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Mitochondrial potassium sequestration and neuroprotection is mediated by connexin43 Kozoriz, Michael Gregory


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.

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