M2-M3 Linker Research Articles

Energy Maps of Acetylcholine Receptor Gating

Occupancy of an AChR binding site by an agonist (A) shifts a pre-existing equilibrium from the Closed-channel towards the Open-channel conformation. We estimated energy changes in adult mouse A2C↔A2O gating (adult mouse) from single-channels. Mutations mainly cause local energy changes that can be mapped onto structures of related proteins. We made maps showing residue reaction progress (phi values) and approximate extents of energy changes in the channel-opening isomerization. Phi values range from 1 to 0, are approximately constant for each position and have a modal distribution (n=5 populations). Residues having similar phi values are clustered, with phi decreasing approximately longitudinally between the binding site and the gate. Two separated regions in the alpha subunits - the transmitter binding sites and M2-M3 linkers in the membrane domain - have the highest phi values (∼0.95), followed by the rest of the extracellular domain (∼0.8), most of the membrane domain (∼0.6) and the gate region of the pore (0.3). Residues at the extracellular-transmembrane domain interface undergo large energy changes in C→O. Largest energy changes occur at the binding sites, and in the alpha subunit at the M2-M3 linker, M1 and the gate. All of these energy changes, including those associated with the affinity change for the agonist, appear to arise from local ‘resettling’ events that do not transfer significant energy over distance. Expansion and flexure of the M2-M3 linkers in the alpha subunit appears to trigger the global allosteric transition, and the hydrophobic gate appears to unlock in three steps. The local character of side chain energy changes and the similar phi values of the binding sites and distant M2-M3 linkers suggest that the gating transition is not strictly a mechanical process.

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.

The Structural Mechanism of the Cys-Loop Receptor Desensitization

The cys-loop receptors are neurotransmitter-operated ion channels, which mediate fast synaptic transmission for communication between neurons. However, prolonged exposure to the neurotransmitter drives the receptor to a desensitization state, which plays an important role in shaping synaptic transmission. Much progress has been made through more than half a century's research since Katz and Thesleff first descried desensitization for muscle nicotinic acetylcholine receptor. In this review, we summarized recent research developments of receptor desensitization. Now, it has been identified that many parts of the receptor, such as the pore domain (including the hinge in the M2-M3 linker), the binding domain, the coupling region, and the intracellular domain, are all involved in the cys-loop receptor desensitization and that uncoupling between the amino-terminal domain and channel lining domain seems to play a central role in desensitization. This uncoupling is mainly governed by the balance between coupling strength and relative tightness of gating machinery and influenced by other parts of the receptor. Agonist binding induces conformational change to overcome the gating barrier to open the channel through the stressed coupling region, which is subsequently broken, causing receptor desensitization. With rapid advancement in structural biology of membrane receptors, final validation of this mechanism is expected to occur in the near future when the high-resolution structure of the desensitized state is available.

Amino acid residues of both the extracellular and transmembrane domains influence binding of the antiparasitic agent milbemycin to Haemonchus contortus AVR-14B glutamate-gated chloride channels

Glutamate-gated chloride (GluCl) channels are pentameric receptors for the inhibitory neurotransmitter glutamate in invertebrates and are a major target for macrolide anthelmintics. Three amino acids in GluCl channels are reported to render macrolide resistance in nematodes and insects. To examine whether these three amino acids are involved in binding of the antiparasitic agent milbemycin (MLM) to the GluCl channels of the nematode parasite Haemonchus contortus, the equivalent amino acids (L256, P316, and G329) of the Hco-AVR-14B subunit were substituted with various amino acids. cDNAs encoding the wild type and mutants of this subunit were transfected into COS-1 cells for transient expression and analysis of GluCl channels. The abilities of these mutant channels to bind [3H]MLM A4 were remarkably decreased when compared with the wild-type channel. In patch clamp analysis, L256F and P316S mutant channels were 37- and 100-fold less sensitive to MLM A4 when compared with the wild-type channel, respectively. These findings indicate that amino acid changes in the β10 strand, the M2–M3 linker, and the M3 region influence MLM A4 binding to the channel. Homology modeling and ligand docking studies suggest the presence of two potential binding sites for MLM A4.

