The calcium-sensitive large-conductance potassium channel (BK/MAXI K) is present in the inner mitochondrial membrane of rat brain

Neuroscience. 2006;139(4):1249-61. doi: 10.1016/j.neuroscience.2006.01.061. Epub 2006 Mar 29.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Blotting, Western / methods
  • Brain / metabolism*
  • Brain / ultrastructure*
  • Citrate (si)-Synthase / metabolism
  • Immunohistochemistry / methods
  • Large-Conductance Calcium-Activated Potassium Channels / metabolism*
  • Membrane Proteins / classification
  • Membrane Proteins / metabolism
  • Microscopy, Electron, Transmission / methods
  • Mitochondrial Membranes / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Subcellular Fractions / metabolism
  • Subcellular Fractions / ultrastructure

Substances

  • Large-Conductance Calcium-Activated Potassium Channels
  • Membrane Proteins
  • Citrate (si)-Synthase