Identification and localization of BK-beta subunits in the distal nephron of the mouse kidney.
Large-conductance, Ca(2+)-activated K(+) channels (BK), comprised of pore-forming alpha- and accessory beta-subunits, secrete K(+) in the distal nephron under high-flow and high-K(+) diet conditions. BK channels are detected by electrophysiology in many nephron segments; however, the accessory beta-subunit associated with these channels has not been determined. We performed RT-PCR, Western blotting, and immunohistochemical staining to determine whether BK-beta1 is localized to the connecting tubule's principal-like cells (CNT) or intercalated cells (ICs), and whether BK-beta2-4 are present in other distal nephron segments. RT-PCR and Western blots revealed that the mouse kidney expresses BK-beta1, BK-beta2, and BK-beta4. Available antibodies in conjunction with BK-beta1(-/-) and BK-beta4(-/-) mice allowed the specific localization of BK-beta1 and BK-beta4 in distal nephron segments. Immunohistochemical staining showed that BK-beta1 is localized in the CNT but not ICs of the connecting tubule. The localization of BK-beta4 was discerned using an anti-BK-beta4 antibody on wild-type tissue and anti-GFP on GFP-replaced BK-beta4 mouse (BK-beta4(-/-)) tissue. Both antibodies (anti-BK-beta4 and anti-GFP) localized BK-beta4 to the thick ascending limb (TAL), distal convoluted tubule (DCT), and ICs of the distal nephron. It is concluded that BK-beta1 is narrowly confined to the apical membrane of CNTs in the mouse, whereas BK-beta4 is expressed in the TAL, DCT, and ICs.
Grimm
P Richard
PR
Dept. of Cellular and Integrative Physiology, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850, USA.
Foutz
Ruth M
RM
Brenner
Robert
R
Sansom
Steven C
SC
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007150:Immunohistochemistry; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000033:anatomy & histology / Q000378:metabolism*; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009399:Nephrons / Q000033:anatomy & histology / Q000378:metabolism*; D024681:Potassium Channels, Calcium-Activated / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
C494843:Kcnmb4 protein, mouse; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D024681:Potassium Channels, Calcium-Activated; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins