RESUMO
The pathophysiological mechanisms underlying the constellation of symptoms that characterize COVID-19 are only incompletely understood. In an effort to fill these gaps, a "nicotinic hypothesis," which posits that nicotinic acetylcholine receptors (AChRs) act as additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptors, has recently been put forth. A key feature of the proposal (with potential clinical ramifications) is the suggested competition between the virus' spike protein and small-molecule cholinergic ligands for the receptor's orthosteric binding sites. This notion is reminiscent of the well-established role of the muscle AChR during rabies virus infection. To address this hypothesis directly, we performed equilibrium-type ligand-binding competition assays using the homomeric human α7-AChR (expressed on intact cells) as the receptor, and radio-labeled α-bungarotoxin (α-BgTx) as the orthosteric-site competing ligand. We tested different SARS-CoV-2 spike protein peptides, the S1 domain, and the entire S1-S2 ectodomain, and found that none of them appreciably outcompete [125I]-α-BgTx in a specific manner. Furthermore, patch-clamp recordings showed no clear effect of the S1 domain on α7-AChR-mediated currents. We conclude that the binding of the SARS-CoV-2 spike protein to the human α7-AChR's orthosteric sites-and thus, its competition with ACh, choline, or nicotine-is unlikely to be a relevant aspect of this complex disease.
Assuntos
COVID-19 , Receptores Nicotínicos , Humanos , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Bungarotoxinas , Nicotina , Receptor Nicotínico de Acetilcolina alfa7 , Ligantes , SARS-CoV-2 , Receptores Nicotínicos/metabolismo , Colinérgicos , ColinaRESUMO
One of the most fundamental questions in the field of Cys-loop receptors (pentameric ligand-gated ion channels, pLGICs) is how the affinity for neurotransmitters and the conductive/nonconductive state of the transmembrane pore are correlated despite the â¼60-Å distance between the corresponding domains. Proposed mechanisms differ, but they all converge into the idea that interactions between wild-type side chains across the extracellular-transmembrane-domain (ECD-TMD) interface are crucial for this phenomenon. Indeed, the successful design of fully functional chimeras that combine intact ECD and TMD modules from different wild-type pLGICs has commonly been ascribed to the residual conservation of sequence that exists at the level of the interfacial loops even between evolutionarily distant parent channels. Here, using mutagenesis, patch-clamp electrophysiology, and radiolabeled-ligand binding experiments, we studied the effect of eliminating this residual conservation of sequence on ion-channel function and cell-surface expression. From our results, we conclude that proper state interconversion ("gating") does not require conservation of sequence-or even physicochemical properties-across the ECD-TMD interface. Wild-type ECD and TMD side chains undoubtedly interact with their surroundings, but the interactions between them-straddling the interface-do not seem to be more important for gating than those occurring elsewhere in the protein. We propose that gating of pLGICs requires, instead, that the overall structure of the interfacial loops be conserved, and that their relative orientation and distance be the appropriate ones for changes in one side to result in changes in the other, in a phenomenon akin to the nonspecific "bumping" of closely apposed domains.
Assuntos
Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/química , Ativação do Canal Iônico , Transdução de Sinais , Substituição de Aminoácidos , Animais , Caenorhabditis elegans , Galinhas , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/genética , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/metabolismo , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Domínios ProteicosRESUMO
Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed in Escherichia coli's inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein-bulk-membrane interface. One of them was consistent with a "regular" diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution in E. coli polar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, "resting" conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.
