Molecular assembly and subcellular distribution of ATP-sensitive potassium channel proteins in rat hearts.
Cardiac ATP-sensitive K(+) (K(ATP)) channels are proposed to contribute to cardio-protection and ischemic preconditioning. Although mRNAs for all subunits of K(ATP) channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti-peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin-solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2-SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2)(4)-(SUR2A)(4), suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac K(ATP) channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning.
Kuniyasu
Akihiko
A
Department of Molecular Cell Function, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Ohe-Honmachi, 862-0973 Kumamoto, Japan.
Kaneko
Kazuyoshi
K
Kawahara
Kohichi
K
Nakayama
Hitoshi
H
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002499:Centrifugation, Density Gradient; D008861:Microsomes / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism; D011233:Precipitin Tests; D021122:Protein Subunits; D051381:Rats; D011955:Receptors, Drug / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D000255:Adenosine Triphosphate