RESUMO
ATP-sensitive potassium channels are an octomeric complex of four pore-forming subunits of the Kir 6.0 family and four sulfonylurea receptors. The Kir 6.0 family consists of two known members, Kir 6.1 and Kir 6.2, with distinct functional properties. The tetrameric structure of the pore-forming domain leads to the possibility that mixed heteromultimers may form. In this study, we examine this by using biochemical and electrophysiological techniques after heterologous expression of these subunits in HEK293 cells. After the coexpression of Kir 6.1 and Kir 6.2, Kir 6.1 can be coimmunoprecipitated with isoform-specific Kir 6.2 antisera and vice versa. Coexpression of SUR2B and Kir 6.2 with Kir 6.1 dominant negatives at a 1:1 expression ratio and vice versa led to a potent suppression of current. Kir 6.1, and Kir 6.2 dominant negative mutants were without effect on an inwardly rectifying potassium channel from a different family, Kir 2.1. Single-channel analysis, after coexpression of SUR2B, Kir 6.1, and Kir 6.2, revealed the existence of five distinct populations with differing single-channel current amplitudes. All channel populations were inhibited by glibenclamide. A dimeric Kir 6.1-Kir 6.2 construct expressed with SUR2B had a single-channel conductance intermediate between that of either Kir 6.2 or Kir 6.1 expressed with SUR2B. In conclusion, Kir 6.1 and Kir 6.2 readily coassemble to produce functional channels, and such phenomena may contribute to the diversity of nucleotide-regulated potassium currents seen in native tissues.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/química , Isoformas de Proteínas/química , Receptores de Droga/química , Potenciais de Ação , Sequência de Aminoácidos , Animais , Biopolímeros , Linhagem Celular , Glibureto/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Rim/citologia , Substâncias Macromoleculares , Microscopia de Fluorescência , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/imunologia , Potássio/metabolismo , Bloqueadores dos Canais de Potássio , Canais de Potássio/genética , Canais de Potássio/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Multimerização Proteica , Subunidades Proteicas , Coelhos , Receptores de Droga/antagonistas & inibidores , Receptores de Droga/genética , Receptores de Droga/imunologia , Proteínas Recombinantes de Fusão/química , Relação Estrutura-Atividade , Receptores de Sulfonilureias , TransfecçãoRESUMO
ATP-sensitive potassium channels form a link between membrane excitability and cellular metabolism. These channels are important in physiological processes such as insulin release and they are an important site of drug action. They are an octomeric complex comprised of four sulfonylurea receptors, a member of the ATP-binding cassette family of proteins, and four Kir 6.0 subunits from the inward rectifier family of potassium channels. We have investigated the nature of the interaction between SUR1 and Kir 6.2 and the domains on the channel responsible for the biochemical and functional manifestations of coupling. The results point to the proximal C terminus determining biochemical interaction in a region that also largely governs homotypic and heterotypic interaction between different Kir family members. While this domain may be necessary for functional communication between the two proteins, it is not sufficient since relative modifications of either the N or C terminus are able to disrupt many aspects of functional coupling mediated by the sulfonylurea receptor.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Trifosfato de Adenosina/farmacologia , Ativação do Canal Iônico , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Receptores de Droga/metabolismo , Sítios de Ligação , Relação Dose-Resposta a Droga , Humanos , Modelos Biológicos , Bloqueadores dos Canais de Potássio , Canais de Potássio/genética , Ligação Proteica , Receptores de Droga/antagonistas & inibidores , Receptores de Droga/genética , Proteínas Recombinantes de Fusão , Proteínas Recombinantes/metabolismo , Receptores de Sulfonilureias , Tolbutamida/farmacologiaRESUMO
This presentation will briefly consider past uses of gold compared with currently available dental materials for the restoration of teeth. A progressive analysis of stresses involved versus the mechanical and behavioural properties of contemporary materials will provide insight into the selection of appropriate materials for specific restorative situations. The place of gold-based restorations will be discussed and the future of these more durable prostheses will become apparent.