Assuntos
Proteínas de Bactérias/química , Escherichia coli/química , Ativação do Canal Iônico , Canais Iônicos de Abertura Ativada por Ligante/química , Bicamadas Lipídicas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Canais Iônicos de Abertura Ativada por Ligante/genética , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The lipid dependence of the nicotinic acetylcholine receptor from the Torpedo electric organ has long been recognized, and one of the most consistent experimental observations is that, when reconstituted in membranes formed by zwitterionic phospholipids alone, exposure to agonist fails to elicit ion-flux activity. More recently, it has been suggested that the bacterial homolog ELIC (Erwinia chrysanthemi ligand-gated ion channel) has a similar lipid sensitivity. As a first step toward the elucidation of the structural basis of this phenomenon, we solved the structures of ELIC embedded in palmitoyl-oleoyl-phosphatidylcholine- (POPC-) only nanodiscs in both the unliganded (4.1-Å resolution) and agonist-bound (3.3 Å) states using single-particle cryoelectron microscopy. Comparison of the two structural models revealed that the largest differences occur at the level of loop C-at the agonist-binding sites-and the loops at the interface between the extracellular and transmembrane domains (ECD and TMD, respectively). On the other hand, the transmembrane pore is occluded in a remarkably similar manner in both structures. A straightforward interpretation of these findings is that POPC-only membranes frustrate the ECD-TMD coupling in such a way that the "conformational wave" of liganded-receptor gating takes place in the ECD and the interfacial M2-M3 linker but fails to penetrate the membrane and propagate into the TMD. Furthermore, analysis of the structural models and molecular simulations suggested that the higher affinity for agonists characteristic of the open- and desensitized-channel conformations results, at least in part, from the tighter confinement of the ligand to its binding site; this limits the ligand's fluctuations, and thus delays its escape into bulk solvent.
Assuntos
Microscopia Crioeletrônica , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Animais , Sítios de Ligação , Ligantes , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Domínios Proteicos , Receptores Nicotínicos/metabolismo , TorpedoRESUMO
Whether synaptic transmission is excitatory or inhibitory depends, to a large extent, on whether the ion channels that open upon binding the released neurotransmitter conduct cations or anions. The mechanistic basis of the opposite charge selectivities of Cys-loop receptors has only recently begun to emerge. It is now clear that ionized side chains-whether pore-facing or buried-in the first α-helical turn of the second transmembrane segments underlie this phenomenon and that the electrostatics of backbone atoms are not critically involved. Moreover, on the basis of electrophysiological observations, it has recently been suggested that not only the sign of charged side chains but also their conformation are crucial determinants of cation-anion selectivity. To challenge these ideas with the chemical and structural rigor that electrophysiological observations naturally lack, we performed molecular dynamics, Brownian dynamics, and electrostatics calculations of ion permeation. To this end, we used structural models of the open-channel conformation of the α1 glutamate-gated Cl- channel and the α1 glycine receptor. Our results provided full support to the notion that the conformation of charged sides chains matters for charge selectivity. Indeed, whereas some rotamers of the buried arginines at position 0' conferred high selectivity for anions, others supported the permeation of cations and anions at similar rates or even allowed the faster permeation of cations. Furthermore, we found that modeling glutamates at position -1' of the anion-selective α1 glycine receptor open-state structure-instead of the five native alanines-switches charge selectivity also in a conformation-dependent manner, with some glutamate rotamers being much more effective at conferring selectivity for cations than others. Regarding pore size, we found that the mere expansion of the pore has only a minimal impact on cation-anion selectivity. Overall, these results bring to light the previously unappreciated impact of side-chain conformation on charge selectivity in Cys-loop receptors.
Assuntos
Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/química , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/metabolismo , Sequência de Aminoácidos , Animais , Ácido Glutâmico , Humanos , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Among neurotransmitter-gated ion channels, the superfamily of pentameric ligand-gated ion channels (pLGICs) is unique in that its members display opposite permeant-ion charge selectivities despite sharing the same structural fold. Although much effort has been devoted to the identification of the mechanism underlying the cation-versus-anion selectivity of these channels, a careful analysis of past work reveals that discrepancies exist, that different explanations for the same phenomenon have often been put forth, and that no consensus view has yet been reached. To elucidate the molecular basis of charge selectivity for the superfamily as a whole, we performed extensive mutagenesis and electrophysiological recordings on six different cation-selective and anion-selective homologs from vertebrate, invertebrate, and bacterial origin. We present compelling evidence for the critical involvement of ionized side chains-whether pore-facing or buried-rather than backbone atoms and propose a mechanism whereby not only their charge sign but also their conformation determines charge selectivity. Insertions, deletions, and residue-to-residue mutations involving nonionizable residues in the intracellular end of the pore seem to affect charge selectivity by changing the rotamer preferences of the ionized side chains in the first turn of the M2 α-helices. We also found that, upon neutralization of the charged residues in the first turn of M2, the control of charge selectivity is handed over to the many other ionized side chains that decorate the pore. This explains the long-standing puzzle as to why the neutralization of the intracellular-mouth glutamates affects charge selectivity to markedly different extents in different cation-selective pLGICs.
