhancement of agonist binding affinity by local anesthetics, and distinct affinities for curariform antagonists. Patch clamp measurements on AM4 cells reveal AChR single channel properties identical to those obtained from BC3H-1 cells, including a single class of channels with a conductance of 56 pS, short and long duration openings at low and high agonist concentrations, brief and intermediate closed duration components at low agonist concentrations, and six distinct closed duration components at high agonist concentrations. The biochemical, pharmacological, and single channel measurements indicate at least 95% of the surface AChRs on AM4 cells are alpha 2 beta gamma delta pentamers.     

The extracellular domain of the beta1 subunit is both necessary and sufficient for beta1-like modulation of sodium channel gating.

The type IIA voltage-gated sodium Na(+) channel from rat brain is composed of a large, pore-forming alpha subunit and the auxiliary subunits beta1 and beta2. When expressed in Xenopus oocytes, the beta1 subunit modulates the gating properties of the type IIA alpha subunit, resulting in acceleration of both inactivation and recovery from inactivation and in a negative shift in the voltage dependence of fast inactivation. The beta1 subunit is composed of an extracellular domain with a single immunoglobulin-like fold, a single transmembrane segment, and a small intracellular domain. A series of chimeras with exchanges of domains between the Na(+) channel beta1 and beta2 subunits and between beta1 and the structurally related protein myelin P0 were constructed and analyzed by two-microelectrode voltage clamp in Xenopus oocytes. Only chimeras containing the beta1 extracellular domain were capable of beta1-like modulation of Na(+) channel gating. Neither the transmembrane segment nor the intracellular domain was required for modulation, although mutation of Glu(158) within the transmembrane domain altered the voltage dependence of steady-state inactivation. A truncated beta1 subunit was engineered in which the beta1 extracellular domain was fused to a recognition sequence for attachment of a glycosylphosphatidylinositol membrane anchor. The beta1(ec)-glycosylphosphatidylinositol protein fully reproduced modulation of Na(+) channel inactivation and recovery from inactivation by wild-type beta1. Our findings demonstrate that extracellular domain of the beta1 subunit is both necessary and sufficient for the modulation of Na(+) channel gating.     

Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation epsilonA411P in the amphipathic helix of the acetylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three unrelated families are either homozygous for epsilonA411P or are heterozygous and harbor a null mutation in the second epsilon allele, indicating that epsilonA411P is recessive. We expressed human AChRs containing wild-type or A411P epsilon subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by epsilonA411P. Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.     

Brain alpha-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily

alpha-Bungarotoxin (alpha Bgt) is a potent, high-affinity antagonist for nicotinic acetylcholine receptors (AChRs) from muscle, but not for AChRs from neurons. Both muscle and neuronal AChRs are thought to be formed from multiple homologous subunits aligned around a central cation channel whose opening is regulated by ACh binding. In contrast, the exact structure and function of high-affinity alpha Bgt binding proteins (alpha BgtBPs) found in avian and mammalian neurons remain unknown. Here we show that cDNA clones encoding alpha BgtBP alpha 1 and alpha 2 subunits define alpha BgtBPs as members of a gene family within the ligand-gated ion channel gene superfamily, but distinct from the gene families of AChRs from muscles and nerves. Subunit-specific monoclonal antibodies raised against bacterially expressed alpha BgtBP alpha 1 and alpha 2 subunit fragments reveal the existence of at least two different alpha BgtBP subtypes in embryonic day 18 chicken brains. More than 75% of all alpha BgtBPs have the alpha 1 subunit, but no alpha 2 subunit, and a minor alpha BgtBP subtype (approximately 15%) has both the alpha 1 and alpha 2 subunits.     

Trafficking and potential assembly patterns of epsilon-containing GABAA receptors

Incorporation of the epsilon subunit into the GABAA receptor has been suggested to confer unusual, but variable, biophysical and pharmacological characteristics to both recombinant and native receptors. Due to their structural similarity with the gamma subunits, epsilon subunits have been assumed to substitute at the single position of the gamma subunit in assembled receptors. However, prior work suggests that functional variability in epsilon-containing receptors may reflect alternative sites of incorporation and of not just one, but possibly multiple epsilon subunits in the pentameric receptor complex. Here we present data indicating that increased expression of epsilon, in conjunction with alpha2 and beta3 subunits, results in expression of GABAA receptors with correspondingly altered rectification, deactivation and levels of spontaneous openings, but not increased total current density. We also provide data that the epsilon subunit, like the beta3 subunit, can self-export and data from chimeric receptors suggesting that similarities between the assembly domains of the beta3 and the epsilon subunits may allow the epsilon subunit to replace the beta, as well as the gamma, subunit. The substitution of an epsilon for a beta, as well as the gamma subunit and formation of receptors with alternative patterns of assembly with respect to epsilon incorporation may underlie the observed variability in both biophysical and pharmacological properties noted not only in recombinant, but also in native receptors.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

Tryptophan substitutions at lipid-exposed positions of the gamma M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor

Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (γM3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltage-clamped oocytes. For all the substitutions the concentration for half-maximal activation, EC ₅₀, is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2- to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized αC418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the γM3/αM4 double mutants, the EC ₅₀s were similar to those measured for the αC418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs. Received: 29 January 2001/Revised: 14 May 2001     

Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha3beta4 AChRs expressed in permanently transfected HEK cells.

We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect alpha3beta4 AChRs. alpha7 AChR function was not detected, yet we estimate that there are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC(50) values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16+/-1 microM) > nicotine (Nic; 48 +/- 7 microM) > or = cytisine (Cyt; 57 +/- 3 microM) = acetylcholine (ACh; 59 +/- 6 microM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 microM ACh; -60 mV). Assays that used mAbs confirmed the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. Although 18% of total alpha3 AChRs contained beta2 subunits, no beta2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface alpha3beta2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells. Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC(50) values) for this line was DMPP (14 +/- 1 microM) = Cyt (18 +/- 1 microM) > Nic (56 +/- 15 microM > ACh (79 +/- 8 microM). The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to alpha3beta4 AChRs.     

Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells

We used transient transfection in COS cells to compare the properties of mouse muscle acetylcholine receptors (AChRs) containing alpha, beta, delta, and either gamma or epsilon subunits. gamma- and epsilon-AChRs had identical association rates for binding 125I-alpha-bungarotoxin, and identical curves for inhibition of toxin binding by d-tubocurarine, but epsilon-AChRs had a significantly longer half-time of turnover in the membrane than gamma-AChRs. A myasthenic serum specific for the embryonic form of the AChR reduced toxin binding to gamma-, but not epsilon-AChRs. The gamma-AChRs had channel characteristics of embryonic AChRs, whereas the major class of epsilon-AChR channels had the characteristics of adult AChRs. Two minor channel classes with smaller conductances were also seen with epsilon-AChR. Thus, some, but not all, of the differences between AChRs at adult endplates and those in the extrasynaptic membrane can be explained by the difference in subunit composition of gamma- and epsilon-AChRs.     

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.