| Autor: |
Segura É; Département de Pharmacologie et Physiologie, Faculté de Médecine, Centre de Recherche de l'Institut de Cardiologie de Montréal, Université de Montréal, Montréal, QC H1T 1C8, Canada., Zhao J; Centre de Recherche de l'Institut de Cardiologie de Montréal, Université de Montréal, Montréal, QC H1T 1C8, Canada., Broszczak M; Centre de Recherche de l'Institut de Cardiologie de Montréal, Université de Montréal, Montréal, QC H1T 1C8, Canada., Audet F; Département de Pharmacologie et Physiologie, Faculté de Médecine, Centre de Recherche de l'Institut de Cardiologie de Montréal, Université de Montréal, Montréal, QC H1T 1C8, Canada., Sauvé R; Département de Pharmacologie et Physiologie, Faculté de Médecine, Université de Montréal, 2900 Bd Édouard-Montpetit, Montréal, QC H3T 1J4, Canada., Parent L; Département de Pharmacologie et Physiologie, Faculté de Médecine, Centre de Recherche de l'Institut de Cardiologie de Montréal, Université de Montréal, Montréal, QC H1T 1C8, Canada. |
| Abstrakt: |
Ca 2+ binding to the ubiquitous Ca 2+ sensing protein calmodulin (CaM) activates the intermediate conductance Ca 2+ -activated SK4 channel. Potential hydrophilic pockets for CaM binding have been identified at the intracellular HA and HB helices in the C-terminal of SK4 from the three published cryo-EM structures of SK4. Single charge reversal substitutions at either site, significantly weakened the pull-down of SK4 by CaM wild-type (CaM), and decreased the TRAM-34 sensitive outward K + current densities in native HEK293T cells when compared with SK4 WT measured under the same conditions. Only the doubly substituted SK4 R352D/R355D (HB helix) obliterated the CaM-mediated pull-down and thwarted outward K + currents. However, overexpression of CaM E84K/E87K, which had been predicted to face the arginine doublet, restored the CaM-mediated pull-down of SK4 R352D/R355D and normalized its whole-cell current density. Virtual analysis of the putative salt bridges supports a unique role for the positively charged arginine doublet at the HB helix into anchoring the interaction with the negatively charged CaM glutamate 84 and 87 CaM. Our findings underscore the unique contribution of electrostatic interactions in carrying CaM binding onto SK4 and support the role of the C-terminal HB helix to the Ca 2+ -dependent gating process. |