RESUMO
Among ion channels, only the nicotinic-receptor superfamily has evolved to generate both cation- and anion-selective members. Although other, structurally unrelated, neurotransmitter-gated cation channels exist, no other type of neurotransmitter-gated anion channel, and thus no other source of fast synaptic inhibitory signals, has been described so far. In addition to the seemingly straightforward electrostatic effect of the presence (in the cation-selective members) or absence (in the anion-selective ones) of a ring of pore-facing carboxylates, mutational studies have identified other features of the amino-acid sequence near the intracellular end of the pore-lining transmembrane segments (M2) that are also required to achieve the high charge selectivity shown by native channels. However, the mechanism underlying this more subtle effect has remained elusive and a subject of speculation. Here we show, using single-channel electrophysiological recordings to estimate the protonation state of native ionizable side chains, that anion-selective-type sequences favour whereas cation-selective-type sequences prevent the protonation of the conserved, buried basic residues at the intracellular entrance of the pore (the M2 0' position). We conclude that the previously unrecognized tunable charge state of the 0' ring of buried basic side chains is an essential feature of these channels' versatile charge-selectivity filter.
Assuntos
Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Animais , Sítios de Ligação , Condutividade Elétrica , Células HEK293 , Humanos , Cinética , Ligantes , Camundongos , Mutação , Prolina/genética , Subunidades Proteicas , Prótons , Ratos , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Receptores Nicotínicos/classificação , Receptores Nicotínicos/genética , Eletricidade EstáticaRESUMO
On the basis of single-channel currents recorded from the muscle nicotinic acetylcholine receptor (AChR), we have recently hypothesized that the conformation adopted by the glutamate side chains at the first turn of the pore-lining α-helices is a key determinant of the rate of ion permeation. In this paper, we set out to test these ideas within a framework of atomic detail and stereochemical rigor by conducting all-atom molecular dynamics and Brownian dynamics simulations on an extensively validated model of the open-channel muscle AChR. Our simulations provided ample support to the notion that the different rotamers of these glutamates partition into two classes that differ markedly in their ability to catalyze ion conduction, and that the conformations of the four wild-type glutamates are such that two of them "fall" in each rotamer class. Moreover, the simulations allowed us to identify the mm (χ(1) â -60°; χ(2) â -60°) and tp (χ(1) â 180°; χ(2) â +60°) rotamers as the likely conduction-catalyzing conformations of the AChR's selectivity-filter glutamates. More generally, our work shows an example of how experimental benchmarks can guide molecular simulations into providing a type of structural and mechanistic insight that seems otherwise unattainable.
Assuntos
Glutamatos/química , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Animais , Eletricidade , Glutamina/genética , Ativação do Canal Iônico , Camundongos , Modelos Moleculares , Músculos/metabolismo , Mutação/genética , Permeabilidade , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Cryoelectron microscopy and X-ray crystallography have recently been used to generate structural models that likely represent the unliganded closed-channel conformation and the fully liganded open-channel conformation of different members of the nicotinic-receptor superfamily. To characterize the structure of the closed-channel conformation in its liganded state, we identified a number of positions in the loop between transmembrane segments 2 (M2) and 3 (M3) of a proton-gated ortholog from the bacterium Gloeobacter violaceus (GLIC) where mutations to alanine reduce the liganded-gating equilibrium constant, and solved the crystal structures of two such mutants (T25'A and Y27'A) at pH ~4.0. At the level of backbone atoms, the liganded closed-channel model presented here differs from the liganded open-channel structure of GLIC in the pre-M1 linker, the M3-M4 loop, and much more prominently, in the extracellular half of the pore lining, where the more pronounced tilt of the closed-channel M2 α-helices toward the pore's long axis narrows the permeation pathway. On the other hand, no differences between the liganded closed-channel and open-channel models could be detected at the level of the extracellular domain, where conformational changes are expected to underlie the low-to-high proton-affinity switch that drives gating of proton-bound channels. Thus, the liganded closed-channel model is nearly indistinguishable from the recently described "locally closed" structure. However, because cross-linking strategies (which could have stabilized unstable conformations) and mutations involving ionizable side chains (which could have affected proton-gated channel activation) were purposely avoided, we favor the notion that this structure represents one of the end states of liganded gating rather than an unstable intermediate.
