Spontaneous thermal motion of the GABA(A) receptor M2 channel-lining segments

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.     

Tryptophan scanning mutagenesis in the alphaM3 transmembrane domain of the Torpedo californica acetylcholine receptor: functional and structural implications

The functional role of the alphaM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within alphaM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [(125)I]-alpha-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipid-exposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the alphaM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC(50). Taken together, our results suggest that alphaM3 contributes to the gating machinery of the nicotinic ACh receptor and that alphaM3 is comprised of a mixture of two types of helical structures.     

Topography of nicotinic acetylcholine receptor membrane-embedded domains

The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized with N-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in alphaM1, alphaM4, gammaM1, and gammaM4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear alpha-helical structure, but not so for M1, for which "classical" models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.     

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.     

Loose protein packing around the extracellular half of the GABA(A) receptor beta1 subunit M2 channel-lining segment

GABA(A) receptors are ligand-gated ion channels formed by the pseudosymmetrical assembly of five homologous subunits around the central channel axis. The five M2 membrane-spanning segments largely line the channel. In the present work we probed the water surface accessibility of the beta(1) subunit M2 segment using the substituted cysteine accessibility method. We assayed the reaction of the negatively charged sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS(-)), by its effect on subsequent currents elicited by EC(50) and saturating GABA concentrations. pCMBS(-), applied with GABA, reacted with 14 of the 19 residues tested. At the M2 cytoplasmic end from 2' to 6' only beta(1)A252C (2') and beta(1)T256C (6') were pCMBS(-)-reactive in the presence of GABA. We infer that the M2 segments are tightly packed in this region. Toward the extracellular half of M2 all residues from beta(1)T262C (12') through beta(1)E270C (20') reacted with pCMBS(-) applied with GABA. We infer that this region is highly mobile and loosely packed against the rest of the protein. Based on differences in pCMBS(-) reaction rates two domains can be distinguished on the putative channel-lining side of M2. A faster reacting domain includes the 2', 9', 12', 13', and 16' residues. The slower reacting face contains the 6', 10', and 14' residues. We hypothesize that these may represent the channel-lining faces in the closed and open states and that gating involves an 80-100 degrees rotation of the M2 segments. These results are consistent with the loose packing of the M2 segments inferred from the structure of the homologous Torpedo nicotinic acetylcholine receptor.     

Conformational dynamics of the alphaM3 transmembrane helix during acetylcholine receptor channel gating

Muscle acetylcholine receptors are synaptic ion channels that "gate" between closed- and open-channel conformations. We used Phi-value analysis to probe the transition state of the diliganded gating reaction with regard to residues in the M3, membrane-spanning helix of the muscle acetylcholine receptor alpha-subunit. Phi (a fraction between 1 and 0) parameterizes the extent to which a mutation changes the opening versus the closing rate constant and, for a linear reaction mechanism, the higher the Phi-value, the "earlier" the gating motion. In the upper half of alphaM3 the gating motions of all five tested residues were temporally correlated (Phi approximately 0.30) and serve to link structural changes occurring at the middle of the M2, pore-lining helix with those occurring at the interface of the extracellular and transmembrane domains. alphaM3 belongs to a complex and diverse set of synchronously moving parts that change structure relatively late in the channel-opening process. The propagation of the gating Brownian conformational cascade has a complex spatial distribution in the transmembrane domain.     

The alphaM1 segment of the nicotinic acetylcholine receptor exhibits conformational flexibility in a membrane environment

The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) is predominantly alpha-helical, and of the four distinctly different transmembrane M-segments, only the helicity of M1 is ambiguous. In this study, we have investigated the conformation of a membrane-embedded synthetic M1 segment by solid-state nuclear magnetic resonance (NMR) methods. A 35-residue peptide representing the extended alphaM1 domain 206-240 of the Torpedo californica nAChR was synthesized with specific 13C - and 15N-labelled amino acids, and was incorporated in different phosphatidylcholine model membranes. The chemical shift of the isotopic labels was resolved by magic angle spinning (MAS) NMR and could be related to the secondary structure of the alphaM1 analog at the labelled sites. Our results show that the membrane-embedded alphaM1 segment forms an unstable alpha-helix, particularly near residue Leu18 (alphaLeu223 in the entire nAChR). This non-helical tendency was most pronounced when the peptide was incorporated in fully hydrated phospholipid bilayers, with an estimated 40-50% of the peptides having an extended conformation at position Leu18. We propose that the conserved proline residue at position 16 in the alphaM1 analog imparts a conformational flexibility on the M1 segments that could enable membrane-mediated modulation of nAChR activity.     

