RESUMEN
Recent evidence using GT1-7 cells indicates that GnRH pulsatility depends on exocytotic-release and gene transcription events. To determine whether calcium or DREAM may play a role in linking these processes, we used an L-type Ca(2+)-blocker (nimodipine) and found that not only GnRH gene expression (GnRH-GE) pulse activity was abolished but also that binding of proteins to OCT1BS-a (essential site for GnRH-GE pulses) was reduced. We further found that only EF-hand forms of DREAM were expressed in GT1-7 and that DREAM was part of the complex binding to OCT1BS-a. Finally, microinjection of DREAM antibody into cells abolished GnRH-GE pulses demonstrating its importance in pulsatility. These results reveal that calcium and DREAM may bridge cytoplasmic and nuclear events enabling temporal coordination of intermittent activity. Expression of DREAM in various cell types coupled with the universal role of calcium raise the possibility that these factors may play similar role in other secretory cells.
Asunto(s)
Señalización del Calcio , Regulación de la Expresión Génica , Hormona Liberadora de Gonadotropina/genética , Proteínas de Interacción con los Canales Kv/metabolismo , Fotones , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/efectos de los fármacos , Canales de Calcio Tipo L , Señalización del Calcio/efectos de los fármacos , ADN Complementario/genética , Ensayo de Cambio de Movilidad Electroforética , Elementos de Facilitación Genéticos/efectos de los fármacos , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Interacción con los Canales Kv/química , Proteínas de Interacción con los Canales Kv/genética , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Nimodipina/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Ratas , Proteínas Represoras/química , Proteínas Represoras/genéticaRESUMEN
The Kv4.2 transient voltage-dependent potassium current contributes to the morphology of the cardiac action potential as well as to neuronal excitability and firing frequency. Here we report profound effects of the Kv4.2 C terminus on the surface expression and activation gating properties of Kv4.2 that are modulated by the direct interaction between KChIP2, an auxiliary regulatory subunit, and the C terminus of Kv4.2. We show that increasingly large truncations of the C terminus of rat Kv4.2 (wild type) cause a progressive decrease of Kv4.2 current along with a shift in voltage-dependent activation that is closely correlated with negative charge deletion. Co-expression of more limited Kv4.2 C-terminal truncation mutants (T588 and T528) with KChIP2 results in a doubling of Kv4.2 protein expression and up to an 8-fold increase in Kv4.2 current amplitude. Pulsechase experiments show that co-expression with KChIP2 slows Kv4.2 wild type degradation 8-fold. Co-expression of KChIP2 with an intermediate-length C-terminal truncation mutant (T474) shifts Kv4.2 activation voltage dependence and enhances expression of Kv4.2 current. The largest truncation mutants (T417 and DeltaC) show an intracellular localization with no measurable currents and no response to KChIP2 co-expression. Co-immunoprecipitation and competitive glutathione S-transferase-binding assays indicate a direct interaction between KChIP2 and the Kv4.2 C terminus with a relative binding affinity comparable with that of the N terminus. Overall, these results suggest that the C-terminal domain of Kv4.2 plays a critical role in voltage-dependent activation and functional expression that is mediated by direct interaction between the Kv4.2 C terminus and KChIP2.