Molecular basis of the facilitation of the heterooligomeric GIRK1/GIRK4 complex by cAMP dependent protein kinase

摘要

G-protein activated inwardly rectifying K+ channels (GIRKs) of the heterotetrameric GIRK1/GIRK4 composition mediate IK + ACh in atrium and are regulated by cAMP dependent protein kinase (PKA). Phosphorylation of GIRK1/GIRK4 complexes promotes the activation of the channel by the G-protein G?|?-dimer ("heterologous facilitation"). Previously we reported that 3 serines/threonines (S/Ts) within the GIRK1 subunit are phosphorylated by the catalytic subunit of PKA (PKA-cs) in-vitro and are responsible for the acute functional effects exerted by PKA on the homooligomeric GIRK1F137S (GIRK1*) channel. Here we report that homooligomeric GIRK4WT and GIRK4S143T (GIRK4*) channels are clearly regulated by PKA phosphorylation. Heterooligomeric channels of the GIRK1S385CS401CT407C/GIRK4 WT composition, where the GIRK1 subunit is devoid of PKA mediated phosphorylation, exhibited reduced but still significant acute effects (reduction during agonist application was ?? 49% compared to GIRK1 WT/GIRK4WT). Site directed mutagenesis of truncated cytosolic regions of GIRK4 revealed four serines/threonines (S/Ts) that were heavily phosphorylated by PKA-cs in vitro. Two of them were found to be responsible for the acute effects exerted by PKA in vivo, since the effect of cAMP injection was reduced by ?? 99% in homooligomeric GIRK4 *T199CS412C channels. Coexpression of GIRK1WT/ GIRK4T199CS412C reduced the acute effect by ?? 65%. Only channels of the GIRK1S385CS401CT407C/GIRK4T199CS412C composition were practically devoid of PKA mediated effects (reduction by ?? 97%), indicating that both subunits contribute to the heterologous facilitation of IK + ACh. ? 2012 Elsevier B.V. All rights reserved.