Assuntos
Canais Iônicos Sensíveis a Ácido/química , Cianobactérias/química , Ativação do Canal Iônico/fisiologia , Modelos Moleculares , Conformação Proteica , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Mutagênese , Técnicas de Patch-ClampRESUMO
It has recently been proposed that post-translational modification of not only the M3-M4 linker but also the M1-M2 linker of pentameric ligand-gated ion channels modulates function in vivo. To estimate the involvement of the M1-M2 linker in gating and desensitization, we engineered a series of mutations to this linker of the human adult-muscle acetylcholine receptor (AChR), the α3ß4 AChR and the homomeric α1 glycine receptor (GlyR). All tested M1-M2 linker mutations had little effect on the kinetics of deactivation or desensitization compared with the effects of mutations to the M2 α-helix or the extracellular M2-M3 linker. However, when the effects of mutations were assessed with 50 Hz trains of â¼1 ms pulses of saturating neurotransmitter, some mutations led to much more, and others to much less, peak-current depression than observed for the wild-type channels, suggesting that these mutations could affect the fidelity of fast synaptic transmission. Nevertheless, no mutation to this linker could mimic the irreversible loss of responsiveness reported to result from the oxidation of the M1-M2 linker cysteines of the α3 AChR subunit. We also replaced the M3-M4 linker of the α1 GlyR with much shorter peptides and found that none of these extensive changes affects channel deactivation strongly or reduces the marked variability in desensitization kinetics that characterizes the wild-type channel. However, we found that these large mutations to the M3-M4 linker can have pronounced effects on desensitization kinetics, supporting the notion that its post-translational modification could indeed modulate α1 GlyR behavior.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais Iônicos de Abertura Ativada por Ligante/fisiologia , Mutação/genética , Receptores Nicotínicos/genética , Acetilcolina/farmacologia , Animais , Relação Dose-Resposta a Droga , Glicina/farmacologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Cloreto de Potássio/farmacologia , Estrutura Secundária de Proteína , Receptores de Glicina , Fatores de TempoRESUMO
One of the great challenges of mechanistic ion-channel biology is to obtain structural information from well-defined functional states. In the case of neurotransmitter-gated ion channels, the open-channel conformation is particularly elusive owing to its transient nature and brief mean lifetime. In this Chapter, we show how the analysis of single-channel currents recorded from mutants engineered to contain single ionizable side chains in the transmembrane region can provide specific information about the open-channel conformation without any interference from the closed or desensitized conformations. The method takes advantage of the fact that the alternate binding and unbinding of protons to and from an ionizable side chain causes the charge of the protein to fluctuate by 1 unit. We show that, in mutant muscle acetylcholine nicotinic receptors (AChRs), this fluctuating charge affects the rate of ion conduction in such a way that individual proton-transfer events can be identified in a most straightforward manner. From the extent to which the single-channel current amplitude is reduced every time a proton binds, we can learn about the proximity of the engineered side chain to the lumen of the pore. And from the kinetics of proton binding and unbinding, we can calculate the side-chain's affinity for protons (pK a), and hence, we can learn about the electrostatic properties of the microenvironment around the introduced ionizable group. The application of this method to systematically mutated AChRs allowed us to identify unambiguously the stripes of the M1, M2 and M3 transmembrane α-helices that face the pore's lumen in the open-channel conformation in the context of a native membrane.
