RESUMEN
The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular junction caused by gain-of-function mutations to the muscle nicotinic acetylcholine (ACh) receptor (AChR). Although it is clear that the slower deactivation time course of the ACh-elicited currents plays a central role in the etiology of this disease, it has been suggested that other abnormal properties of these mutant receptors may also be critical in this respect. We characterized the kinetics of a panel of five SCCMS AChRs (alphaS269I, betaV266M, epsilonL221F, epsilonT264P, and epsilonL269F) at the ensemble level in rapidly perfused outside-out patches. We found that, for all of these mutants, the peak-current amplitude decreases along trains of nearly saturating ACh pulses delivered at physiologically relevant frequencies in a manner that is consistent with enhanced entry into desensitization during the prolonged deactivation phase. This suggests that the increasingly reduced availability of activatable AChRs upon repetitive stimulation may well contribute to the fatigability and weakness of skeletal muscle that characterize this disease. Also, these results emphasize the importance of explicitly accounting for entry into desensitization as one of the pathways for burst termination, if meaningful mechanistic insight is to be inferred from the study of the effect of these naturally occurring mutations on channel function. Applying a novel single-channel-based approach to estimate the contribution of Ca(2+) to the total cation currents, we also found that none of these mutants affects the Ca(2+)-conduction properties of the AChR to an extent that seems to be of physiological importance. Our estimate of the Ca(2+)-carried component of the total (inward) conductance of wild-type and SCCMS AChRs in the presence of 150 mM Na(+), 1.8 mM Ca(2+), and 1.7 mM Mg(2+) on the extracellular side of cell-attached patches turned out be in the 5.0-9.4 pS range, representing a fractional Ca(2+) current of approximately 14%, on average. Remarkably, these values are nearly identical to those we estimated for the NR1-NR2A N-methyl-d-aspartate receptor (NMDAR), which has generally been considered to be the main neurotransmitter-gated pathway of Ca(2+) entry into the cell. Our estimate of the rat NMDAR Ca(2+) conductance (using the same single-channel approach as for the AChR but in the nominal absence of extracellular Mg(2+)) was 7.9 pS, corresponding to a fractional Ca(2+) current of 13%.
Asunto(s)
Acetilcolina/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Síndromes Miasténicos Congénitos/genética , Receptores Nicotínicos , Animales , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Cinética , Ratones , Fibras Musculares Esqueléticas/fisiología , Proteínas Mutantes/metabolismo , Síndromes Miasténicos Congénitos/metabolismo , Técnicas de Placa-Clamp , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Relación Estructura-Actividad , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genéticaRESUMEN
Although the muscle nicotinic receptor (AChR) desensitizes almost completely in the steady presence of high concentrations of acetylcholine (ACh), it is well established that AChRs do not accumulate in desensitized states under normal physiological conditions of neurotransmitter release and clearance. Quantitative considerations in the framework of plausible kinetic schemes, however, lead us to predict that mutations that speed up channel opening, slow down channel closure, and/or slow down the dissociation of neurotransmitter (i.e., gain-of-function mutations) increase the extent to which AChRs desensitize upon ACh removal. In this paper, we confirm this prediction by applying high-frequency trains of brief ( approximately 1 ms) ACh pulses to outside-out membrane patches expressing either lab-engineered or naturally occurring (disease-causing) gain-of-function mutants. Entry into desensitization was evident in our experiments as a frequency-dependent depression in the peak value of succesive macroscopic current responses, in a manner that is remarkably consistent with the theoretical expectation. We conclude that the comparatively small depression of the macroscopic currents observed upon repetitive stimulation of the wild-type AChR is due, not to desensitization being exceedingly slow but, rather, to the particular balance between gating, entry into desensitization, and ACh dissociation rate constants. Disruption of this fine balance by, for example, mutations can lead to enhanced desensitization even if the kinetics of entry into, and recovery from, desensitization themselves are not affected. It follows that accounting for the (usually overlooked) desensitization phenomenon is essential for the correct interpretation of mutagenesis-driven structure-function relationships and for the understanding of pathological synaptic transmission at the vertebrate neuromuscular junction.
