Grimm P Richard, Foutz Ruth M, Brenner Robert, Sansom Steven C
Dept. of Cellular and Integrative Physiology, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850, USA.
Am J Physiol Renal Physiol. 2007 Jul;293(1):F350-9. doi: 10.1152/ajprenal.00018.2007. Epub 2007 Apr 25.
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.
大电导钙激活钾通道（BK）由形成孔道的α亚基和辅助性β亚基组成，在高流量和高钾饮食条件下，在远端肾单位分泌钾离子。通过电生理学方法在许多肾单位节段检测到了BK通道；然而，与这些通道相关的辅助性β亚基尚未确定。我们进行了逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹和免疫组织化学染色，以确定BK-β1是否定位于连接小管的主细胞样细胞（CNT）或闰细胞（ICs），以及BK-β2 - 4是否存在于其他远端肾单位节段。RT-PCR和蛋白质免疫印迹显示，小鼠肾脏表达BK-β1、BK-β2和BK-β4。利用现有的抗体以及BK-β1基因敲除小鼠和BK-β4基因敲除小鼠，可将BK-β1和BK-β4在远端肾单位节段中特异性定位。免疫组织化学染色显示，BK-β1定位于连接小管的CNT而非ICs。使用抗BK-β4抗体对野生型组织以及抗绿色荧光蛋白（GFP）抗体对GFP替代的BK-β4小鼠（BK-β4基因敲除小鼠）组织进行检测，从而确定BK-β4的定位。两种抗体（抗BK-β4和抗GFP）均将BK-β4定位于远端肾单位的髓袢升支粗段（TAL）、远曲小管（DCT）和ICs。结论是，BK-β1在小鼠中狭窄地局限于CNT的顶膜，而BK-β4在TAL、DCT和ICs中表达。
