Modulation of a GIRK1 Active Mutant Subunit by Protein Kinase C Isoforms

Abstract

G-protein gated inwardly rectifying potassium channels (GIRK)are activated by the Gᵦᵧ dimer of Gi/o-protein coupled receptors to inhibit excitability. There are four distinct subunits, GIRK1-GIRK4, which exist as heterotetramers or homotetramers throughout the body. Unlike GIRK1 and GIRK3 subunits that do not form functional homotetramers, GIRK2 and GIRK4 can each form homotetramers but can also assemble with the other subunits to form heterotetramers. These channels are activated by Na+, ethanol and PIP2, and are modulated by various factors like Protein kinase A and C (PKC). Phosphorylation of the channels by PKC has been widely reported to inhibit channel activity, however the effects of the various PKC isoforms on channel activity has yet to be fully defined. The 14 different isoforms of PKC are categorized into conventional, novel and atypical PKC's. In this study, we aimed to understand the various effects of two isoforms of PKC, the conventional PKCα and novel PKCε on GIRK1 through GIRK1(F137S), an active mutant of GIRK1 that can form functional homotetrameric channels. PKCε augmented GIRK1(F137S)activity, while PKCα inhibited GIRK1(F137S)activity. Due to the differential effect of these PKC isoforms on GIRK1, we sought to identify distinct phosphorylation sites on this subunit. Phosphorylation sites for both PKC isoforms on GIRK1(F137S) were predicted using the Group-based Prediction 3.0 software, and a site-directed mutagenesis approach is being employed to study alanine mutants of the channel in two-electrode voltage clamp experiments. Additionally, PIP2 dependence of PKC-mediated GIRK1 channel activity will be determined by depleting PIP2. This work aims to inform the elucidation of PKC modulation of GIRK1/2 and GIRK1/4 heterotetramers and the relevance of these effects on phosphorylation-mediated channelopathies.