Asunto(s)
Emparejamiento Cromosómico/genética , Emparejamiento Cromosómico/fisiología , Músculos/fisiología , Mutación/genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/fisiología , Animales , Estimulación Eléctrica , Electrofisiología , Humanos , Riñón/citología , Riñón/embriología , Riñón/fisiología , Matemática , Ratones , Neurotransmisores/fisiología , Técnicas de Placa-Clamp , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Factores de Tiempo , TransfecciónRESUMEN
Steady-state single channel activity from NMDA receptors was recorded at a range of concentrations of both glutamate and glycine. The results were fitted with several plausible mechanisms that describe both binding and gating. The mechanisms we have tested were based on our present understanding of receptor structure, or based on previously proposed mechanisms for these receptors. The steady-state channel properties appear to have virtually no dependence on the concentration of either ligand, other than the frequency of channel activations. This limited the ability to discriminate detail in the mechanism, and, along with the persistence of open-shut correlations in high agonist concentrations, suggests that NMDA channels, unlike other neurotransmitter receptors, cannot open unless all binding sites are occupied. As usual for analyses of NMDA channels, the applicability of our results to physiological observations is limited by uncertainties in synaptic zinc and hydrogen ion concentrations, both of these being known to affect the receptor. The mechanism that we propose, on the basis of steady-state single channel recordings, predicts with fair accuracy the apparent open and shut-time distributions in different concentrations of agonists, correlations between open and shut times, and both the rising and falling phases of the macroscopic response to concentration jumps, and can therefore account for the main features of synaptic currents.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales Iónicos/química , Canales Iónicos/fisiología , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Interpretación Estadística de Datos , Ácido Edético/farmacología , Femenino , Ácido Glutámico/farmacología , Ácido Glutámico/fisiología , Glicina/farmacología , Glicina/fisiología , Concentración de Iones de Hidrógeno , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Funciones de Verosimilitud , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Probabilidad , Unión Proteica , Receptores de Glutamato/fisiología , Receptores de Glicina/fisiología , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Sistemas de Mensajero Secundario/fisiología , Factores de Tiempo , Xenopus laevisRESUMEN
The effect of GABA on membrane potential and ionic currents of X-organ neurons isolated from the crayfish eyestalk was investigated. Under voltage-clamp conditions, GABA elicited an inward Na+ current followed by a sustained outward chloride current. Sodium current was partially blocked in a dose-dependent manner by antagonists of GABA plasma membrane transporters such as beta-alanine, nipecotic acid, 1-[2([(diphenylmethylene)imino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO 711), and SKF89976-A at concentrations between 1 and 100 microm. This current was totally blocked by the combined application of NO 711 (5 microm) and beta-alanine (50 microm). We obtained an EC(50) of 5 microm and a Hill coefficient of 0.97 for the GABA transport mediated response. These results together with studies of immunolocalization using antibodies against neuronal vertebrate GABA transporters (GATs) indicate the presence of GAT-1- and GAT-3-like proteins in X-organ neurons. To isolate the sustained outward Cl- current, extracellular free sodium solution was used to minimize the contribution of GAT activity. We concluded that this current was caused by the activation of GABA(A)-like receptors with an EC50 of 10 microm and a Hill number of 1.7. To assign a functional role to the GATs in the X-organ sinus gland system, we determine the GABA concentration (0.46-0.15 microm) in hemolymph samples using HPLC. In summary, our results suggest that a sodium-dependent electrogenic GABA uptake mechanism has a direct influence on the excitability of the X-organ neurons, maintaining an excitatory tone that is dependent on the circulating GABA level.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Proteínas de Transporte de Membrana , Neuronas/metabolismo , Sistemas Neurosecretores/metabolismo , Transportadores de Anión Orgánico , Animales , Astacoidea , Cloruros/metabolismo , Femenino , Antagonistas del GABA/farmacología , Proteínas Transportadoras de GABA en la Membrana Plasmática , Hemolinfa/química , Inmunohistoquímica , Transporte Iónico/efectos de los fármacos , Masculino , Neuronas/efectos de los fármacos , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/efectos de los fármacos , Técnicas de Placa-Clamp , Receptores de GABA-A/metabolismo , Sodio/metabolismo , Ácido gamma-Aminobutírico/análisis , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Nicotinic ACh receptor channels (AChRs) exposed to high concentrations of ACh adopt 'desensitized' conformations that have a high affinity for the transmitter and no measurable ion conductance. Single-channel currents elicited by 0.1 or 1 mM ACh were recorded from human embryonic kidney (HEK) cells that had been transiently transfected with mouse alpha, beta, delta, and epsilon subunits. On the time scale of approximately 0.1 ms to approximately 1 h, apparent open intervals are described by a single exponential component, and shut intervals associated with desensitization are described by the sum of four or five exponential components. The kinetic behaviour appeared to be stationary and homogeneous. Desensitization rate constants were estimated by kinetic modelling of currents from cell-attached and outside-out patches (where the number of channels in the patch was measured). A single AChR recovered from the longest-lived desensitized state only after approximately 5 min. The occupancy of an AChR for each of the desensitized states was calculated as a function of time after the continuous application of a pulse of saturating ACh. The longest-lived desensitized state accounted for 90 % of the total only after several seconds. The fractional recovery from desensitization (during a 200 ms wash period) decreased as the duration of the desensitizing pulse increased, suggesting that recovery is slower from the longer-lived desensitized states. The free energy landscape for the AChR desensitization reaction in cell-attached patches exhibited an initial destabilization, followed by a plateau region of gradually increasing stability, followed by a deep well.
Asunto(s)
Receptores Nicotínicos/efectos de los fármacos , Animales , Línea Celular , Humanos , Riñón/metabolismo , Cinética , Ligandos , Modelos Lineales , Potenciales de la Membrana/fisiología , Ratones , Técnicas de Placa-Clamp , Proteínas Recombinantes/metabolismoRESUMEN
Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a "ball-and-chain" mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. Long transjunctional voltage pulses caused virtual disappearance of the residual current at steady state. Single channel data revealed disappearance of the residual state, increase in the mean open time, and slowing of the transition times between open and closed states. Coexpression of CxM257 with Cx43CT in a separate fragment restored the lower conductance state. We propose that Cx43CT is an effector of fast voltage gating. Truncation of Cx43CT limits channel transitions to those occurring across the higher energy barrier that separates open and closed states. We further propose that a ball-and-chain interaction provides the fast component of voltage-dependent gating between CT domain and a receptor affiliated with the pore.