Abstract 1595: Orientation of Connexin 43 in the Inner Mitochondrial Membrane and its Role in Respiration
At the sarcolemma, six connexin 43 (Cx43) proteins form hemichannels, which contribute to volume regulation. The open-probability of these hemichannels is regulated in part via the phosphorylation of Cx43 at residues predominantly present in the cytosolic carboxy-terminal domain. Cx43 is also present at the inner membrane of cardiomyocyte mitochondria. The aim of the present study was to analyze
the orientation of Cx43 at the inner mitochondrial membrane, and to study
whether or not the hemichannel blocker 18 α glycyrrhetinic acid (18αGA) influences mitochondrial respiration.
Mitochondria were isolated from rat left ventricular myocardium and were treated with different concentrations of proteinase K, which has a broad spectrum of actions on peptide bonds. Western blot analysis was performed for marker proteins of different mitochondrial compartments (ATP synthase α: inner mitochondrial membrane (IMM), subunit in the matrix; uncoupling protein 3 (UCP3): IMM, corresponding epitope in the intermembrane space; translocase of the outer membrane 20 (Tom20): outer mitochondrial membrane; manganese superoxide dismutase (MnSOD): matrix). At a proteinase K concentration of 50 μg/ml, the immunoreactivities of the analyzed proteins were almost completely lost compared to untreated mitochondria, which were set as 100%. The use of 5 μg/ml proteinase K resulted in similarly reduced immunoreactivities for Cx43 (19.4±5.8 %, n=6), UCP3 (23.0±4 %, n=7) and Tom20 (14.4±5.5 %, n=7), whereas the immunoreactivities of ATP synthase α were 49.1±6.4 % (n=7) and of MnSOD 79.9±17.4 % (n=6). Addition 10 μM and 100 μM, but not 1 μM of the hemichannel blocker 18αGA to rat ventricular mitochondria induced a loss of mitochondrial autofluorescence and membrane potential, indicating irreversible damage of the mitochondria. 1 μM 18αGA reduced the ADP-stimulated respiration to 77.3±4.2 % (n=13, p<0.05) compared to 102.1±8.1 % with DMSO (n=19). In conclusion, the C-terminus of mitochondrial Cx43 is directed towards the intermembrane space, where it can be the target of kinases known to interact with Cx43. The reduction of mitochondrial respiration by the hemichannel blocker 18αGA is in accordance with the existence of Cx43 hemichannels in the inner mitochondrial membrane.