Connexin43 in cardiomyocyte mitochondria contributes to mitochondrial potassium uptake

Connexin43 is present at the inner membrane of cardiomyocyte mitochondria (mCx43), but its function remains unknown. In this study we verified the presence of mCx43 by a mass spectrometry-based proteomic approach in purified mitochondrial preparations from mouse myocardium and determined by western blot analysis that the C-terminus of mCx43 is oriented towards the intermembrane space. Cross-linking studies with dimethylsuberimidate indicated the presence of Cx43 hexamers in mitochondrial membranes. The contribution of Cx43 to both mitochondrial dye uptake and K+ flux was assessed in wild-type mice using hemichannel blockers and Cx43KI32 mice in which Cx43 had been replaced by Cx32. Uptake of the Cx43 hemichannel-permeant dye Lucifer Yellow was reduced in mitochondria from wild-type mice by two hemichannel blockers (carbenoxolone and heptanol) and in Cx43KI32 compared with wild-type mice. Mitochondrial K+ influx (PBFI fluorescence) was decreased in digitonin-permeabilized cardiomyocytes from Cx32 mutants compared with wild-type mice, and addition of the Cx43 hemichannel blocker 18 alpha-glycyrrhetinic acid had an inhibitory effect on mitochondrial K+ influx in wild-type cardiomyocytes, but not in cardiomyocytes from Cx32 mutants. These results indicate that mCx43 contributes to mitochondrial K+ flux in cardiomyocytes, potentially by forming hemichannel-like structures.