Title

Modulatory activation of an invertebrate Kir channel by protein kinase C: Investigation of an interaction with PIP2

ORCiD

Carlos A. Villalba-Galea: 0000-0002-6489-4651

Document Type

Poster

Conference Title/Conference Publication

American Society for Biochemistry and Molecular Biology Annual Meeting

Organization

American Society for Biochemistry and Molecular Biology

Location

San Diego, CA

Conference Dates

April 2-6, 2016

Date of Presentation

4-4-2016

ISSN

2572-6803

Abstract

All vertebrate inwardly rectifying potassium (Kir) channels require for activity the membrane lipid, phosphatidyl inositol 4,5-bisphosphate or PIP2. Evidence from several labs points to possible interactions between PIP2-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 PIP2 . 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 PIP2 sensitivity. Furthermore, Kir1.1 mutants with reduced PIP2 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 PIP2. 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 PIP2. 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 PIP2, 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 PIP2 binding residues, we are testing for functional interactions between phosphorylation and PIP2. To examine this, we are evaluating how PMA alters the kinetics of onset and recovery from voltage-sensitive phosphatase-mediated reduction of PIP2 in wild-type AqKir and mutant channels with high affinity for PIP2. In addition, we are using our published homology model to examine the energetic effects of channel phosphorylation on PIP2 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 InformationSupported by NIH grant 2R15-GM096142 to Linda Boland.

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