RESUMO
We used Xenopus oocytes co-expressing thyrotropin-releasing hormone (TRH) receptors and human ether-a-go-go-related gene (HERG) K+ channel variants carrying different amino-terminal modifications to check the relevance of the proximal domain for hormonal regulation of the channel. Deletion of the whole proximal domain (Delta 138-373) eliminates TRH-induced modifications in activation and deactivation parameters. TRH effects on activation are also suppressed with channels lacking the second half of the proximal domain or only residues 326-373. However, normal responses to TRH are obtained with Delta 346-373 channels. Thus, whereas residues 326-345 are required for the hormonal modulation of HERG activation, different proximal domain sequences contribute to set HERG gating characteristics and its regulation by TRH.
Assuntos
Proteínas de Transporte de Cátions , Proteínas de Ligação a DNA , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Transativadores , Fosfolipases Tipo C/metabolismo , Animais , Linhagem Celular , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Mutagênese Sítio-Dirigida , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/química , Canais de Potássio/genética , Estrutura Terciária de Proteína/fisiologia , Receptores do Hormônio Liberador da Tireotropina/genética , Deleção de Sequência , Relação Estrutura-Atividade , Hormônio Liberador de Tireotropina/farmacologia , Regulador Transcricional ERG , Transfecção , XenopusRESUMO
The biochemical cascade linking activation of phospholipase C-coupled thyrotropin-releasing hormone (TRH) receptors to rat ERG (r-ERG) channel modulation was studied in situ using perforated-patch clamped adenohypophysial GH3 cells and pharmacological inhibitors. To check the recent suggestion that Rho kinase is involved in the TRH-induced r-ERG current suppression, the hormonal effects were studied in cells pretreated with the Rho kinase inhibitors Y-27632 and HA-1077. The TRH-induced r-ERG inhibition was not significantly modified in the presence of the inhibitors. Surprisingly, the hormonal effects became irreversible in the presence of HA-1077 but not in the presence of the more potent Rho kinase inhibitor Y-27632. Further experiments indicated that the effect of HA-1077 correlated with its ability to inhibit protein kinase C (PKC). The hormonal effects also became irreversible in cells in which PKC activity was selectively impaired with GF109203X, Gö6976 or long-term incubation with phorbol esters. Furthermore, the reversal of the effects of TRH, but not its ability to suppress r-ERG currents, was blocked if diacylglycerol generation was prevented by blocking phospholipase C activity with U-73122. Our results suggest that a pathway involving an as yet unidentified protein kinase is the main cause of r-ERG inhibition in perforated-patch clamped GH3 cells. Furthermore, they demonstrate that although not necessary to trigger the ERG current reductions induced by TRH, an intracellular signal cascade involving phosphatidylinositol-4,5-bisphosphate hydrolysis by phospholipase C, activation of an alpha/betaII conventional PKC and one or more dephosphorylation steps catalysed by protein phosphatase 2A, mediates recovery of ERG currents following TRH withdrawal.