GSK3β Transfers Cytoprotective Signaling through Connexin 43 onto Mitochondrial ATP-Sensitive K+ Channels

Abstract

Background: Ischemic preconditioning involves activation of signaling pathways, including activation of protein kinase C (PKC) and inhibition of glycogen synthase kinase 3beta (GSK3β). Opening of mitochondrial ATP-sensitive K+ (mitoKATP) channels has been proposed to play a critical role in ischemic preconditioning. Recently, we demonstrated that connexin 43 (Cx43) found in the inner mitochondrial membrane, is essential for cytoprotective signal transduction onto mitoKATP channels.Objectives: To dissect signal transduction targeted at mitoKATP channels and to possibly identify a functional link between mitoKATP channel activation, mitochondrial Cx43 and GSK3β activity.Methods: We used direct single-channel patch-clamp recordings of cardiac mitoplasts from C57/Bl6J mice and homozygous GSK3β-S9A mice with cardiac-specific expression of a constitutively active form of GSK3β, and performed Western blots and co-immunoprecipitation of isolated mitochondria.Results: MitoKATP channel activity was stimulated by the GSK3β small molecule inhibitor SB216763, which increased the open probability compared to control. The Cx43 inhibitor carbenoxolone and the Cx43 mimetic peptide 43GAP27 significantly reduced mitoKATP channel activation by SB216763, supporting the notion that GSK3 transfers cytoprotective signaling via Cx43 onto mitoKATP channels. In GSK3β-S9A mitoplasts mitoKATP currents could be recorded with similar single-channel properties as in wildtype mitochondria. Single-channel activation of mitoKATP by the PKC activator PMA was significantly attenuated in GSK3β-S9A mitoplasts. Western blot analysis revealed reduced phosphorylation of mitochondrial Cx43 at the PKC phosphorylation site Ser368 upon PMA application in mitochondria from GSK3β-S9A mice versus wildtype. Furthermore Immunoprecipitation of Cx43 from isolated mitochondria revealed a signal for GSK3β, while immunoprecipitation of GSK3β also showed a signal for Cx43, indicating an association of these proteins.Conclusions: PKC transmits a signal onto mitoKATP channels through GSK3β. This GSK3β signal transduction on mitoKATP channels is transferred via Cx43 within a multiprotein signaling module.