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Glycogen synthase kinase 3? transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K+ channels.

ABSTRACT: Despite compelling evidence supporting key roles for glycogen synthase kinase 3? (GSK3?), mitochondrial adenosine triphosphate-sensitive K(+) (mitoK(ATP)) channels, and mitochondrial connexin 43 (Cx43) in cytoprotection, it is not clear how these signaling modules are linked mechanistically. By patch-clamping the inner membrane of murine cardiac mitochondria, we found that inhibition of GSK3? activated mitoK(ATP). PKC activation and protein phosphatase 2a inhibition increased the open probability of mitoK(ATP) channels through GSK3?, and this GSK3? signal was mediated via mitochondrial Cx43. Moreover, (i) PKC-induced phosphorylation of mitochondrial Cx43 was reduced in GSK3?-S9A mice; (ii) Cx43 and GSK3? proteins associated in mitochondria; and (iii) SB216763-mediated reduction of infarct size was abolished in Cx43 KO mice in vivo, consistent with the notion that GSK3? inhibition results in mitoK(ATP) opening via mitochondrial Cx43. We therefore directly targeted mitochondrial Cx43 by the Cx43 C-terminal binding peptide RRNYRRNY for cardioprotection, circumventing further upstream pathways. RRNYRRNY activated mitoK(ATP) channels via Cx43. We directly recorded mitochondrial Cx43 channels that were activated by RRNYRRNY and blocked by the Cx43 mimetic peptide (43)GAP27. RRNYRRNY rendered isolated cardiomyocytes in vitro and the heart in vivo resistant to ischemia/reperfusion injury, indicating that mitochondrial Cx43- and/or mitoK(ATP)-mediated reduction of infarct size was not undermined by RRNYRRNY-related opening of sarcolemmal Cx43 channels. Our results demonstrate that GSK3? transfers cytoprotective signaling through mitochondrial Cx43 onto mitoK(ATP) channels and that Cx43 functions as a channel in mitochondria, being an attractive target for drug treatment against cardiomyocyte injury.

SUBMITTER: Rottlaender D

PROVIDER: S-EPMC3277155 | biostudies-other | 2012 Jan

REPOSITORIES: biostudies-other

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Glycogen synthase kinase 3β transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K+ channels.

Rottlaender Dennis D Boengler Kerstin K Wolny Martin M Schwaiger Astrid A Motloch Lukas J LJ Ovize Michel M Schulz Rainer R Heusch Gerd G Hoppe Uta C UC

Proceedings of the National Academy of Sciences of the United States of America 20120111 5

Despite compelling evidence supporting key roles for glycogen synthase kinase 3β (GSK3β), mitochondrial adenosine triphosphate-sensitive K(+) (mitoK(ATP)) channels, and mitochondrial connexin 43 (Cx43) in cytoprotection, it is not clear how these signaling modules are linked mechanistically. By patch-clamping the inner membrane of murine cardiac mitochondria, we found that inhibition of GSK3β activated mitoK(ATP). PKC activation and protein phosphatase 2a inhibition increased the open probabilit ...[more]

PMID: 22238425

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Project description:Glycolytic cancer cells produce large quantities of lactate that must be removed to sustain metabolism in the absence of oxidative phosphorylation. The only venting mechanism described to do this at an adequate rate is H+-coupled lactate efflux on monocarboxylate transporters (MCTs). Outward MCT activity is, however, thermodynamically inhibited by extracellular acidity, a hallmark of solid tumours. This inhibition would feedback unfavourably on metabolism and growth, raising the possibility that other venting mechanisms become important in under-perfused tumours. We investigated connexin-assembled gap junctions as an alternative route for discharging lactate from pancreatic ductal adenocarcinoma (PDAC) cells. Diffusive coupling (calcein transmission) in vitro was strong between Colo357 cells, weaker yet hypoxia-inducible between BxPC3 cells, and very low between MiaPaCa2 cells. Coupling correlated with levels of connexin-43 (Cx43), a protein previously linked to late-stage disease. Evoked lactate dynamics, imaged in Colo357 spheroids using cytoplasmic pH as a read-out, indicated that lactate anions permeate gap junctions faster than highly-buffered H+ ions. At steady-state, junctional transmission of lactate (a chemical base) from the spheroid core had an alkalinizing effect on the rim, producing therein a milieu conducive for growth. Metabolite assays demonstrated that Cx43 knockdown increased cytoplasmic lactate retention in Colo357 spheroids (diameter ~150 μm). MiaPaCa2 cells, which are Cx43 negative in monolayer culture, showed markedly increased Cx43 immunoreactivity at areas of invasion in orthotopic xenograft mouse models. These tissue areas were associated with chronic extracellular acidosis (as indicated by the marker LAMP2 near/at the plasmalemma), which can explain the advantage of junctional transmission over MCT in vivo. We propose that Cx43 channels are important conduits for dissipating lactate anions from glycolytic PDAC cells. Furthermore, lactate entry into the better-perfused recipient cells has a favourable alkalinizing effect and supplies substrate for oxidative phosphorylation. Cx43 is thus a novel target for influencing metabolite handling in junctionally-coupled tumours.

Dataset Information

Glycogen synthase kinase 3? transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K+ channels.

Publications

Glycogen synthase kinase 3β transfers cytoprotective signaling through connexin 43 onto mitochondrial ATP-sensitive K+ channels.

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