Differential Regulation of PI(4,5)P2 Sensitivity of Kv7.2 and Kv7.3 Channels by Calmodulin.

Carolina Gomis-Perez,Pilar Areso,Maria V Soldovieri,Covadonga Malo,Alvaro Villarroel,Maurizio Taglialatela,Paolo Ambrosino

doi:10.3389/fnmol.2017.00117

Abstract

HIGHLIGHTS - Calmodulin-dependent Kv7.2 current density without the need of binding calcium.- Kv7.2 current density increase is accompanied with resistance to PI(4,5)P2 depletion.- Kv7.3 current density is insensitive to calmodulin elevation.- Kv7.3 is more sensitive to PI(4,5)P2 depletion in the presence of calmodulin.- Apo-calmodulin influences PI(4,5)P2 dependence in a subunit specific manner.The identification and understanding of critical factors regulating M-current functional density, whose main components are Kv7.2 and Kv7.3 subunits, has profound pathophysiological impact given the important role of the M-current in neuronal excitability control. We report the increase in current density of Kv7.2 channels by calmodulin (CaM) and by a mutant CaM unable to bind Ca2+ (CaM1234) revealing that this potentiation is calcium independent. Furthermore, after co-expressing a CaM binding protein (CaM sponge) to reduce CaM cellular availability, Kv7.2 current density was reduced. Current inhibition after transient depletion of the essential Kv7 co-factor phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) by activating Danio rerio voltage sensitive phosphatase (DrVSP) was blunted by co-expressing CaM1234 or the CaM sponge. In addition, CaM-dependent potentiation was occluded by tonic elevation of PI(4,5)P2 levels by PI(4)P5-kinase (PIP5K) expression. In contrast to the effect on homomeric Kv7.2 channels, CaM1234 failed to potentiate heteromeric Kv7.2/3 or homomeric Kv7.3 channels. Sensitivity to PI(4,5)P2 depletion of Kv7.2/3 channels was increased after expression of CaM1234 or the CaM sponge, while that of homomeric Kv7.3 was unaltered. Altogether, the data reveal that apo-CaM influences PI(4,5)P2 dependence of Kv7.2, Kv7.2/3, and of Kv7.3 channels in a subunit specific manner.

Highlights

Calmodulin (CaM) is a small acidic protein of 148 residues that confers Ca2+ sensitivity to a large variety of intracellular proteins, Ca2+ independent roles for CaM are emerging (Zhang et al, 1995; Jurado et al, 1999; Villarroel et al, 2014)
We observed that CaM co-expression potentiated the maximal current density of Kv7.2 isoform 3 channels expressed in HEK293T cells (Figure 1) and in CHO cells (Ambrosino et al, 2015); similar results were achieved when the human isoform 4 of Kv7.2 was expressed
Based on the similarity of the effects observed with CaM and CaM1234 and the changes in current density under conditions that result in tonic elevation or transient reduction of PI(4,5)P2 levels, we propose that CaM increases the efficacy of PI(4,5)P2 action on Kv7.2 channels without the need of binding Ca2+

Summary

Introduction

Calmodulin (CaM) is a small acidic protein of 148 residues that confers Ca2+ sensitivity to a large variety of intracellular proteins, Ca2+ independent roles for CaM are emerging (Zhang et al, 1995; Jurado et al, 1999; Villarroel et al, 2014). The importance of CaM in the central nervous system (CNS) function is reflected in the Differential Apocalmodulin-PIP2 Regulation of Kv7 Channels extraordinary high concentration of this protein (from 10 to 100 μM) in different brain areas (Xia and Storm, 2005). CaM mediates Ca2+-dependent M-current regulation by bradykinin activation in sympathetic neurons (Gamper and Shapiro, 2003; Greene and Hoshi, 2017) and influences the action of the essential co-factor phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (Kosenko et al, 2012; Kang et al, 2014; Alberdi et al, 2015). The reduction in free CaM in hippocampal neurons decreases M-current density and increases neuronal excitability (Shahidullah et al, 2005; Zhou et al, 2016), whereas elevation of CaM levels in sympathetic neurons prevents bradykinin M-current suppression (Zaika et al, 2007)

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