The energetic consequences of loop 9 gating motions in acetylcholine receptor‐channels

Acetylcholine receptor-channels (AChRs) mediate fast synaptic transmission between nerve and muscle. In order to better-understand the mechanism by which this protein assembles and isomerizes between closed- and open-channel conformations we measured changes in the diliganded gating equilibrium constant (E(2)) consequent to mutations of residues at the C-terminus of loop 9 (L9) in the α and ε subunits of mouse neuromuscular AChRs. These amino acids are close to two interesting interfaces, between the extracellular and transmembrane domain within a subunit (E–T interface) and between primary and complementary subunits (P–C interface). Most α subunit mutations modestly decreased E(2) (mainly by slowing the channel-opening rate constant) and sometimes produced AChRs that had heterogeneous gating kinetic properties. Mutations in the ε subunit had a larger effect and could either increase or decrease E(2), but did not induce kinetic heterogeneity. There are broad-but-weak energetic interactions between αL9 residues and others at the αE–T interface, as well as between the εL9 residue and others at the P–C interface (in particular, the M2–M3 linker). These interactions serve, in part, to maintain the structural integrity of the AChR assembly at the E–T interface. Overall, the energy changes of L9 residues are significant but smaller than in other regions of the protein.

Engineering a Prokaryotic Cys-loop Receptor with a Third Functional Domain

Prokaryotic members of the Cys-loop receptor ligand-gated ion channel superfamily were recently identified. Previously, Cys-loop receptors were only known from multicellular organisms (metazoans). Contrary to the metazoan Cys-loop receptors, the prokaryotic ones consist of an extracellular (ECD) and a transmembrane domain (TMD), lacking the large intracellular domain (ICD) present in metazoa (between transmembrane segments M3 and M4). Using a chimera approach, we added the 115-amino acid ICD from mammalian serotonin type 3A receptors (5-HT(3A)) to the prokaryotic proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC). We created 12 GLIC-5-HT(3A)-ICD chimeras by replacing a variable number of amino acids in the short GLIC M3M4 linker with the entire 5-HT(3A)-ICD. Two-electrode voltage clamp recordings after expression in Xenopus laevis oocytes showed that only two chimeras were functional and produced currents upon acidification. The pH(50) was comparable with wild-type GLIC. 5-HT(3A) receptor expression can be inhibited by the chaperone protein RIC-3. We have shown previously that the 5-HT(3A)-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT(3A) receptors in X. laevis oocytes. Expression of both functional 5-HT(3A) chimeras was inhibited by RIC-3 co-expression, indicating appropriate folding of the 5-HT(3A)-ICD in the chimeras. Our results indicate that the ICD can be considered a separate domain that can be removed from or added to the ECD and TMD while maintaining the overall structure and function of the ECD and TMD.

Desensitization of α7 Nicotinic Receptor Is Governed by Coupling Strength Relative to Gate Tightness

Binding of a neurotransmitter to its membrane receptor opens an integral ion conducting pore. However, prolonged exposure to the neurotransmitter drives the receptor to a refractory state termed desensitization, which plays an important role in shaping synaptic transmission. Despite intensive research in the past, the structural mechanism of desensitization is still elusive. Using mutagenesis and voltage clamp in an oocyte expression system, we provide several lines of evidence supporting a novel hypothesis that uncoupling between binding and gating machinery is the underlying mechanism for α7 nicotinic receptor (nAChR) desensitization. First, the decrease in gate tightness was highly correlated to the reduced desensitization. Second, nonfunctional mutants in three important coupling loops (loop 2, loop 7, and the M2-M3 linker) could be rescued by a gating mutant. Furthermore, the decrease in coupling strength in these rescued coupling loop mutants reversed the gating effect on desensitization. Finally, coupling between M1 and hinge region of the M2-M3 linker also influenced the receptor desensitization. Thus, the uncoupling between N-terminal domain and transmembrane domain, governed by the balance of coupling strength and gate tightness, underlies the mechanism of desensitization for the α7 nAChR.

