RESUMO
Nicotinic acetylcholine receptors are allosteric proteins that generate membrane currents by isomerizing ("gating") between resting and active conformations under the influence of neurotransmitters. Here, to explore the mechanisms that link the transmitter-binding sites (TBSs) with the distant gate, we use mutant cycle analyses to measure coupling between residue pairs, phi value analyses to sequence domain rearrangements, and current simulations to reproduce a microsecond shut component ("flip") apparent in single-channel recordings. Significant interactions between amino acids separated by >15 Å are rare; an exception is between the αM2-M3 linkers and the TBSs that are â¼30 Å apart. Linker residues also make significant, local interactions within and between subunits. Phi value analyses indicate that without agonists, the linker is the first region in the protein to reach the gating transition state. Together, the phi pattern and flip component suggest that a complete, restingâactive allosteric transition involves passage through four brief intermediate states, with brief shut events arising from sojourns in all or a subset. We derive energy landscapes for gating with and without agonists, and propose a structure-based model in which restingâactive starts with spontaneous rearrangements of the M2-M3 linkers and TBSs. These conformational changes stabilize a twisted extracellular domain to promote transmembrane helix tilting, gate dilation, and the formation of a "bubble" that collapses to initiate ion conduction. The energy landscapes suggest that twisting is the most energetically unfavorable step in the restingâactive conformational change and that the rate-limiting step in the reverse process is bubble formation.
Assuntos
Ativação do Canal Iônico/fisiologia , Receptores Nicotínicos/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Conformação Molecular , Mutação , Receptores Nicotínicos/genéticaRESUMO
A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αε (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔGB1). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔGB1 is â¼-2 kcal/mol more favorable at αγ compared with at αε and αδ. Only three of the aromatics contribute significantly to ΔGB1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔGB1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αε and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αε and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse.
Assuntos
Músculos/metabolismo , Neurotransmissores/química , Receptores Colinérgicos/fisiologia , Acetilcolina/metabolismo , Animais , Sítios de Ligação , Colina/química , Simulação por Computador , Eletrofisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Hidrólise , Ligantes , Lymnaea , Camundongos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Receptores Colinérgicos/química , Transmissão Sináptica , Termodinâmica , Torpedo , Triptofano/químicaRESUMO
Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of αY93, αG147, αW149, αY190, αY198, εW55, and δW57, the change in LA binding energy was approximately half that in HA binding energy. The results were analyzed as a linear free energy relationship between LA and HA agonist binding, the slope of which (κ) gives the fraction of the overall binding chemical potential where the LA complex is established. The linear correlation between LA and HA binding energies suggests that the overall binding process is by an integrated mechanism (catch-and-hold). For the agonist and the above mutations, κ â¼ 0.5, but side-chain substitutions of two residues had a slope that was significantly higher (0.90; αG153) or lower (0.25; εP121). The results suggest that backbone rearrangements in loop B, loop C, and the non-α surface participate in both LA binding and the LA â HA affinity switch. It appears that all of the intermediate steps in AChR activation comprise a single, energetically coupled process.
Assuntos
Acetilcolina/metabolismo , Simulação de Acoplamento Molecular , Receptores Nicotínicos/química , Acetilcolina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMO
Synaptic receptors are allosteric proteins that switch on and off to regulate cell signalling. Here, we use single-channel electrophysiology to measure and map energy changes in the gating conformational change of a nicotinic acetylcholine receptor. Two separated regions in the α-subunits--the transmitter-binding sites and αM2-αM3 linkers in the membrane domain--have the highest Ï-values (change conformation the earliest), followed by the extracellular domain, most of the membrane domain and the gate. Large gating-energy changes occur at the transmitter-binding sites, α-subunit interfaces, the αM1 helix and the gate. We hypothesize that rearrangements of the linkers trigger the global allosteric transition, and that the hydrophobic gate unlocks in three steps. The mostly local character of side-chain energy changes and the similarly high Ï-values of separated domains, both with and without ligands, suggest that gating is not strictly a mechanical process initiated by the affinity change for the agonist.