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.     

Identification of the sites of incorporation of [3H]ethidium diazide within the Torpedo nicotinic acetylcholine receptor ion channel

The binding sites of ethidium, a noncompetitive antagonist of the nicotinic acetylcholine receptor (nAChR), have been localized in the Torpedo nAChR in the desensitized state by use of a photoactivatible derivative, [(3)H]ethidium diazide. At 10 microM [(3)H]ethidium diazide, incorporation into the alpha-, beta-, and delta-subunits was inhibited by the presence of phencyclidine (PCP). Within the alpha-subunit, the incorporation was mapped to a 20-kDa fragment beginning at alphaSer-173 and containing the first three transmembrane segments, alphaM1, alphaM2, and alphaM3. Further digestion of this fragment generated two fragments with PCP-inhibitable incorporation, one containing alphaM1 and one containing both alphaM2 and alphaM3. Within alphaM2, specific incorporation was present in alphaLeu-251 and alphaSer-252, residues that have been previously shown to line the lumen of the ion channel. Digestion of the delta-subunit with S. aureus V8 protease generated a 14-kDa and a 20-kDa fragment, both of which began at Ile-192 and contained PCP-inhibitable labeling. The 14-kDa fragment, containing deltaM1 and deltaM2, was further digested to generate a 3-kDa fragment, containing deltaM2 alone, with PCP-inhibitable incorporation. Digestion of the 20-kDa fragment, which contained deltaM1, deltaM2, and deltaM3, generated two fragments with incorporation, one containing the deltaM1 segment and the other containing deltaM2 and deltaM3. These results establish that in the desensitized state of the nAChR, the high-affinity binding site of ethidium is within the lumen of the ion channel and that the bound drug is in contact with amino acids from both the M1 and M2 hydrophobic segments.     

Gating at the mouth of the acetylcholine receptor channel: energetic consequences of mutations in the alphaM2-cap

Gating of nicotinic acetylcholine receptors from a C(losed) to an O(pen) conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where C<-->O gating motions generate a low<-->high change in the affinity for agonists and in the ionic conductance, respectively. We have measured the energy changes and relative timing of gating movements for residues that lie between these two locations, in the C-terminus of the pore-lining M2 helix of the alpha subunit ('alphaM2-cap'). This region contains a binding site for non-competitive inhibitors and a charged ring that influences the conductance of the open pore. alphaM2-cap mutations have large effects on gating but much smaller effects on agonist binding, channel conductance, channel block and desensitization. Three alphaM2-cap residues (alphaI260, alphaP265 and alphaS268) appear to move at the outset of channel-opening, about at the same time as those at the transmitter binding site. The results suggest that the alphaM2-cap changes its secondary structure to link gating motions in the extracellular domain with those in the channel that regulate ionic conductance.     

Role of a key cysteine residue in the gating of the acetylcholine receptor

We have examined changes in single-channel behavior that result from conservative amino acid substitutions at the Cys230 residue in the putative first transmembrane region (M1) of the murine nicotinic acetylcholine receptor. Mutations made in the gamma subunit altered the energy barrier for a single closing rate constant in proportion to the size of the substituted side chain. One of these substitutions, when made in the alpha subunits, had no effect on gating. No mutations altered permeation. We conclude that the region surrounding the M1 Cys is involved in the gating of the nicotinic acetylcholine receptor and that the gamma subunit contributes significantly to the control of channel closure.     

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 alpha and non-alpha subunits, or homo-pentameric, composed of alpha7 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 alpha1 and epsilon or alpha7 subunits. The replacement of M3 in alpha1 by epsilonM3 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 epsilon chimeric subunit. The duration of the open state decreases with the increase in the number of alpha1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each alpha1M3 segment decreases the energy barrier of the closing process by approximately 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of alpha1 sequence by alpha7 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.     

