Modulation of GIRK channels by protein kinase C & its role in atrial fibrillation

Abstract

Atrial fibrillation (AF) affects 1 in 4 people greater than 65 years of age. It is characterized by an irregular heart rate that can lead to blood clots, stroke, and heart failure. The etiopathogenesis of AF has not been clearly defined, however, the constitutive activity of the G-protein gated inwardly-rectifying potassium channel - GIRK1/4 - in atrial myocytes has been implicated. GIRK channels are crucial for the maintenance of the resting membrane potential and inhibitory post-synaptic potentials in the body. GIRK channel activity is increased by PIP2, Na+, Gβγ-subunits and is modulated by several factors including protein kinase C (PKC). PKC is widely reported to inhibit GIRK channels and has 14 different isoforms which are divided into conventional, novel, and atypical sub-types. An imbalance in the expression of conventional (α) and novel (ε) subtypes of PKC in atrial fibrillation has been reported. We evaluated PKC isoform-specific effects on GIRK active mutant, homomeric subunits - GIRK1(F137S), GIRK4(S143T) and GIRK2(E152D), referred to as GIRKx, - and wild-type heteromers - GIRK1/2 and GIRK1/4 expressed in Xenopus oocytes and HEK293T cells. The conventional isoform, PKCα, inhibited the activity of all active mutant, homomeric channels. The novel isoform, PKCε, augmented the basal activity of GIRK1, GIRK4* and GIRK1/4 as well as channel activity in response to agonist stimulation of the Gi-coupled, muscarinic M2 receptor (M2R)/dopamine D2 receptor (D2R). Conversely, PKCε inhibited GIRK1/2 and GIRK2* basal activity and diminished inward currents evoked through dopamine stimulation of D2R. Similar inhibitory effects by PKCε were obtained with mutants of the more distantly related IRK (Kir2) family members. The effect of PKCε on the GIRK2 active mutant and GIRK4 suggest that these subunits dominate over GIRK1 in heteromeric GIRK1/2 and GIRK1/4 channels. Due to the novel, stimulatory effect of PKCε on GIRK4, we sought to identify distinct, putative phosphorylation sites for PKCε on the GIRK4 channel subunit. Phosphorylation sites for PKCε on GIRK4 were predicted using the Group-based Prediction System software and represented on homology models of GIRK4 based on the GIRK2 crystal structure. To converge on the phosphorylation site(s) on GIRK4, chimeras between GIRK4(S143T) and IRK1 were employed concurrently with site-directed mutagenesis. Mutations of GIRK4(S418) either abolished (Ala) or mimicked (Glu) the effects of PKC potentiation of the wild-type GIRK4 currents. Further, the mechanism of PKC phosphorylation-mediated channel modulation was elucidated through an optogenetic probe that depletes PIP2. We determined that PKCε strengthened the interactions of the GIRK1/4 heteromer, GIRK4 and GIRK1* with PIP2. PKCα inhibited GIRK1/4 and GIRK1* channels by weakening channel-PIP2 interactions. Moreover, the GIRK4(S418A) mutant reversed the strengthening of channel-PIP2 interactions induced by PKC -mediated phosphorylation. The insight into the mechanism of increase in channel activity after PKCε phosphorylation is guiding the design of allosteric, small-molecule channel modulator scaffolds, like GAT1578 to reverse the PKCε-mediated GIRK overactivity that leads to atrial fibrillation.--Author's abstract