RESUMEN
We recently reported that a histidine (H191) in the S3-S4 loop of domain I is critical for nickel inhibition of the Cav3.2 T-type Ca2+ channel. As in Cav3.2, two histidine residues are commonly found in the IS3-IS4 loops of mammalian Cav2.3 Ca2+ channels, which are also blocked by low micromolar concentrations of nickel. We show here by site-directed mutagenesis and electrophysiology that both residues contribute to the nickel sensitivity of Cav2.3, with H183 being more critical than H179. These findings strongly suggest that both H179 and H183 in the IS3-IS4 loop are essential structural determinants required for nickel sensitive inhibition of the Cav2.3.
Asunto(s)
Canales de Calcio Tipo R/química , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/química , Histidina/metabolismo , Níquel/farmacología , Secuencia de Aminoácidos , Animales , Canales de Calcio Tipo R/metabolismo , Proteínas de Transporte de Catión/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Activación del Canal Iónico/efectos de los fármacos , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estructura Secundaria de Proteína , Alineación de Secuencia , Relación Estructura-Actividad , XenopusRESUMEN
T-type Ca2+ channels play essential roles in numerous cellular processes. Recently, we reported that phorbol-12-myristate-13-acetate (PMA) potently enhanced the current amplitude of Cav3.2 T-type channels reconstituted in Xenopus oocytes. Here, we have compared PMA modulation of the activities of Cav3.1, Cav3.2 and Cav3.3 channels, and have investigated the underlying mechanism. PMA augmented the current amplitudes of the three T-type channel isoforms, but the fold stimulations and time courses differed. The augmentation effects were not mimicked by 4alpha-PMA, an inactive stereoisomer of PMA, but were abolished by preincubation with protein kinase C (PKC) inhibitors, indicating that PMA augmented T-type channel currents via activation of oocyte PKC. The stimulation effect on Cav3.1 channel activity by PKC was mimicked by endothelin when endothelin receptor type A was coexpressed with Cav3.1 in the Xenopus oocyte system. Pharmacological studies combined with fluorescence imaging revealed that the surface density of Cav3.1 T-type channels was not significantly changed by activation of PKC. The PKC effect on Cav3.1 was localized to the cytoplasmic II-III loop using chimeric channels with individual cytoplasmic loops of Cav3.1 replaced by those of Cav2.1.
Asunto(s)
Canales de Calcio Tipo T/efectos de los fármacos , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Calcio/metabolismo , Canales de Calcio Tipo T/análisis , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Relación Dosis-Respuesta a Droga , Endotelina-1/farmacología , Activación Enzimática/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Microinyecciones , Mutación , Oocitos/química , Oocitos/metabolismo , Técnicas de Placa-Clamp , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Receptor de Endotelina A/efectos de los fármacos , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Xenopus laevisRESUMEN
Molecular cloning studies have revealed that heterogeneity of T-type Ca2+ currents in native tissues arises from the three isoforms of Ca(v)3 channels: Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3. From pharmacological analysis of the recombinant T-type channels, low concentrations (<50 microM) of nickel were found to selectively block the Ca(v)3.2 over the other isoforms. To date, however, the structural element(s) responsible for the nickel block on the Ca(v)3.2 T-type Ca2+ channel remain unknown. Thus, we constructed chimeric channels between the nickel-sensitive Ca(v)3.2 and the nickel-insensitive Ca(v)3.1 to localize the region interacting with nickel. Systematic assaying of serial chimeras suggests that the region preceding domain I S4 of Ca(v)3.2 contributes to nickel block. Point mutations of potential nickel-interacting sites revealed that H191Q in the S3-S4 loop of domain I significantly attenuated the nickel block of Ca(v)3.2, mimicking the nickel-insensitive blocking potency of Ca(v)3.1. These findings indicate that His-191 in the S3-S4 loop is a critical residue conferring nickel block to Ca(v)3.2 and reveal a novel role for the S3-S4 loop to control ion permeation through T-type Ca2+ channels.