Mitochondrial potassium sequestration and neuroprotection is mediated by connexin43

Reduction in the expression of the astrocyte gap junction protein connexin43 (Cx43) increases infarct volume following middle cerebral artery occlusion (MCAO). A potential mechanism for this effect is disruption of ion buffering. During ischemia, extracellular potassium concentration ([K⁺]₀) rises, leading to a variety of potentially detrimental effects on neuronal function. It is known that astrocytes contribute to the clearance of K⁺o to neighbouring cells through Cx43 based gap junctions, but mitochondria also contain Cx43 which could play a role in K⁺ uptake. Mitochondrial K⁺ sequestration was examined by loading astrocytes with the fluorescent K⁺ indicator PBFI. Release of K⁺ from mitochondria into the cytoplasm was examined after uncoupling the mitochondrial membrane potential with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Transient applications of elevated [K⁺]₀ led to increases in K⁺ within mitochondria, as assessed by increases in the magnitudes of cytoplasmic [K⁺] ([K⁺]i) transients evoked by brief exposures to CCCP. When mitochondrial K⁺ sequestration was impaired by prolonged application of CCCP, there was a robust increase in [K⁺]i upon exposure to elevated [K⁺]₀. Blockade of plasmalemmal K⁺ uptake routes by ouabain, Ba²⁺, or a cocktail of voltage-activated K⁺ channel inhibitors reduced K⁺ uptake into mitochondria. Reductions in mitochondrial K⁺ uptake occurred in the presence of the mitoKATP channel inhibitor 5-hydroxydecanoic acid. Rises in [K⁺]i evoked by brief applications of CCCP following exposure to high-[K⁺]₀ were also reduced by gap junction blockers and in astrocytes isolated from Cx43-null mice, suggesting that connexins also play a role in K⁺ uptake into astrocyte mitochondria. In a second study, the carboxy-terminal (CT) region of Cx43, a region important for channel activity, was explored in mutant mice expressing a truncated form of Cx43 (Cx43ΔCT mice). These mice exhibit enhanced cerebral injury following MCAO. In the peri-infarct region, astrogliosis was reduced and inflammatory cell invasion was increased. Cultured astrocytes from Cx43ΔCT mice were less coupled, and displayed alterations in channel gating, hemichannel activity, Ca²⁺ wave properties and showed impairment of mitochondrial K⁺ sequestration. These results suggest that astrocytic Cx43 contributes to the mitochondrial sequestration of K⁺ and that the CT region plays an important role in protection following stroke.Medicine, Faculty ofGraduat