Voltage-Dependent Accessibility of Phosphorylation Sites at the Carboxy-Terminal Domain of Kv1.3 Channels Determines Kv1.3-Induced Cell Proliferation

Abstract

The plasticity of vascular smooth muscle cells that can switch from a contractile to a proliferative phenotype associates with several cardiovascular diseases. We have previously demonstrated that changes in the expression of voltage-dependent potassium (Kv) channels associated with this proliferative phenotype. These results were validated in transfected HEK293 cells: Kv1.5 decreased proliferation and Kv1.3 increased cell proliferation independently of ion flux (Cidad et al. ATVB 2012, 32:1299-307). Our data also suggested that the movement of the voltage-sensor of Kv1.3 channels could be coupled to signaling pathways leading to proliferation.Here we explore Kv1.3 regions involved in the signaling mechanism(s) associated with proliferation, by constructing chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels without consensus phosphorylation sites. These constructs were expressed as GFP- or cherry-fusion proteins, and we studied their functional expression, their effects on proliferation and their phosphorylation state in HEK cells combining immunocytochemical, electrophysiological and EdU incorporation studies.The molecular determinants of Kv1.3-induced proliferation are located at the carboxy-terminal domain. We identified two close residues (Y447 and S459) whose mutation abolished proliferation. Moreover, proliferation was increased with chimeric Kv1.5 mutants containing this segment (YS segment) but only when located at specific place within the channel molecule. Besides the selective MEK/ERK blocker PD98059 was able to inhibit Kv1.3-induced proliferation and tyrosine phosphorylation of the channel, having no effect on Y447A mutant channel. Finally, voltage-dependent transitions of Kv1.3 from close to open conformation increased pTyr labelling.We conclude that the signaling pathway linking Kv1.3 channels with proliferation involves voltage-induced conformational changes promoting tyrosine phosphorylation. Both phosphorylation and proliferation increase by facilitating the closed to open transition, being diminished in mutant channels that reside in the inactivated state. Supported by ISCIII-FEDER RD12/0042/0006, and BFU2013-45867-R grants.