The calcium-sensitive large-conductance potassium channel (BK/MAXI K) is present in the inner mitochondrial membrane of rat brain.
Large-conductance voltage- and calcium-sensitive channels are known to be expressed in the plasmalemma of central neurons; however, recent data suggest that large-conductance voltage- and calcium-sensitive channels may also be present in mitochondrial membranes. To determine the subcellular localization and distribution of large-conductance voltage- and calcium-sensitive channels, rat brain fractions obtained by Ficoll-sucrose density gradient centrifugation were examined by Western blotting, immunocytochemistry and immuno-gold electron microscopy. Immunoblotting studies demonstrated the presence of a consistent signal for the alpha subunit of the large-conductance voltage- and calcium-sensitive channel in the mitochondrial fraction. Double-labeling immunofluorescence also demonstrated that large-conductance voltage- and calcium-sensitive channels are present in mitochondria and co-localize with mitochondrial-specific proteins such as the translocase of the inner membrane 23, adenine nucleotide translocator, cytochrome c oxidase or complex IV-subunit 1 and the inner mitochondrial membrane protein but do not co-localize with calnexin, an endoplasmic reticulum marker. Western blotting of discrete subcellular fractions demonstrated that cytochrome c oxidase or complex IV-subunit 1 was only expressed in the mitochondrial fraction whereas actin, acetylcholinesterase, cadherins, calnexin, 58 kDa Golgi protein, lactate dehydrogenase and microtubule-associated protein 1 were not, demonstrating the purity of the mitochondrial fraction. Electron microscopic examination of the mitochondrial pellet demonstrated gold particle labeling within mitochondria, indicative of the presence of large-conductance voltage- and calcium-sensitive channels in the inner mitochondrial membrane. These studies provide concrete morphological evidence for the existence of large-conductance voltage- and calcium-sensitive channels in mitochondria: our findings corroborate the recent electrophysiological evidence of mitochondrial large-conductance voltage- and calcium-sensitive channels in glioma and cardiac cells.
Douglas
R M
RM
Department of Pediatrics (Division of Respiratory Medicine), Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Lai
J C K
JC
Bian
S
S
Cummins
L
L
Moczydlowski
E
E
Haddad
G G
GG
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure*; D002950:Citrate (si)-Synthase / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D008565:Membrane Proteins / Q000145:classification / Q000378:metabolism; D046529:Microscopy, Electron, Transmission / Q000379:methods; D051336:Mitochondrial Membranes / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure
D051036:Large-Conductance Calcium-Activated Potassium Channels; D008565:Membrane Proteins; D002950:Citrate (si)-Synthase