Identity and function of a cardiac mitochondrial small conductance Ca²⁺-activated K⁺ channel splice variant.

We provide evidence for location and function of a small conductance, Ca²⁺-activated K⁺ (SK_Ca) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SK_Ca agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SK_Ca antagonists worsened IR injury. Intravenous infusion of a SK_Ca channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the N-terminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca²⁺ calmodulin binding domain is required for Ca²⁺ sensing to induce mK⁺ influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK⁺ influx was driven by adding CaCl₂. Moreover, there was a greater fall in membrane potential (ΔΨ_m), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SK_Ca channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SK_Ca channel opening remains unclear. SK_Ca channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa²⁺ overload.

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