Mechanism of PKCε regulation of cardiac GIRK channel gating

Abstract

G-protein-gated inwardly rectifying potassium (GIRK) channel activity is regulated by the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PI 4,5P 2 ). Constitutive activity of cardiac GIRK channels in atrial myocytes, that is implicated in atrial fibrillation (AF), is mediated via a protein kinase C-ε (PKCε)-dependent mechanism. The novel PKC isoform, PKCε, is reported to enhance the activity of cardiac GIRK channels. Here, we report that PKCε stimulation leads to activation of GIRK channels in mouse atria and in human stem cell-derived atrial cardiomyocytes (iPSCs). We identified residue GIRK4(S418) which when mutated to Ala abolished, or to Glu, mimicked the effects of PKCε on GIRK currents. PKCε strengthened the interactions of the cardiac GIRK isoforms, GIRK4 and GIRK1/4 with PIP 2 , an effect that was reversed in the GIRK4(S418A) mutant. This mechanistic insight into the PKCε-mediated increase in channel activity because of GIRK4(S418) phosphorylation, provides a precise druggable target to reverse AF-related pathologies due to GIRK overactivity.

The W101C KCNJ5 Mutation Induces Slower Pacing by Constitutively Active GIRK Channels in hiPSC-Derived Cardiomyocytes

“…Among others, the novel PKC isoform, PKCε, which is enhanced by AF [47], may cause an increased open channel probability of cardiac GIRK channels by phosphorylation of the channel. The cytosolic C-terminal end of the GIRK4 channel, and especially Ser418, is critical for basal activity and the PKCε-mediated augmentation, as recently demonstrated by Gada et al [48], thus excluding such a mechanism for the constitutively active GIRK due to the W101C KCNJ5 mutation. In addition, Logothetis and colleagues created various other mutations in GIRK4 [49,50] and they demonstrated that S143T GIRK4, which is within the pore region of the channel, gives rise to highly active homomeric channels.…”

PI(4,5)P2 regulates the gating of NaV1.4 channels

“…To study the effects of dephosphorylating PI(4,5)P 2 at the 5-position, we used CRY2-5P OCRL , a well-established optogenetic version of the inositol 5-phosphatase region of Lowe’s oculocerebrorenal protein, OCRL ( Fig. 3 A ; Idevall-Hagren et al, 2012 ; Xu et al, 2020 ; Ningoo et al, 2021 ; Xu et al, 2022 ; Gada et al, 2023a ; Gada et al, 2023c ). TIRFM studies with iRFP-PH PLCδ1 showed that photoactivation of CRY2-5P OCRL evoked rapid redistribution of the PI(4,5)P 2 -biosensor away from the cell membrane, with total depletion occurring within ∼225 s ( Fig.…”

“…The headgroup carries negatively charged phosphates at the 4 and 5 positions that interact with basic residues on partner proteins with a range of functional consequences ( Dickson and Hille, 2019 ). PI(4,5)P 2 is necessary in the gating process of many channel types, but also acts as an allosteric nexus that can regulate channel function by mediating the effects of drugs, post translational modifications, or protein partners ( Liang et al, 2014 ; Xu et al, 2020 ; Gada and Logothetis, 2022 ; Xu et al, 2022 ; Gada et al, 2023a ). PI(4,5)P 2 levels are regulated by the activities of specific kinases and PI-phosphatases, which contribute to a dynamic equilibrium that modulates cell signaling and excitable behavior.…”