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

Carmen Müllner^*, Daniel Yakubovich, Carmen W. Dessauer, Dieter Platzer^* and Wolfgang Schreibmayer^*

^* Institute for Medical Physics and Biophysics, Graz University, Austria; Institute for Physiology and Pharmacology, Sackler Medical School, Tel Aviv University, Israel; and Department of Integrative Biology & Pharmacology, University of Texas, Houston Medical School, Houston, Texas USA

Correspondence: Address reprint requests to Wolfgang Schreibmayer, Institute for Medical Physics and Biophysics, Graz University, Harrachgasse 21/4, A-8010 Graz, Austria. Tel.: 43 316 380 4155; Fax: 43 316 380 9660; E-mail: wolfgang.schreibmayer{at}kfunigraz.ac.at.

G-Protein activated, inwardly rectifying potassium channels (GIRKs) are important effectors of G-protein ß/-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 (PP₂A). 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 m₂-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_ß1/2, used to activate the channel, P_o was significantly reduced in patches treated with PP₂A, 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 C₅ state was responsible for facilitation of GIRK1/GIRK4 channels by protein phosphorylation. Dephosphorylation by PP₂A led to an increase of G_ß1/2 concentration required for full activation of the channel and hence to a reduction of the apparent affinity of GIRK1/GIRK4 for G_ß1/2. (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 PP₂A, indicating that they constitute an essential part of the off-switch.

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