Naringin directly activates inwardly rectifying potassium channels at an overlapping binding site to tertiapin-Q.

G protein-coupled inwardly rectifying potassium (K(IR) 3) channels are important proteins that regulate numerous physiological processes including excitatory responses in the CNS and the control of heart rate. Flavonoids have been shown to have significant health benefits and are a diverse source of compounds for identifying agents with novel mechanisms of action. The flavonoid glycoside, naringin, was evaluated on recombinant human K(IR) 3.1-3.4 and K(IR) 3.1-3.2 expressed in Xenopus oocytes using two-electrode voltage clamp methods. In addition, we evaluated the activity of naringin alone and in the presence of the K(IR) 3 channel blocker tertiapin-Q (0.5 nM, 1 nM and 3 nM) at recombinant K(IR) 3.1-3.4 channels. Site-directed mutagenesis was used to identify amino acids within the M1-M2 loop of the K(IR) 3.1(F137S) mutant channel important for naringin's activity. Naringin (100 µM) had minimal effect on uninjected oocytes but activated K(IR) 3.1-3.4 and K(IR) 3.1-3.2 channels. The activation by naringin of K(IR) 3.1-3.4 channels was inhibited by tertiapin-Q in a competitive manner. An alanine-scan performed on the K(IR) 3.1(F137S) mutant channel, replacing one by one aromatic amino acids within the M1-M2 loop, identified tyrosines 148 and 150 to be significantly contributing to the affinity of naringin as these mutations reduced the activity of naringin by 20- and 40-fold respectively. These results show that naringin is a direct activator of K(IR) 3 channels and that tertiapin-Q shares an overlapping binding site on the K(IR) 3.1-3.4. This is the first example of a ligand that activates K(IR) 3 channels by binding to the extracellular M1-M2 linker of the channel.

On the Mechanism Underlying the Irreversible Desensitization of Human Ganglionic Nicotinic Receptors

alpha3-containing nicotinic acetylcholine receptors (AChRs) are expressed in autonomic ganglia and are thought to mediate fast synaptic transmission. These receptors have largely eluded rigorous kinetic characterization due to a combination of poor expression in heterologous systems and the total loss of receptor responsiveness during the course of excised-patch electrophysiology experiments. Recently, it has been demonstrated that a cysteine-to-alanine mutation at the −4’ position of the cytosolic M1-M2 linker can prevent this rundown in whole-cell patches of rodent superior cervical ganglion neurons (Campanucci et al., 2010). This work also suggested that this rundown, which plays a pivotal role in diabetic neuropathy, is due to oxidation of these cysteine residues. Here, we heterologously expressed human alpha3-subunit-containing AChRs in HEK-293 cells and found that the −4’ cysteine mutation cannot prevent the irreversible rundown of currents observed in outside-out patches of membrane. Furthermore, we found that the irreversible loss of receptor responsiveness does not occur in the whole-cell or cell-attached configurations, implying that patch excision leads to this phenomenon. We also investigated the effects of the reciprocal −4’ cysteine mutation in all five subunits of the human muscle AChRs, which do not natively have cysteines at this position. We found that introduction of the cysteines does not lead to an irreversible rundown, although it does slow down the rate of recovery from desensitization. Taken together, our results thus far suggest that the mechanism underlying the rundown of currents mediated by alpha3-containing AChRs in outside-out patches is fundamentally different from that mediated by cysteine oxidation in whole-cell recordings.