Assuntos
Ativação do Canal Iônico , Engenharia de Proteínas , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Sítios de Ligação , Genótipo , Humanos , Transporte de Íons , Cinética , Potenciais da Membrana , Modelos Moleculares , Mutação , Fenótipo , Ligação Proteica , Estrutura Secundária de Proteína , Prótons , Receptores Muscarínicos/química , Receptores Muscarínicos/genética , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Eletricidade Estática , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinic-receptor superfamily.
Assuntos
Proteínas de Bactérias/genética , Dickeya chrysanthemi/genética , Canais Iônicos de Abertura Ativada por Ligante/genética , Mutação , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Cristalografia por Raios X , Cisteamina/farmacologia , Dickeya chrysanthemi/fisiologia , Feminino , Células HEK293 , Humanos , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Modelos Moleculares , Oócitos/metabolismo , Oócitos/fisiologia , Conformação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Fatores de Tempo , Xenopus laevisRESUMO
In ion channels, 'rings' of ionized side chains that decorate the walls of the permeation pathway often lower the energetic barrier to ion conduction. Using single-channel electrophysiological recordings, we studied the poorly understood ring of four glutamates (and one glutamine) that dominates this catalytic effect in the muscle nicotinic acetylcholine receptor ('the intermediate ring of charge'). We show that all four wild-type glutamate side chains are deprotonated in the range of 6.0-9.0 pH, that only two of them contribute to the size of the single-channel current, that these side chains must be able to adopt alternate conformations that either allow or prevent their negative charges from increasing the rate of cation conduction and that the location of these glutamate side chains squarely at one of the ends of the transmembrane pore is critical for their largely unshifted pK(a) values and for the unanticipated impact of their conformational flexibility on cation permeation.
Assuntos
Glutamatos/química , Receptores Nicotínicos/química , Adulto , Sequência de Aminoácidos , Catálise , Cátions/metabolismo , Óxido de Deutério/metabolismo , Eletroquímica , Glutamina/química , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Conformação Molecular , Músculo Esquelético/enzimologia , Técnicas de Patch-Clamp , PorosidadeRESUMO
Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are by no means new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled α-bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric α7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two longstanding questions in the field: our results suggest that ligand-binding affinities are insensitive to binding-site occupancy and that mutations to amino-acid residues in the transmembrane domain are unlikely to affect the channel's affinities for ligands that bind to the extracellular domain.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Sítios de Ligação , Humanos , Transporte de Íons , Canais Iônicos de Abertura Ativada por Ligante/química , Ligantes , Modelos MolecularesRESUMO
Changes in synaptic strength allow synapses to regulate the flow of information in the neural circuits in which they operate. In particular, changes lasting from milliseconds to minutes ('short-term changes') underlie a variety of computational operations and, ultimately, behaviours. Most studies thus far have attributed the short-term type of plasticity to activity-dependent changes in the dynamics of neurotransmitter release (a presynaptic mechanism) while largely dismissing the role of the loss of responsiveness of postsynaptic receptor channels to neurotransmitter owing to entry into desensitization. To better define the response of the different neurotransmitter-gated ion channels (NGICs) to repetitive stimulation without interference from presynaptic variables, we studied eight representative members of all three known superfamilies of NGICs in fast-perfused outside-out patches of membrane. We found that the responsiveness of all tested channels (two nicotinic acetylcholine receptors, two glycine receptors, one GABA receptor, two AMPA-type glutamate receptors and one purinergic receptor) declines along trains of brief neurotransmitter pulses delivered at physiologically relevant frequencies to an extent that suggests that the role of desensitization in the synaptic control of action-potential transmission may be more general than previously thought. Furthermore, our results indicate that a sizable fraction (and, for some NGICs, most) of this desensitization occurs during the neurotransmitter-free interpulse intervals. Clearly, an incomplete clearance of neurotransmitter from the synaptic cleft between vesicle-fusion events need not be invoked to account for NGIC desensitization upon repetitive stimulation.
