Phosphorylation in the C-terminus of the rat connexin46 (rCx46) and regulation of the conducting activity of the formed connexons

To analyse the role of PKC-dependent phosphorylation in the C-terminus of rCx46 in regulation of rCx46 connexons, truncated mutants rCx46_45.3 and rCx46_44.2 which end before and after PKC-dependent phosphorylation sites respectively were generated. Both rCx46_45.3 and rCx46_44.2 formed connexons in Xenopus oocytes similar to Cx46_wt-connexons. They were activated by depolarisation above −40 mV and at voltages above 50 mV, inactivation was spontaneously observed or induced by PKC activator TPA, suggesting that inactivation does not require PKC-dependent phosphorylation in the C-terminus. Three casein-kinase-II-(CKII)-dependent phosphorylation sites were also identified. rCx46_37.7 and rCx46_28.2 respectively without two or all of these sites were generated. rCx46_37.7-connexons were similar to rCx46_wt-connexons. rCx46_28.2-connexons comparable to rCx46_wt-connexons were observed after injection of 50 times more rCx46_28.2-mRNA (25 ng per oocyte). CKII-blocker inhibited depolarisation-evoked currents in oocytes injected with 0.5 ng per oocyte rCx46_37.7-mRNA or rCx46_wt-mRNA. Injection of 25 ng per oocyte rCx46_37.7-mRNA or rCx46_wt-mRNA overcame the effect of CKII-inhibitor. We propose that CKII-dependent phosphorylation in the C-terminus accelerates formation of rCx46-connexons.