Role of the extracellular transmembrane domain interface in gating and pharmacology of a heteromeric neuronal nicotinic receptor

Nicotinic acetylcholine receptors (nAChRs) transmit the agonist signal to the channel gate through a number of extracellular domains. We have previously shown that particular details of the process of coupling binding to gating could be quantitative and qualitatively different in muscle and neuronal type nAChRs. We have extended previous studies on homomeric alpha7 nAChRs to heteromeric alpha3beta4 nAChRs, by mutating residues located at loops 2 and 7, and M2-M3 linker of both alpha3 and beta4 subunits which, in order to monitor surface expression, were modified to bind alpha-bungarotoxin, and expressed in Xenopus oocytes. We show that, in general, mutations in these domains of both alpha3 and beta4 subunits affect the gating function, although the effects are slightly larger if they are inserted in the alpha3 subunit. However, the involvement of a previously reported intrasubunit interaction in coupling (Gln48-Ile130) seems to be restricted to the beta4 subunit. We also show that mutations at these domains, particularly loop 2 of the alpha3 subunit, change the pharmacological profile of alpha3beta4 nAChRs, decreasing nicotine's and increasing cytisine's effectiveness relative to acetylcholine. It is concluded that, unlike muscle nAChRs, the non-alpha subunits play a relevant role in the coupling process of neuronal alpha3beta4 nAChRs.

Role of the extracellular transmembrane domain interface in gating and pharmacology of a heteromeric neuronal nicotinic receptor

J. Neurochem. (2010) 113, 1036–1045. Abstract Nicotinic acetylcholine receptors (nAChRs) transmit the agonist signal to the channel gate through a number of extracellular domains. We have previously shown that particular details of the process of coupling binding to gating could be quantitative and qualitatively different in muscle and neuronal type nAChRs. We have extended previous studies on homomeric α7 nAChRs to heteromeric α3β4 nAChRs, by mutating residues located at loops 2 and 7, and M2–M3 linker of both α3 and β4 subunits which, in order to monitor surface expression, were modified to bind α-bungarotoxin, and expressed in Xenopus oocytes. We show that, in general, mutations in these domains of both α3 and β4 subunits affect the gating function, although the effects are slightly larger if they are inserted in the α3 subunit. However, the involvement of a previously reported intrasubunit interaction in coupling (Gln48-Ile130) seems to be restricted to the β4 subunit. We also show that mutations at these domains, particularly loop 2 of the α3 subunit, change the pharmacological profile of α3β4 nAChRs, decreasing nicotine’s and increasing cytisine’s effectiveness relative to acetylcholine. It is concluded that, unlike muscle nAChRs, the non-α subunits play a relevant role in the coupling process of neuronal α3β4 nAChRs.

Isomerization of the proline in the M2–M3 linker is not required for activation of the human 5‐HT3A receptor

Each subunit of the cation-selective members of the Cys-loop family of ligand-gated ion channels contains a conserved proline residue in the extracellular loop between the second and third transmembrane domains. In the mouse homomeric 5-hydroxytryptamine type 3A (5-HT(3)A) receptor, the effects of substitution of this proline by unnatural amino acids led to the suggestion that trans-cis isomerization of the protein backbone at this position is integral to agonist-induced channel opening [Nature (2005) vol. 438, pp. 248-252]. We explored the generality of this conclusion using natural amino acid mutagenesis of the homologous human 5-HT(3)A receptor. The conserved proline (P303) was substituted by either a histidine or tryprophan and the mutant receptors were expressed in Xenopus oocytes. These mutations did not significantly affect the magnitude of agonist-mediated currents, compromise channel gating by 5-HT or inhibition of 5-HT-induced currents by either picrotoxin or d-tubocurarine. The mutations did, however, result in altered dependence on extracellular Ca(2+) concentration and a 10-fold increase in the rate of receptor desensitization. These results demonstrate an important role for P303 in 5-HT(3)A receptor function but indicate that trans-cis isomerization at this proline is unlikely to be a general mechanism underlying the gating process.

