Connexin43 in cardiomyocyte mitochondria contributes to mitochondrial potassium uptake.

Elisabet Miro-Casas, Marisol Ruiz-Meana, Esperanza Agullo, Sabine Stahlhofen, Antonio Rodríguez-Sinovas, Alberto Cabestrero, Inmaculada Jorge, Iratxe Torre, Jesus Vazquez, Kerstin Boengler, Rainer Schulz, Gerd Heusch, David Garcia-Dorado

Index: Cardiovasc. Res. 83 , 747-56, (2009)

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Abstract

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dye uptake and K(+) flux was assessed in wild-type mice using hemichannel blockers and Cx43KI32 mice in which Cx43 had been replaced by Cx32.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 18alpha-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.