RESUMEN
Scaffolding growth factor receptor-bound (Grb) adaptor proteins are components of macromolecular signaling complexes at the plasma membrane and thus are putative regulators of ion channel activity. The present study aimed to define the impact of Grb adaptor proteins on the function of cardiac K(+) channels. To this end channel proteins were coinjected with the adaptor proteins in Xenopus oocytes and channel activity analyzed with two-electrode voltage-clamp. It is shown that coexpression of Grb adaptor proteins can reduce current amplitudes of coexpressed channels. Grb7 and 10 significantly inhibited functional currents generated by hERG, Kv1.5 and Kv4.3 channels. Only Grb10 significantly inhibited KCNQ1/KCNE1 K(+) channels, and only Grb7 reduced Kir2.3 activity, whereas neither Grb protein significantly affected the closely related Kir2.1 and Kir2.2 channels. The present observations for the first time provide evidence for a selective and modulatory role of Grb adaptor proteins in the functional expression of cardiac K(+) channels.
Asunto(s)
Proteína Adaptadora GRB10/metabolismo , Proteína Adaptadora GRB7/metabolismo , Canales de Potasio de Rectificación Interna/biosíntesis , Canales de Potasio con Entrada de Voltaje/biosíntesis , Animales , Proteína Adaptadora GRB10/genética , Proteína Adaptadora GRB7/genética , Humanos , Miocardio/metabolismo , Oocitos , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio con Entrada de Voltaje/genética , Transfección , Xenopus laevisRESUMEN
The weak inward rectifier potassium channel ROMK is important for water and salt reabsorption in the kidney. Here we identified Golgin-160 as a novel interacting partner of the ROMK channel. By using yeast two-hybrid assays and co-immunoprecipitations from transfected cells, we demonstrate that Golgin-160 associates with the ROMK C-terminus. Immunofluorescence microscopy confirmed that both proteins are co-localized in the Golgi region. The interaction was further confirmed by the enhancement of ROMK currents by the co-expressed Golgin-160 in Xenopus oocytes. The increase in ROMK current amplitude was due to an increase in cell surface density of ROMK protein. Golgin-160 also stimulated current amplitudes of the related Kir2.1, and of voltage-gated Kv1.5 and Kv4.3 channels, but not the current amplitude of co-expressed HERG channel. These results demonstrate that the Golgi-associated Golgin-160 recognizes the cytoplasmic C-terminus of ROMK, thereby facilitating the transport of ROMK to the cell surface. However, the stimulatory effect on the activity of more distantly-related potassium channels suggests a more general role of Golgin-160 in the trafficking of plasma membrane proteins.
Asunto(s)
Autoantígenos/metabolismo , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/genética , Sitios de Unión , Transporte Biológico Activo , Células COS , Línea Celular , Chlorocebus aethiops , Femenino , Biblioteca de Genes , Proteínas de la Matriz de Golgi , Humanos , Técnicas In Vitro , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Complejos Multiproteicos , Oocitos/metabolismo , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/genética , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas del Sistema de Dos Híbridos , Xenopus laevisRESUMEN
Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) that contributes to action potential repolarization of human atrial myocytes. Block of these channels has been proposed as a treatment for atrial arrhythmias. Here we report a novel and potent inhibitor of Kv1.5 potassium channels, N-benzyl-N-pyridin-3-yl-methyl-2-(toluene-4-sulfonylamino)-benzamide hydrochloride (S0100176), which exhibits features consistent with preferential block of the open state. The IC50 of S0100176 for Kv1.5 expressed in Xenopus oocytes was 0.7 microm. Ala-scanning mutagenesis within the pore helix and the S6 segment, regions that form the walls of the central cavity, was combined with voltage clamp analysis to identify point mutations that altered drug affinity. This approach identified Thr-479, Thr-480, Val-505, Ile-508, and Val-512 as the most important residues for block by S0100176. Mutations of these key residues to Ala or other amino acids caused marked changes in the IC50 of S0100176 (p<0.01). For example, the IC50 of S0100176 increased 362-fold for T480A, 26-fold for V505A, 150-fold for I508A, and 99-fold for V512A. We used modeling to dock S0100176 into the inner cavity of a Kv1.5 pore homology model that was generated based on the crystal structure of KcsA. The docking predicted that the five residues identified by the Ala scan were positioned less than 4.5 A from the compound. Based on the homology models, the positions of the five amino acids identified to interact with S0100176 face toward the central cavity and overlap with putative binding sites for other blockers and voltage-gated potassium channels.
Asunto(s)
Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/fisiología , Sulfonamidas/farmacología , ortoaminobenzoatos/farmacología , Sustitución de Aminoácidos , Animales , Sitios de Unión , Humanos , Canal de Potasio Kv1.5 , Potenciales de la Membrana/efectos de los fármacos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oocitos , Canales de Potasio/química , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/genética , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMEN
The HCN4 gene encodes a hyperpolarization-activated cation current contributing to the slow components of the pacemaking currents I(f) in the sinoatrial node and I(h) or I(q) in the thalamus. Heterologous expression studies of individual HCN channels have, however, failed to reproduce fully the diversity of native I(f/h/q) currents, suggesting the presence of modulating auxiliary subunits. Consistent with this is the recent description of KCNE2, which is highly expressed in the sinoatrial node, as a beta-subunit of rapidly activating HCN1 and HCN2 channels. To determine whether KCNE2 can also modulate the slow component of native I(f/h/q) currents, we co-expressed KCNE2 with HCN4 in Xenopus oocytes and in Chinese hamster ovary (CHO) cells and analysed the resulting currents using two-electrode voltage-clamp and patch-clamp techniques, respectively. In both cell types, co-expressed KCNE2 enhanced HCN4-generated current amplitudes, slowed the activation kinetics and shifted the voltage for half-maximal activation of currents to more negative voltages. In contrast, the related family members KCNE1, KCNE3 and KCNE4 did not change current characteristics of HCN4. Consistent with these electrophysiological results, the carboxy-terminal tail of KCNE2, but not of other KCNE subunits, interacted with the carboxy-terminal tail of HCN4 in yeast two-hybrid assays. KCNE2, by modulating I(f) or I(h) currents, might thus contribute to the electrophysiological diversity of known pacemaking currents in the heart and brain.