Distinct structural elements in the first membrane-spanning segment of the epithelial sodium channel

Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an alpha2betagamma or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate site within alphaM1. We performed site-directed mutagenesis at this site and found that, although the mutants reduced channel activity, ENaC protein expression at the plasma membrane was unaffected. To deduce the role of alphaM1 in the pore structure of ENaC, we performed tryptophan-scanning mutagenesis throughout alphaM1 (residues 110-130). We found that mutations within the amino-terminal part of alphaM1 had effects on activity and selectivity with a periodicity consistent with a helical structure but no effect on channel surface expression. We also observed that mutations within the carboxyl-terminal part of alphaM1 had effects on activity and selectivity but with no apparent periodicity. Additionally, these mutants reduced channel surface expression. Our data support a model in which the amino-terminal half of alphaM1 is alpha-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of alphaM1 may be helical or assume a different conformation and may be involved in tertiary interactions essential to proper channel folding or assembly. Together, our data suggest that alphaM1 is divided into two distinct regions.     

Nicotinic receptor fourth transmembrane domain: hydrogen bonding by conserved threonine contributes to channel gating kinetics

The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association with the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated alphaT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of alphaT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of alphaT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.     

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.     

Energetic Contributions to Channel Gating of Residues in the Muscle Nicotinic Receptor β1 Subunit

In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit.     

Lipid matters: nicotinic acetylcholine receptor-lipid interactions (Review)

Ligand-gated ion channels mediate fast intercellular communication in response to endogenous neurotransmitters. The nicotinic acetylcholine receptor (AChR) is the archetype molecule in the superfamily of these membrane proteins. Early electron spin resonance studies led to the discovery of a lipid fraction in direct contact with the AChR, with rotational dynamics 50-fold slower than those of the bulk lipids. This AChR-vicinal lipid region has since been postulated to be a possible site of lipid modulation of receptor function. The polarity and molecular dynamics of solvent dipoles-mainly water-of AChR-vicinal lipids in the membrane have been studied with Laurdan extrinsic fluorescence, and Forster-type resonance energy transfer (FRET) was introduced to characterize the receptor-associated lipid microenvironment. FRET enabled one to discriminate between the bulk lipid and the AChR-vicinal lipid. The latter is in a liquid-ordered phase and exhibits a higher degree of order than the bulk bilayer lipid. Changes in FRET efficiency induced by fatty acids, phospholipids and cholesterol also led to the identification of discrete sites for these lipids on the AChR protein. After delineating the topography of the AChR membrane-embedded domains with fluorescence methods, sites for steroids are being explored with site-directed mutagenesis and patch-clamp recording. Pyrene-labelled Cys residues in alphaM1, alphaM4, gammaM1 and gammaM4 transmembrane regions were found to lie in a shallow position. For M4 segments, this is in agreement with a canonical linear alpha-helix; for M1, it is necessary to postulate a substantial amount of non-helical structure, and/or of kinks, to rationalize the shallow location of Cys residues. Mutations of Thr422, a residue close to the extracellular-facing membrane hemilayer in alphaM4, affect the steroid modulation of AChR function, suggesting its involvement in steroid-AChR interactions.     

Effects of substitution of putative transmembrane segments on nicotinic acetylcholine receptor function

Mutants of the Torpedo nicotinic acetylcholine receptor in which each of the putative transmembrane segments of the alpha-subunit is replaced by the hydrophobic transmembrane segment of the vesicular stomatitis virus glycoprotein or of the human interleukin-2 receptor have been produced in Xenopus oocytes by cDNA manipulations. Functional analysis of these mutants shows that the hydrophobic segment M4 can be replaced by foreign transmembrane sequences without loss of channel activity. It is also suggested that the hydrophobic segments M1, M2 and M3 and the amphipathic segment MA are important for efficient expression of the acetylcholine receptor on the cell surface and that the specific amino acid sequence of segment M2 may be involved in channel activity.     

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.     

Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy

The structures of functional peptides corresponding to the predicted channel-lining M2 segments of the nicotinic acetylcholine receptor (AChR) and of a glutamate receptor of the NMDA subtype (NMDAR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. Both M2 segments form straight transmembrane alpha-helices with no kinks. The AChR M2 peptide inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminal side of the membrane, which is assigned to be intracellular. A model built from these solid-state NMR data, and assuming a symmetric pentameric arrangement of M2 helices, results in a funnel-like architecture for the channel, with the wide opening on the N-terminal intracellular side.