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

Abstract

Large-conductance, Ca2+-activated K+ channels (BK), comprised of pore-forming α- and accessory β-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 β-subunit associated with these channels has not been determined. We performed RT-PCR, Western blotting, and immunohistochemical staining to determine whether BK-β1 is localized to the connecting tubule's principal-like cells (CNT) or intercalated cells (ICs), and whether BK-β2-4 are present in other distal nephron segments. RT-PCR and Western blots revealed that the mouse kidney expresses BK-β1, BK-β2, and BK-β4. Available antibodies in conjunction with BK-β1−/− and BK-β4−/− mice allowed the specific localization of BK-β1 and BK-β4 in distal nephron segments. Immunohistochemical staining showed that BK-β1 is localized in the CNT but not ICs of the connecting tubule. The localization of BK-β4 was discerned using an anti-BK-β4 antibody on wild-type tissue and anti-GFP on GFP-replaced BK-β4 mouse (BK-β4−/−) tissue. Both antibodies (anti-BK-β4 and anti-GFP) localized BK-β4 to the thick ascending limb (TAL), distal convoluted tubule (DCT), and ICs of the distal nephron. It is concluded that BK-β1 is narrowly confined to the apical membrane of CNTs in the mouse, whereas BK-β4 is expressed in the TAL, DCT, and ICs.