Inverse Effects on Gating and Modulation Caused by a Mutation in the M2-M3 Linker of the GABAA Receptor γ Subunit

M2-M3 linkers are receptor subunit domains known to be critical for the normal function of cysteine-loop ligand-gated ion channels. Previous studies of alpha and beta subunits of type "A" GABA receptors suggest that these linkers couple extracellular elements involved in GABA binding to the transmembrane segments that control the opening of the ion channel. To study the importance of the gamma subunit M2-M3 linker, we examined the macroscopic and single-channel effects of an engineered gamma2(L287A) mutation on GABA activation and propofol modulation. In the macroscopic analysis, we found that the gamma2(L287A) mutation decreased GABA potency but increased the ability of propofol to enhance both GABA potency and efficacy compared with wild-type receptors. Indeed, although propofol had significant effects on GABA potency in wild-type receptors, we found that propofol produced no corresponding increase in GABA efficacy. At the single-channel level, mutant receptors showed a loss in the longest of three open-time components compared with wild-type receptors under GABA activation. Furthermore, propofol reduced the duration of one closed-time component, increased the duration of two open-time components, and generated a third open component with a longer lifetime in mutant compared with wild-type receptors. Taken together, we conclude that although the gamma subunit is not required for the binding of GABA or propofol, the M2-M3 linker of this subunit plays a critical role in channel gating by GABA and allosteric modulation by propofol. Our results also suggest that in wild-type receptors, propofol exerts its enhancing effects by mechanisms extrinsic to channel gating.

Allosteric activation mechanism of the cys-loop receptors

Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a beta sheet of amino-terminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

Binding to Gating Transduction in Nicotinic Receptors: Cys-Loop Energetically Couples to Pre-M1 and M2–M3 Regions

The nicotinic acetylcholine receptor (AChR) transduces binding of nerve-released ACh into opening of an intrinsic ion channel, yet the intraprotein interactions behind transduction remain to be fully elucidated. Attention has focused on the region of the AChR in which the beta1-beta2 and Cys-loops from the extracellular domain project into a cavity framed by residues preceding the first transmembrane domain (pre-M1) and the linker spanning transmembrane domains M2 and M3. Previous studies identified a principal transduction pathway in which the pre-M1 domain is coupled to the M2-M3 linker through the beta1-beta2 loop. Here we identify a parallel pathway in which the pre-M1 domain is coupled to the M2-M3 linker through the Cys-loop. Mutagenesis, single-channel kinetic analyses and thermodynamic mutant cycle analyses reveal energetic coupling among alphaLeu 210 from the pre-M1 domain, alphaPhe 135 and alphaPhe 137 from the Cys-loop, and alphaLeu 273 from the M2-M3 linker. Residues at equivalent positions of non-alpha-subunits show negligible coupling, indicating these interresidue couplings are specific to residues in the alpha-subunit. Thus, the extracellular beta1-beta2 and Cys-loops bridge the pre-M1 domain and M2-M3 linker to transduce agonist binding into channel gating.

An Ion Selectivity Filter in the Extracellular Domain of Cys-loop Receptors Reveals Determinants for Ion Conductance

Neurotransmitter binding to Cys-loop receptors promotes a prodigious transmembrane flux of several million ions/s, but to date, structural determinants of ion flux have been identified flanking the membrane-spanning region. Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO4(2-) with a ring of lysines at a position equivalent to 24 A above the lipid membrane in homologous Cys-loop receptors. Analysis of multiple sequence alignments revealed that residues equivalent to the ring of lysines are negatively charged in cation-selective receptors but are positively charged in anion-selective receptors. Charge reversal of side chains at homologous positions in the nicotinic receptor from the motor end plate decreases unitary conductance up to 80%. Selectivity filters stemming from transmembrane alpha-helices have similar pore diameters and compositions of amino acids. These findings establish that when the channel opens under a physiological electrochemical gradient, permeant ions are initially stabilized within the extracellular vestibule of Cys-loop receptors, and this stabilization is a major determinant of ion conductance.

