Cx45 regulation by kinases and impact of expression in heart failure

Abstract

Phosphorylation plays a crucial role in connexin regulation by modulating gap junction intercellular communication (GJIC), localization, stability, and interactions with signaling proteins. Few kinases are known to phosphorylate Cx45, and their target residues remain unknown. A phosphorylation screen identified several Cx45-targeting kinases activated in heart disease, among which c-Src was found by mass spectroscopy to phosphorylate residues Y324 and Y356. Unlike Cx43, c-Src phosphorylation of Cx45 did not impair GJIC, alter junctional localization, or affect interactions with cytoskeletal proteins β-tubulin, Drebrin, and ZO-1. In LA-25 cells where Cx43 is internalized after temperature sensitive activation of v-Src, expression of Cx45 unexpectedly maintained Cx43 at the plasma membrane. Phospho-specific antibodies helped identify that while Cx43 had a tyrosine phosphorylation pattern favoring turnover, the serine phosphorylation pattern was conducive for GJIC. Furthermore, in a rat model of heart failure, Cx45 was expressed in the ventricle and co-localized with Cx43, leading to altered dye coupling indicative of a shift toward Cx45-like channel permeability. Altogether, our data suggests that in heart failure, c-Src activation on its own would not have an adverse effect on Cx45 function and that aberrant Cx45 expression helps Cx43 transport to and maintain at the intercalated disc. Yet the dominant effect of Cx45 in heteromeric channels could ultimately make Cx45 a key driver of cardiac dysfunction. Finally, the observation that Cx45-mediated coupling remains functional even in the same pathological environment where Cx43-mediated communication is inhibited suggests that kinase regulation of connexins is isoform-specific and not universally predictable.