Assuntos
Receptores Nicotínicos/química , Algoritmos , Sítio Alostérico , Animais , Sítios de Ligação , Cristalografia por Raios X , Eletrofisiologia , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
The neuromuscular acetylcholine (ACh) receptor has two conserved prolines in loop D of the complementary subunit at each of its two transmitter-binding sites (α-ε and α-δ). We used single-channel electrophysiology to estimate the energy changes caused by mutations of these prolines with regard to unliganded gating (ΔG0) and the affinity change for ACh that increases the open channel probability (ΔGB). The effects of mutations of ProD2 (εPro-121/δPro-123) were greater than those of its neighbor (εPro-120/δPro-122) and were greater at α-ε versus α-δ. The main consequence of the congenital myasthenic syndrome mutation εProD2-L was to impair the establishment of a high affinity for ACh and thus make ΔGB less favorable. At both binding sites, most ProD2 mutations decreased constitutive activity (increased ΔG0). LRYHQG and RL substitutions reduced substantially the net binding energy (made ΔGB(ACh) less favorable) by ≥2 kcal/mol at α-ε and α-δ, respectively. Mutant cycle analyses were used to estimate energy coupling between the two ProD2 residues and between each ProD2 and glycine residues (αGly-147 and αGly-153) on the primary (α subunit) side of each binding pocket. The distant binding site prolines interact weakly. ProD2 interacts strongly with αGly-147 but only at α-ε and only when ACh is present. The results suggest that in the low to-high affinity change there is a concerted inter-subunit strain in the backbones at εProD2 and αGly-147. It is possible to engineer receptors having a single functional binding site by using a α-ε or α-δ ProD2-R knock-out mutation. In adult-type ACh receptors, the energy from the affinity change for ACh is approximately the same at the two binding sites (approximately -5 kcal/mol).
Assuntos
Prolina/química , Prolina/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Prolina/genética , Receptores Nicotínicos/genéticaRESUMO
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 (ET interface) and between primary and complementary subunits (PC 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 αET interface, as well as between the εL9 residue and others at the PC interface (in particular, the M2M3 linker). These interactions serve, in part, to maintain the structural integrity of the AChR assembly at the ET interface. Overall, the energy changes of L9 residues are significant but smaller than in other regions of the protein.
Assuntos
Acetilcolina/genética , Acetilcolina/metabolismo , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Transformada , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Células HEK293 , Humanos , Cinética , Camundongos , Conformação Molecular , Músculos/metabolismo , Músculos/fisiologia , Mutação Puntual , Estrutura Terciária de Proteína , Subunidades ProteicasRESUMO
The temperature dependence of agonist binding and channel gating were measured for wild-type adult neuromuscular acetylcholine receptors activated by acetylcholine, carbamylcholine, or choline. With acetylcholine, temperature changed the gating rate constants (Q(10) ≈ 3.2) but had almost no effect on the equilibrium constant. The enthalpy change associated with gating was agonist-dependent, but for all three ligands it was approximately equal to the corresponding free-energy change. The equilibrium dissociation constant of the resting conformation (K(d)), the slope of the rate-equilibrium free-energy relationship (Φ), and the acetylcholine association and dissociation rate constants were approximately temperature-independent. In the mutant αG153S, the choline association and dissociation rate constants were temperature-dependent (Q(10) ≈ 7.4) but K(d) was not. By combining two independent mutations, we were able to compensate for the catalytic effect of temperature on the decay time constant of a synaptic current. At mouse body temperature, the channel-opening and -closing rate constants are â¼400 and 16 ms(-1). We hypothesize that the agonist dependence of the gating enthalpy change is associated with differences in ligand binding, specifically to the open-channel conformation of the protein.
Assuntos
Agonistas Colinérgicos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Receptores Colinérgicos/metabolismo , Temperatura , Acetilcolina/farmacologia , Animais , Carbacol/farmacologia , Células HEK293 , Humanos , Cinética , Camundongos , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
Nicotinic acetylcholine receptors (AChRs) are synaptic ion channels that spontaneously isomerize (i.e., gate) between resting and active conformations. We used single-molecule electrophysiology to measure the temperature dependencies of mouse neuromuscular AChR gating rate and equilibrium constants. From these we estimated free energy, enthalpy, and entropy changes caused by mutations of amino acids located between the transmitter binding sites and the middle of the membrane domain. The range of equilibrium enthalpy change (13.4 kcal/mol) was larger than for free energy change (5.5 kcal/mol at 25°C). For two residues, the slope of the rate-equilibrium free energy relationship (Φ) was approximately constant with temperature. Mutant cycle analysis showed that both free energies and enthalpies are additive for energetically independent mutations. We hypothesize that changes in energy associated with changes in structure mainly occur close to the site of the mutation, and, hence, that it is possible to make a residue-by-residue map of heat exchange in the AChR gating isomerization. The structural correlates of enthalpy changes are discussed for 12 different mutations in the protein.