Assuntos
Neurotransmissores/metabolismo , Receptores de AMPA/metabolismo , Receptores Purinérgicos/metabolismo , Substituição de Aminoácidos , Animais , Estimulação Elétrica , Humanos , Técnicas In Vitro , Ativação do Canal Iônico , Cinética , Camundongos , Modelos Neurológicos , Mutagênese Sítio-Dirigida , Plasticidade Neuronal , Técnicas de Patch-Clamp , Receptores de AMPA/química , Receptores de AMPA/genética , Receptores de Glicina/química , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores Purinérgicos/química , Receptores Purinérgicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transmissão SinápticaRESUMO
As a step toward gaining a better understanding of the physicochemical bases of pK(a)-value shifts in ion channels, we have previously proposed a method for estimating the proton affinities of systematically engineered ionizable side chains from the kinetic analysis of single-channel current recordings. We reported that the open-channel current flowing through mutants of the (cation-selective) muscle nicotinic acetylcholine receptor (AChR) engineered to bear single basic residues in the transmembrane portion of the pore domain fluctuates between two levels of conductance. Our observations were consistent with the idea that these fluctuations track directly the alternate protonation-deprotonation of basic side chains: protonation of the introduced basic group would attenuate the single-channel conductance, whereas its deprotonation would restore the wild-type-like level. Thus, analysis of the kinetics of these transitions was interpreted to yield the pK(a) values of the substituted side chains. However, other mechanisms can be postulated that would also be consistent with some of our findings but according to which the kinetic analysis of the fluctuations would not yield true pK(a)s. Such mechanisms include the pH-dependent interconversion between two conformations of the channel that, while both ion permeable, would support different cation-conduction rates. In this article, we present experimental evidence for the notion that the fluctuations of the open-channel current observed for the muscle AChR result from the electrostatic interaction between fixed charges and the passing cations rather than from a change in conformation. Hence, we conclude that bona fide pK(a) values can be obtained from single-channel recordings.
Assuntos
Canais Iônicos/química , Canais Iônicos/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Ácidos , Aminoácidos , Cátions , Linhagem Celular , Fenômenos Eletrofisiológicos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Técnicas de Patch-Clamp , Subunidades Proteicas , Proteínas/química , Proteínas/metabolismo , Prótons , Eletricidade EstáticaRESUMO
Although membrane proteins often rely on ionizable residues for structure and function, their ionization states under physiological conditions largely elude experimental estimation. To gain insight into the effect of the local microenvironment on the proton affinity of ionizable residues, we have engineered individual lysines, histidines and arginines along the alpha-helical lining of the transmembrane pore of the nicotinic acetylcholine receptor. We can detect individual proton binding-unbinding reactions electrophysiologically at the level of a single proton on a single side chain as brief blocking-unblocking events of the passing cation current. Kinetic analysis of these fluctuations yields the position-dependent rates of proton transfer, from which the corresponding pK(a) values and shifts in pK(a) can be calculated. Here we present a self-consistent, residue-by-residue description of the microenvironment around the pore-lining transmembrane alpha-helices (M2) in the open-channel conformation, in terms of the excess free energy that is required to keep the engineered basic side chains protonated relative to bulk water. A comparison with closed-channel data leads us to propose that the rotation of M2, which is frequently invoked as a hallmark of the gating mechanism of Cys-loop receptors, is minimal, if any.
Assuntos
Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Prótons , Ácidos/química , Ácidos/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Canais Iônicos/genética , Cinética , Lisina/química , Lisina/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Termodinâmica , TitulometriaRESUMO
In many parts of the central nervous system, including the retina, it is unclear whether cholinergic transmission is mediated by rapid, point-to-point synaptic mechanisms, or slower, broad-scale 'non-synaptic' mechanisms. Here, we characterized the ultrastructural features of cholinergic connections between direction-selective starburst amacrine cells and downstream ganglion cells in an existing serial electron microscopy data set, as well as their functional properties using electrophysiology and two-photon acetylcholine (ACh) imaging. Correlative results demonstrate that a 'tripartite' structure facilitates a 'multi-directed' form of transmission, in which ACh released from a single vesicle rapidly (~1 ms) co-activates receptors expressed in multiple neurons located within ~1 µm of the release site. Cholinergic signals are direction-selective at a local, but not global scale, and facilitate the transfer of information from starburst to ganglion cell dendrites. These results suggest a distinct operational framework for cholinergic signaling that bears the hallmarks of synaptic and non-synaptic forms of transmission.
