Modulation of GIRK Channels by Protein Kinase C

Abstract

G protein-gated inwardly rectifying potassium channels (GIRK or Kir3) 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, sodium ions and Gβγ-subunits and is modulated by several factors. Protein Kinase C (PKC), which is widely reported to inhibit GIRK channels, has 14 different isoforms which are divided into conventional, novel and atypical sub-types. In this study, we evaluated PKC isoform-specific effects on GIRK active mutant subunits - GIRK1(F137S), GIRK4(S143T) and GIRK2(E152D) - and wild-type heteromers - GIRK1/2 and GIRK1/4 expressed in Xenopus oocytes and HEK293T cells. The conventional isoform, PKC-alpha, inhibited the activity of GIRK2(E152D) and GIRK4(S143T) homomeric channels. The novel isoform, PKC-epsilon, augmented the basal activity of GIRK1(F137S) and GIRK4(S143T) as well as channel activity in response to agonist stimulation of the Gi-coupled, muscarinic M2 receptor (M2R)/dopamine D2 receptor (D2R). Conversely, PKC-epsilon inhibited GIRK2(E152D) basal activity and diminished inward currents evoked through dopamine stimulation of D2R. Similar inhibitory effects by PKC-epsilon were obtained with the more distantly related IRK (Kir2) family members. The effect of PKC-epsilon on GIRK2, GIRK4 predominated over GIRK1 in heteromeric GIRK1/2 and GIRK1/4 channels. Due to the sequence homology between GIRK2 and GIRK4, and the differential effect of PKC-epsilon activity, we sought distinct, putative phosphorylation sites on each channel subunit. Phosphorylation sites for both PKC isoforms on GIRK2 and GIRK4 were predicted using the Group-based Prediction System software and represented on a model for the GIRK2 homomer and a homology model of GIRK4 based on the GIRK2 crystal structure. To converge on the phosphorylation site(s) on GIRK4, chimeras between GIRK4(S143T) and IRK1 are being employed concurrently with site-directed mutagenesis.