RESUMEN
Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.
Asunto(s)
Canal de Potasio Kv1.3/metabolismo , Canales de Potasio con Entrada de Voltaje , Venenos de Escorpión/metabolismo , Animales , Simulación por Computador , Electrofisiología , Femenino , Canal de Potasio Kv1.3/química , Canal de Potasio Kv1.3/genética , Microinyecciones , Modelos Moleculares , Conformación Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Oocitos/metabolismo , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Estructura Secundaria de Proteína , ARN Complementario/administración & dosificación , Venenos de Escorpión/química , Escorpiones , Electricidad Estática , XenopusRESUMEN
Oxygen-sensitive K(+) channels are important elements in the cellular response to hypoxia. Although much progress has been made in identifying their molecular composition, the structural components associated to their O(2)-sensitivity are not yet understood. Recombinant Kv1.2 currents expressed in Xenopus oocytes are inhibited by a decrease in O(2) availability. On the contrary, heterologous Kv2.1 channels are O(2)-insensitive. To elucidate the protein segment responsible for the O(2)-sensitivity of Kv1.2 channels, we analyzed the response to anoxia of Kv1.2/Kv2.1 chimeric channels. Expression of chimeric Kv2.1 channels each containing the S4, the S1-S3 or the S6-COOH segments of Kv1.2 polypeptide resulted in a K(+) current insensitive to anoxia. In contrast, transferring the S5-S6 segment of Kv1.2 into Kv2.1 produced an O(2)-sensitive K(+) current. Finally, mutating a redox-sensitive methionine residue (M380) of Kv1.2 polypeptide did not affect O(2)-sensitivity. Thus, the pore and its surrounding regions of Kv1.2 polypeptide confer its hypoxic inhibition. This response is independent on the redox modulation of methionine residues in this protein segment.
Asunto(s)
Canales de Potasio con Entrada de Voltaje , Canales de Potasio/química , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Animales , Electrofisiología , Hipoxia , Canal de Potasio Kv.1.2 , Datos de Secuencia Molecular , Oocitos/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Péptidos/química , Mutación Puntual , Potasio/metabolismo , Canales de Potasio/metabolismo , Estructura Terciaria de Proteína , ARN Complementario/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , XenopusRESUMEN
The cardiac pacemaker current I(f) is a major determinant of diastolic depolarization in sinus nodal cells and has a key role in heartbeat generation. Therefore, we hypothesized that some forms of "idiopathic" sinus node dysfunction (SND) are related to inherited dysfunctions of cardiac pacemaker ion channels. In a candidate gene approach, a heterozygous 1-bp deletion (1631delC) in exon 5 of the human HCN4 gene was detected in a patient with idiopathic SND. The mutant HCN4 protein (HCN4-573X) had a truncated C-terminus and lacked the cyclic nucleotide-binding domain. COS-7 cells transiently transfected with HCN4-573X cDNA indicated normal intracellular trafficking and membrane integration of HCN4-573X subunits. Patch-clamp experiments showed that HCN4-573X channels mediated I(f)-like currents that were insensitive to increased cellular cAMP levels. Coexpression experiments showed a dominant-negative effect of HCN4-573X subunits on wild-type subunits. These data indicate that the cardiac I(f) channels are functionally expressed but with altered biophysical properties. Taken together, the clinical, genetic, and in vitro data provide a likely explanation for the patient's sinus bradycardia and the chronotropic incompetence.
Asunto(s)
Arritmia Sinusal/diagnóstico , Fibrilación Atrial/diagnóstico , Bradicardia/diagnóstico , Canales Iónicos/genética , Proteínas Musculares/genética , Anciano , Animales , Arritmia Sinusal/complicaciones , Arritmia Sinusal/genética , Fibrilación Atrial/complicaciones , Fibrilación Atrial/genética , Bradicardia/complicaciones , Bradicardia/genética , Células COS , AMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Análisis Mutacional de ADN , Electrocardiografía , Técnicas Electrofisiológicas Cardíacas , Electrofisiología , Exones , Femenino , Frecuencia Cardíaca/genética , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Intrones , Canales Iónicos/biosíntesis , Proteínas Musculares/biosíntesis , Mutación , Técnicas de Placa-Clamp , Canales de Potasio , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Transporte de Proteínas , Síncope/etiología , TransfecciónRESUMEN
The KCNE genes encode small, single transmembrane domain peptides that associate with pore-forming potassium channel subunits to form mixed complexes with unique characteristics. We have identified a novel member of the human KCNE gene family, hKCNE4. The hKCNE4 gene encodes 170 amino acid protein and is localized to chromosome 2q35-36. The protein sequence shows 90% homology to mouse KCNE4 and 38% identity to human KCNE1. Northern blot analysis revealed that hKCNE4 is expressed strongly in heart, skeletal muscle, and kidney, less in placenta, lung, and liver, and weakly in brain and blood cells. Electrophysiological study showed that hKCNE4 modulates the activation of the KCNQ1 channel.