Invariant Aspartic Acid in Muscle Nicotinic Receptor Contributes Selectively to the Kinetics of Agonist Binding

We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant αD89 forms a highly conserved interdomain contact near αT148, αW149, and αT150. Patch-clamp recordings show that the mutation αD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither αT148L, αT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor αD89. However substituting a negative charge at αT148, but not at αT150, counteracts the effect of αD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring αW149 as principal hydrogen bond donors for αD89. The specific effect of αD89N on ACh association suggests that interdomain hydrogen bonding positions αW149 for optimal interaction with ACh

Initial Coupling of Binding to Gating Mediated by Conserved Residues in the Muscle Nicotinic Receptor

We examined functional consequences of intrasubunit contacts in the nicotinic receptor α subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues αK145, αD200, and αY190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, primarily by slowing the rate of channel opening. The combined mutations αD200N and αK145Q impair channel gating to the same extent as either single mutation, while αK145E counteracts the impaired gating due to αD200K, further suggesting electrostatic interaction between these residues. Interpreted in light of the crystal structure of acetylcholine binding protein (AChBP) with bound carbamylcholine (CCh), the results suggest in the absence of ACh, αK145 and αD200 form a salt bridge associated with the closed state of the channel. When ACh binds, αY190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches αK145, which in turn loosens its contact with αD200. The positional changes of αK145 and αD200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of β-strand 7, which harbors αK145 and is part of the signature Cys-loop, and the second is of β-strand 10, which harbors αD200 and connects to the M1 domain. Thus, interplay between these three conserved residues relays the initial conformational change from the ACh binding site toward the ion channel

Potentiation of a neuronal nicotinic receptor via pseudo-agonist site

Neuronal nicotinic receptors containing α4 and β2 subunits assemble in two pentameric stoichiometries, (α4)3(β2)2 and (α4)2(β2)3, each with distinct pharmacological signatures; (α4)3(β2)2 receptors are strongly potentiated by the drug NS9283, whereas (α4)2(β2)3 receptors are unaffected. Despite this stoichiometry-selective pharmacology, the molecular identity of the target for NS9283 remains elusive. Here, studying (α4)3(β2)2 receptors, we show that mutations at either the principal face of the β2 subunit or the complementary face of the α4 subunit prevent NS9283 potentiation of ACh-elicited single-channel currents, suggesting the drug targets the β2–α4 pseudo-agonist sites, the α4–α4 agonist site, or both sites. To distinguish among these possibilities, we generated concatemeric receptors with mutations at specified subunit interfaces, and monitored the ability of NS9283 to potentiate ACh-elicited single-channel currents. We find that a mutation at the principal face of the β2 subunit at either β2–α4 pseudo-agonist site suppresses potentiation, whereas mutation at the complementary face of the α4 subunit at the α4–α4 agonist site allows a significant potentiation. Thus, monitoring potentiation of single concatemeric receptor channels reveals that the β2–α4 pseudo-agonist sites are required for stoichiometry-selective drug action. Together with the recently determined structure of the (α4)3(β2)2 receptor, the findings have implications for structure-guided drug design

Mechanism of Tacrine Block at Adult Human Muscle Nicotinic Acetylcholine Receptors

We used single-channel kinetic analysis to study the inhibitory effects of tacrine on human adult nicotinic receptors (nAChRs) transiently expressed in HEK 293 cells. Single channel recording from cell-attached patches revealed concentration- and voltage-dependent decreases in mean channel open probability produced by tacrine (IC50 4.6 μM at −70 mV, 1.6 μM at −150 mV). Two main effects of tacrine were apparent in the open- and closed-time distributions. First, the mean channel open time decreased with increasing tacrine concentration in a voltage-dependent manner, strongly suggesting that tacrine acts as an open-channel blocker. Second, tacrine produced a new class of closings whose duration increased with increasing tacrine concentration. Concentration dependence of closed-times is not predicted by sequential models of channel block, suggesting that tacrine blocks the nAChR by an unusual mechanism. To probe tacrine's mechanism of action we fitted a series of kinetic models to our data using maximum likelihood techniques. Models incorporating two tacrine binding sites in the open receptor channel gave dramatically improved fits to our data compared with the classic sequential model, which contains one site. Improved fits relative to the sequential model were also obtained with schemes incorporating a binding site in the closed channel, but only if it is assumed that the channel cannot gate with tacrine bound. Overall, the best description of our data was obtained with a model that combined two binding sites in the open channel with a single site in the closed state of the receptor

Nicotinic Receptor Interloop Proline Anchors β1-β2 and Cys loops in Coupling Agonist Binding to Channel Gating

