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MODULATORY ACTIVATION OF AN INVERTEBRATE KIR CHANNEL BY PROTEIN KINASE C: INVESTIGATION OF AN INTERACTION WITH PIP 2
Author(s) -
Larry Trevor M.,
Butler Julian M.,
Leggett Elizabeth M.,
Rockwell Nathan M.,
Bell Jessica K.,
VillalbaGalea Carlos A.,
Boland Linda M.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.878.8
Subject(s) - protein kinase c , potassium channel , activator (genetics) , staurosporine , xenopus , chemistry , inward rectifier potassium ion channel , threonine , phosphorylation , patch clamp , serine , ion channel , microbiology and biotechnology , biophysics , biochemistry , biology , receptor , gene
All vertebrate inwardly rectifying potassium (Kir) channels require for activity the membrane lipid, phosphatidyl inositol 4,5‐bisphosphate or PIP 2 . Evidence from several labs points to possible interactions between PIP 2 ‐mediated regulation of Kir channel activity and channel phosphorylation. For example, vertebrate Kir2.3 channels are inhibited by the protein kinase C (PKC) activator, phorbol 12‐myristate‐13‐acetate (PMA), but are made insensitive to PKC activation by a mutation that increases the channel's apparent affinity for PIP 2 . Also, PMA treatment enhances Kir1.1 channel sensitivity to internal pH by a mechanism that depends on phosphorylation of a threonine residue which also regulates PIP 2 sensitivity. Furthermore, Kir1.1 mutants with reduced PIP 2 affinity have an increased sensitivity to inhibition by PMA. Together, the data suggest that Kir channel inhibition by PKC activation is inversely correlated with the channel's affinity for interaction with PIP 2 . To further explore this interaction, we studied PKC regulation of AqKir, an inwardly rectifying potassium channel cloned from the marine sponge Amphimedon queenslandica. We recently reported that this invertebrate Kir channel has a very low affinity for PIP 2 . Using two‐electrode voltage clamp, PMA (0.3 –1 μM) enhanced the AqKir K + ‐current by more than 2‐fold in Xenopus oocytes. The specific PKC inhibitor, bis‐indolylmaleimide type I (6–10 μM), and the non‐specific kinase inhibitor, staurosporine (150 nM), each blocked the effect of PMA on AqKir. We identified a threonine residue as a possible phosphoacceptor site for kinase‐mediated current activation in AqKir; substitution of alanine at this site impaired PMA activation while substitution of serine retained modulation by PMA. Mutations in AqKir that restored a vertebrate level of high affinity regulation by PIP 2 , as assessed by electrophysiological excised patches recording and molecular dynamic simulations, impaired the ability of PMA to activate the AqKir channel. Because the postulated PKC phosphorylation residue is spatially juxtaposed to the PIP 2 binding residues, we are testing for functional interactions between phosphorylation and PIP 2 . To examine this, we are evaluating how PMA alters the kinetics of onset and recovery from voltage‐sensitive phosphatase‐mediated reduction of PIP 2 in wild‐type AqKir and mutant channels with high affinity for PIP 2 . In addition, we are using our published homology model to examine the energetic effects of channel phosphorylation on PIP 2 binding in WT and mutant AqKir channels. Future work aims to understand the interaction between the identified phosphoacceptor sites and kinase‐mediated modulation of AqKir channels and provide comparative data for improving our understanding of the modulation of vertebrate Kir channel activity. Support or Funding Information Supported by NIH grant 2R15‐GM096142 to Linda Boland.