RESUMO
The gating behaviour and pharmacological sensitivity of HERG are remarkably different from the corresponding properties of M-eag, a structurally similar member of the Eag family of potassium channels. In contrast to HERG, M-eag exhibits no apparent inactivation and little rectification, and is insensitive to the class III antiarrhythmic drug E-4031. We generated chimeric channels of HERG and M-eag sequences and made point mutations to identify the region necessary for rapid inactivation in HERG. This region includes the P region and half of the S6 putative transmembrane domain, including sites not previously associated with inactivation and rectification in HERG. Transfer of a small segment of the HERG polypeptide to M-eag, consisting largely of the P region and part of the S6 transmembrane domain, is sufficient to confer rapid inactivation and E-4031 sensitivity to M-eag. This region differs from the corresponding region in M-eag by only fifteen residues. Previous hypotheses that rapid inactivation of HERG channels occurs by a C-type inactivation mechanism are supported by the parallel effects on rates of HERG inactivation and Shaker C-type inactivation by a series of mutations at two equivalent sites in the polypeptide sequences. In addition to sites homologous to those previously described for C-type inactivation in Shaker, inactivation in HERG involves a residue in the upstream P region not previously associated with C-type inactivation. Although this site is equivalent to one implicated in P-type inactivation in Kv2.1 channels, our data are most consistent with a single, C-type inactivation mechanism.
Assuntos
Antiarrítmicos/farmacologia , Proteínas de Transporte de Cátions , Proteínas de Ligação a DNA , Piperidinas/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Piridinas/farmacologia , Transativadores , Sequência de Aminoácidos , Animais , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Bloqueadores dos Canais de Potássio , Canais de Potássio/química , Conformação Proteica , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Regulador Transcricional ERGAssuntos
Baculoviridae/genética , DNA Recombinante/isolamento & purificação , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Proteínas Virais/genética , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Ágar , Regulação da Expressão Gênica , Dados de Sequência Molecular , Proteínas de Matriz de Corpos de Inclusão , Reação em Cadeia da Polimerase , Moldes Genéticos , Proteínas Estruturais ViraisRESUMO
The nuclear accessory protein in porcine intestinal nuclear extracts that activates the binding of the vitamin D receptor to its vitamin D response elements has been highly purified. It contains a protein that binds 9-cis-[3H]retinoic acid, was detected on immunoblots with an anti-retinoid X receptor (RXR) peptide antibody, and supports the binding of retinoic acid receptor gamma to the retinoic acid receptor beta gene response element. Most important, the two specific complexes formed by porcine nuclear extract with the vitamin D response elements from either the osteocalcin gene or the rat 24-hydroxylase gene are shifted to a larger complex by both an anti-vitamin D receptor antibody and an anti-RXR antibody, leaving no doubt that in vivo the nuclear accessory factor for the vitamin D receptor in the intestine is an RXR protein.
Assuntos
Sistema Enzimático do Citocromo P-450 , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Calcitriol/metabolismo , Tretinoína/metabolismo , Animais , Anticorpos , Sequência de Bases , Núcleo Celular/metabolismo , Cromatografia de Afinidade , DNA/química , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/isolamento & purificação , Oligodesoxirribonucleotídeos , Osteocalcina/genética , Osteocalcina/metabolismo , Ratos , Fases de Leitura , Receptores do Ácido Retinoico/imunologia , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Esteroide Hidroxilases/genética , Suínos , Fatores de Transcrição/imunologia , Vitamina D3 24-Hidroxilase , Receptor gama de Ácido RetinoicoRESUMO
The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.