Assuntos
Acetilcolina/metabolismo , Sistema Nervoso Central/fisiologia , Transmissão Sináptica/fisiologia , Células Amácrinas/fisiologia , Células Amácrinas/ultraestrutura , Animais , Dendritos/fisiologia , Dendritos/ultraestrutura , Cinética , Camundongos Endogâmicos C57BL , Fótons , Células Ganglionares da Retina/ultraestruturaRESUMO
The phenomenon of ligand-induced ion channel gating hinges upon the ability of a receptor channel to bind ligand molecules with conformation-specific affinities. However, our understanding of this fundamental phenomenon is notably limited, not only because the changes in binding site structure and ligand conformation that occur upon gating are largely unknown but, also, because the strength of these ligand-receptor interactions are experimentally elusive. Both high- and low-efficacy ligands pose a number of analytical and experimental challenges that can render the estimation of their conformation-specific binding affinities impossible. In this paper, we present a novel assay that overcomes some of the hurdles presented by weak agonists of the muscle nicotinic receptor and allows the estimation of their closed-state affinities. The method, which we have termed the "activation-competition" assay, consists of a single-channel concentration-response assay performed in the presence of a binary mixture of ligands of widely different efficacies. By plotting the channel response (i.e., the open probability) as a function of the concentration of each agonist in the mixture, interpreting the observed response in the framework of a plausible kinetic scheme, and fitting the open probability surface with the corresponding function, the affinities of the closed receptor for the two agonists can be simultaneously extracted as free parameters. Here, we applied this methodology to estimate the closed-state affinity of the muscle nicotinic receptor for choline (a very weak agonist) using acetylcholine (ACh) as the partner in the mixture. We estimated the dissociation equilibrium constant of choline (K(D)) from the wild type's closed state to be 4.1 +/- 0.5 mM (and that of ACh to be 106 +/- 6 microM). We also discuss the use of accurate estimates of affinities for low-efficacy agonists as a tool to discriminate between binding and gating effects of mutations, and in the context of the rational design of therapeutic drugs.
Assuntos
Agonistas Nicotínicos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Linhagem Celular , Relação Dose-Resposta a Droga , Ligantes , Camundongos , Agonistas Nicotínicos/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores Nicotínicos/genética , Fatores de TempoRESUMO
Ion channel block in muscle acetylcholine nicotinic receptors (AChRs) is an extensively reported phenomenon. Yet, the mechanisms underlying the interruption of ion flow or the interaction of the blocker with the channel's gates remain incompletely characterized. In this paper, we studied fast channel block by choline, a quaternary-ammonium cation that is also an endogenous weak agonist of this receptor, and a valuable tool in structure-function studies. Analysis of the single-channel current amplitude as a function of both choline concentration and voltage revealed that extracellular choline binds to the open-channel pore with millimolar apparent affinity (K(B) congruent with 12 mM in the presence of approximately 155 mM monovalent and 3.5 mM divalent, inorganic cations), and that it permeates the channel faster than acetylcholine. This, together with its relatively small size ( approximately 5.5 A along its longest axis), suggests that the pore-blocking choline binding site is the selectivity filter itself, and that current blockages simply reflect the longer-lived sojourns of choline at this site. Kinetic analysis of single-channel traces indicated that increasing occupancy of the pore-blocking site by choline (as judged from the reduction of the single-channel current amplitude) is accompanied by the lengthening of (apparent) open interval durations. Consideration of a number of possible mechanisms firmly suggests that this prolongation results from the local effect of choline interfering with the operation of the activation gate (closure of blocked receptors is slower than that of unblocked receptors by a factor of approximately 13), whereas closure of the desensitization gate remains unaffected. Thus, we suggest that these two gates act as distinct molecular entities. Also, the detailed understanding gained here on how choline distorts the observed open-time durations can be used to compensate for this artifact during activation assays. This correction is necessary if we are to understand how choline binds to and gates the AChR.