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This Article Full Text Full Text (PDF) Supplemental data All Versions of this Article: 577/1/17    most recent jphysiol.2006.118299v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ljubkovic, M. Articles by Bienengraeber, M. Search for Related Content PubMed PubMed Citation Articles by Ljubkovic, M. Articles by Bienengraeber, M. Related Collections Cellular

Department of
¹ Anaesthesiology
² Physiology
³ Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
⁴ Department of Anaesthesiology and Intensive Care Medicine, Medical University of Graz, Austria

Selective K⁺ transport in the inner mitochondrial membrane has been attributed to at least three different channel types: ATP-sensitive, Ca²⁺-regulated and voltage-dependent K⁺ channels. Studies utilizing their selective modulators have suggested that an increased activity of these channels plays an important role in the cellular protection from metabolic stress. However, direct evidence for this effect is largely absent, and recent findings on the lack of specificity for several channel openers and blockers have questioned the actual contribution of the mitochondrial K⁺ channels in the preservation of cellular viability. In order to directly investigate the role of enhanced mitochondrial K⁺ uptake in cellular protection, we selectively expressed the inward rectifying K⁺ channel Kir6.2 in the mitochondria of HEK293 and HL-1 cells. Targeted Kir6.2 expression was achieved by cloning the Kir6.2 gene in pCMV/mito/GFP vector and the proper trafficking to mitochondria was confirmed by colocalization studies and Western blot. An increased K⁺ influx to mitochondria overexpressing Kir6.2, as evidenced by using the K⁺-sensitive PBFI AM fluorescent dye, substantially improved the cellular viability after hypoxic stress, which was assessed by lactate dehydrogenase (LDH) release. In parallel, monitoring of mitochondrial Ca²⁺ during stress, via the specific indicator rhod-2, revealed a significant attenuation of Ca²⁺ accumulation in mitochondria overexpressing K⁺ channels. This effect was abolished in mitochondria expressing an inactive mutant of Kir6.2. Mitochondria expressing Kir6.2 K⁺ channel also exhibited a significant degree of depolarization that became even more pronounced during the stress. In conclusion, this study provides the first non-pharmacological evidence that an increased K⁺ influx to mitochondria protects against hypoxic stress by preventing detrimental effects of Ca²⁺ overload.

(Received 29 July 2006; accepted after revision 31 August 2006; first published online 7 September 2006)
Corresponding author M. Bienengraeber: Department of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. Email: mbieneng{at}mcw.edu

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