Nicotinic acetylcholine receptors (AChRs) mediate rapid excitatory synaptic transmission throughout the peripheral and central nervous systems. They transduce binding of nerve-released ACh into opening of an intrinsic channel, yet the structural basis underlying transduction is not fully understood. Previous studies revealed a principal transduction pathway in which αArg 209 of the pre-M1 domain and αGlu 45 of the β1–β2 loop functionally link the two regions, positioning αVal 46 of the β1–β2 loop in a cavity formed by αPro 272 through αSer 269 of the M2–M3 loop. Here we investigate contributions of residues within and proximal to this pathway using single-channel kinetic analysis, site-directed mutagenesis, and thermodynamic mutant cycle analysis. We find that in contributing to channel gating, αVal 46 and αVal 132 of the signature Cys loop couple energetically to αPro 272. Furthermore, these residues are optimized in both their size and hydrophobicity to mediate rapid and efficient channel gating, suggesting naturally occurring substitutions at these positions enable a diverse range of gating rate constants among the Cys-loop receptor superfamily. The overall results indicate that αPro 272 functionally couples to flanking Val residues extending from the β1–β2 and Cys loops within the ACh binding to channel opening transduction pathway

alpha 4 beta 2 Nicotinic Acetylcholine Receptors: Relationships Between Subunit Stoichiometry and Function at the Single Channel Level

Acetylcholine receptors comprising alpha 4 and beta 2 subunits are the most abundant class of nicotinic acetylcholine receptor in the brain. They contribute to cognition, reward, mood, and nociception and are implicated in a range of neurological disorders. Previous measurements of whole-cell macroscopic currents showed that alpha 4 and beta 2 subunits assemble in two predominant pentameric stoichiometries, which differ in their sensitivity to agonists, antagonists, and allosteric modulators. Here we compare agonist-elicited single channel currents from receptors assembled with an excess of either the alpha 4 or beta 2 subunit, forming receptor populations biased toward one or the other stoichiometry, with currents from receptors composed of five concatemeric subunits in which the subunit stoichiometry is predetermined. Our results associate each subunit stoichiometry with a unique single channel conductance, mean open channel lifetime, and sensitivity to the allosteric potentiator 3-[3-(3-pyridinyl)-1,2,4-oxadiazol-5-yl] benzonitrile (NS-9283). Receptors with the composition (alpha 4 beta 2)(2)alpha 4 exhibit high single channel conductance, brief mean open lifetime, and strong potentiation by NS-9283, whereas receptors with the composition (alpha 4 beta 2)(2)beta 2 exhibit low single channel conductance and long mean open lifetime and are not potentiated by NS-9283. Thus single channel current measurements reveal bases for the distinct functional and pharmacological properties endowed by different stoichiometries of alpha 4 and beta 2 subunits and establish pentameric concatemers as a means to delineate interactions between subunits that confer these properties

Targeted Molecular Dynamics Study of C-Loop Closure and Channel Gating in Nicotinic Receptors

The initial coupling between ligand binding and channel gating in the human α7 nicotinic acetylcholine receptor (nAChR) has been investigated with targeted molecular dynamics (TMD) simulation. During the simulation, eight residues at the tip of the C-loop in two alternating subunits were forced to move toward a ligand-bound conformation as captured in the crystallographic structure of acetylcholine binding protein (AChBP) in complex with carbamoylcholine. Comparison of apo- and ligand-bound AChBP structures shows only minor rearrangements distal from the ligand-binding site. In contrast, comparison of apo and TMD simulation structures of the nAChR reveals significant changes toward the bottom of the ligand-binding domain. These structural rearrangements are subsequently translated to the pore domain, leading to a partly open channel within 4 ns of TMD simulation. Furthermore, we confirmed that two highly conserved residue pairs, one located near the ligand-binding pocket (Lys145 and Tyr188), and the other located toward the bottom of the ligand-binding domain (Arg206 and Glu45), are likely to play important roles in coupling agonist binding to channel gating. Overall, our simulations suggest that gating movements of the α7 receptor may involve relatively small structural changes within the ligand-binding domain, implying that the gating transition is energy-efficient and can be easily modulated by agonist binding/unbinding

Mechanism of calcium potentiation of the α7 nicotinic acetylcholine receptor

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration.Fil: Natarajan, Kathiresan. Mayo Clinic Cancer Center; Estados UnidosFil: Mukhtasimova, Nuriya. Mayo Clinic Cancer Center; Estados UnidosFil: Corradi, Jeremias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Lasala, Matías Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Bouzat, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Sine, Steven M.. Mayo Clinic Cancer Center; Estados Unido

Control of Cation Permeation through the Nicotinic Receptor Channel

We used molecular dynamics (MD) simulations to explore the transport of single cations through the channel of the muscle nicotinic acetylcholine receptor (nAChR). Four MD simulations of 16 ns were performed at physiological and hyperpolarized membrane potentials, with and without restraints of the structure, but all without bound agonist. With the structure unrestrained and a potential of −100 mV, one cation traversed the channel during a transient period of channel hydration; at −200 mV, the channel was continuously hydrated and two cations traversed the channel. With the structure restrained, however, cations did not traverse the channel at either membrane potential, even though the channel was continuously hydrated. The overall results show that cation selective transport through the nAChR channel is governed by electrostatic interactions to achieve charge selectivity, but ion translocation relies on channel hydration, facilitated by a trans-membrane field, coupled with dynamic fluctuations of the channel structure