γ-Aminobutyric Acid (GABA) and Pentobarbital Induce Different Conformational Rearrangements in the GABAA Receptor α1 and β2 Pre-M1 Regions

γ-Aminobutyric acid (GABA) binding to GABAA receptors (GABAARs) triggers conformational movements in the α1 and β2 pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABAAR channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABAAR pre-M1 regions. α1β2 GABAARs containing cysteine substitutions in the pre-M1 α1 (K219C, K221C) and β2 (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC50 values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto α1K219C and α1K221C affected GABA- and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For α1K221Cβ2 receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For α1β2K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA- and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA- and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.

Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the Cys-Loop and M2–M3 Linker

Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a “gate” in the transmembrane domain (TMD). We used Φ-value analysis to probe the relative timing of the gating motions of α-subunit residues located near the ECD–TMD interface. Mutation of four of the seven amino acids in the M2–M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (Keq) by up to 10,000-fold (P272 > I274 > A270 > G275). The average Φ-value for the whole linker was ∼0.64. One interpretation of this result is that the gating motions of the M2–M3 linker are approximately synchronous with those of much of M2 (∼0.64), but occur after those of the transmitter binding site region (∼0.93) and loops 2 and 7 (∼0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed Keq by 2800-, 10-, and 18-fold, respectively, and with an average Φ-value of 0.74, similar to those of other cys-loop residues. Even though V132 and I274 are close, the energetic coupling between I and V mutants of these positions was small (≤0.51 kcal mol−1). The M2–M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an ∼16-Å border and involve about a dozen residues.

Non‐charged amino acids from three different domains contribute to link agonist binding to channel gating in α7 nicotinic acetylcholine receptors

Binding of agonists to nicotinic acetylcholine receptors results in channel opening. Previously, we have shown that several charged residues at three different domains of the alpha7 nicotinic receptor are involved in coupling binding and gating, probably through a network of electrostatic interactions. This network, however, could also be integrated by other residues. To test this hypothesis, non-charged amino acids were mutated and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Asn47 and Gln48 (loop 2), Ile130, Trp134, and Gln140 (loop 7), and Thr264 (M2-M3 linker) showed poor or null functional responses, despite significant membrane expression. By contrast, mutants F137A and S265A exhibited a gain of function effect. In all cases, changes in dose-response relationships were small, EC(50) values being between threefold smaller and fivefold larger, arguing against large modifications of agonist binding. Peak currents decayed at the same rate in all receptors except two, excluding large effects on desensitization. Thus, the observed changes could be mostly caused by alterations of the gating characteristics. Moreover, analysis of double mutants showed an interconnection between some residues in these domains, especially Gln48 with Ile130, suggesting a potential coupling between agonist binding and channel gating through these amino acids.

Evolutionarily Conserved Allosteric Network in the Cys Loop Family of Ligand-gated Ion Channels Revealed by Statistical Covariance Analyses

The Cys loop family of ligand-gated ion channels mediate fast synaptic transmission for communication between neurons. They are allosteric proteins, in which binding of a neurotransmitter to its binding site in the extracellular amino-terminal domain triggers structural changes in distant transmembrane domains to open a channel for ion flow. Although the locations of binding site and channel gating machinery are well defined, the structural basis of the activation pathway coupling binding and channel opening remains to be determined. In this paper, by analyzing amino acid covariance in a multiple sequence alignment, we have identified an energetically interconnected network in the Cys loop family of ligand-gated ion channels. Statistical coupling and correlated mutational analyses along with clustering revealed a highly coupled cluster. Mapping the positions in the cluster onto a three-dimensional structural model demonstrated that these highly coupled positions form an interconnected network linking experimentally identified binding domains through the coupling region to the gating machinery. In addition, these highly coupled positions are also condensed in the transmembrane domains, which are a recent focus for the sites of action of many allosteric modulators. Thus, our results revealed a genetically interconnected network that potentially plays an important role in the allosteric activation and modulation of the Cys loop family of ligand-gated ion channels.