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+(SKCa) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SKCaagonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SKCaantagonists worsened IR injury. Intravenous infusion of a SKCachannel 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 (ΔΨm), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SKCachannel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SKCachannel opening remains unclear. SKCachannels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa2+overload.
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, USA.
MeiYing
M
Yang
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, USA; Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI, USA.
Amadou K S
AKS
Camara
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, USA.
Mohammed
M
Aldakkak
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, USA; Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA; Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI, USA.
Wai-Meng
WM
Kwok
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, USA; Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA; Department of Biomedical Engineering, Medical College of Wisconsin and Marquette University, Milwaukee, WI, USA; Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI, USA; Research Service, Zablocki VA Medical Center, Milwaukee, WI, USA. Electronic address: dfstowe@mcw.edu.
David F
DF
Stowe
D015057:1-Naphthylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology / Q000627:therapeutic use; D002122:Calcium Chloride / Q000494:pharmacology; D015687:Cell Hypoxia; D002460:Cell Line; D006168:Guinea Pigs; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008929:Mitochondria, Heart / Q000737:chemistry / Q000378:metabolism*; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000517:prevention & control; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D020033:Protein Isoforms / Q000502:physiology; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000819:agonists / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000502:physiology*
C515166:(R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine; C507351:5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; D001562:Benzimidazoles; C406328:KCNN3 protein, human; C496901:Kcnn3 protein, rat; D026902:Potassium Channel Blockers; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D051640:Small-Conductance Calcium-Activated Potassium Channels; D015057:1-Naphthylamine; D002122:Calcium Chloride; D011188:Potassium
Cardiac mitochondria; Cell signaling; Inner mitochondrial membrane; Ischemia reperfusion injury; Oxidant stress; Small conductance Ca(2+)-sensitive K(+) channel