Single Channel Analysis of the Regulation of GIRK1/GIRK4 Channels by Protein Phosphorylation

G-Protein activated, inwardly rectifying potassium channels (GIRKs) are important effectors of G-protein β/γ-subunits, playing essential roles in the humoral regulation of cardiac activity and also in higher brain functions. G-protein activation of channels of the GIRK1/GIRK4 heterooligomeric composition is controlled via phosphorylation by cyclic AMP dependent protein kinase (PKA) and dephosphorylation by protein phosphatase 2A (PP(2)A). To study the molecular mechanism of this unprecedented example of G-protein effector regulation, single channel recordings were performed on isolated patches of plasma membranes of Xenopus laevis oocytes. Our study shows that: (i) The open probability (P(o)) of GIRK1/GIRK4 channels, stimulated by coexpressed m(2)-receptors, was significantly increased upon addition of the catalytic subunit of PKA to the cytosolic face of an isolated membrane patch. (ii) At moderate concentrations of recombinant G(β1/γ2), used to activate the channel, P(o) was significantly reduced in patches treated with PP(2)A, when compared to patches with PKA-cs. (iii) Several single channel gating parameters, including modal gating behavior, were significantly different between phosphorylated and dephosphorylated channels, indicating different gating behavior between the two forms of the protein. Most of these changes were, however, not responsible for the marked difference in P(o) at moderate G-protein concentrations. (iv) An increase of the frequency of openings (f(o)) and a reduction of dwell time duration of the channel in the long-lasting C(5) state was responsible for facilitation of GIRK1/GIRK4 channels by protein phosphorylation. Dephosphorylation by PP(2)A led to an increase of G(β1/γ2) concentration required for full activation of the channel and hence to a reduction of the apparent affinity of GIRK1/GIRK4 for G(β1/γ2). (v) Although possibly not directly the target of protein phosphorylation/dephosphorylation, the last 20 C-terminal amino acids of the GIRK1 subunit are required for the reduction of apparent affinity for the G-protein by PP(2)A, indicating that they constitute an essential part of the off-switch

Distribution and localization of a G protein-coupled inwardly rectifying K+ channel in the rat.

AbstractThe cellular distribution of the mRNA of the inwardly rectifying K+ channel KGA (GIRK1) was investigated in rat tissue by in situ hybridization. KGA was originally cloned from the heart and represents the first G protein-activated K+ channel identified. It is expressed in peripheral tissue solely in the atrium, but not in the ventricle, skeletal muscle, lung and kidney. In the central nervous system KGA is most prominently expressed in the Ammon's horn and dentate gyrus of the hippocampus, neocortical layers II–VI, cerebellar granular layer, olfactory bulb, anterior pituitary, thalamic nuclei and several distinct nuclei of the lower brainstem. The abundant expression of KGA in many CNS neurons support its important role as a major target channel for G protein mediated receptor function