Identification and localization of BK-beta subunits in the distal nephron of the mouse kidney

Abstract

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.