Identification and localization of BK-β subunits in the distal nephron of the mouse kidney
Identification and localization of BK-β subunits in the distal nephron of the mouse kidney
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DOI：
10.1152/ajprenal.00018.2007
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发表时间：
2007-07-01
影响因子：
4.2
通讯作者：
Sansom, Steven C.
中科院分区：
文献类型：
--
作者：
Grimm, P. Richard;Foutz, Ruth M.;Sansom, Steven C.
大电导 Ca2+ 激活 K+ 通道 (BK) 由成孔 α 亚基和辅助 β 亚基组成，在高流量和高 K+ 饮食条件下在远端肾单位中分泌 K+。 BK 通道可通过电生理学在许多肾单位节段中检测到；然而，与这些通道相关的辅助β亚基尚未确定。我们进行了 RT-PCR、蛋白质印迹和免疫组织化学染色，以确定 BK-β 1 是否定位于连接小管的主样细胞 (CNT) 或闰细胞 (IC)，以及 BK-β 2-4 是否存在于其他远端肾单位段中。 RT-PCR 和蛋白质印迹显示小鼠肾脏表达 BK-β 1、BK-β 2 和 BK-β 4。与 BK-β 1(-/-) 和 BK-β 4(-/-) 小鼠结合的可用抗体允许 BK-β 1 和 BK-β 4 在远端肾单位段中特异性定位。免疫组织化学染色显示 BK-β1 位于 CNT 中，但不位于连接小管的 IC 中。使用野生型组织上的抗 BK-β 4 抗体和 GFP 替换的 BK-β 4 小鼠 (BK-β 4(-/-)) 组织上的抗 GFP 来辨别 BK-β 4 的定位。两种抗体（抗 BK-β 4 和抗 GFP）将 BK-β 4 定位于粗升肢 (TAL)、远端曲管 (DCT) 和远端肾单位的 IC。结论是，BK-β 1 狭窄地局限于小鼠 CNT 的顶膜，而 BK-β 4 在 TAL、DCT 和 IC 中表达。
Large-conductance, Ca2+-activated K+ channels (BK), comprised of poreforming 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-beta 1 is localized to the connecting tubule's principal-like cells (CNT) or intercalated cells (ICs), and whether BK-beta 2-4 are present in other distal nephron segments. RT-PCR and Western blots revealed that the mouse kidney expresses BK-beta 1, BK-beta 2, and BK-beta 4. Available antibodies in conjunction with BK-beta 1(-/-) and BK-beta 4(-/-) mice allowed the specific localization of BK-beta 1 and BK-beta 4 in distal nephron segments. Immunohistochemical staining showed that BK-beta 1 is localized in the CNT but not ICs of the connecting tubule. The localization of BK-beta 4 was discerned using an anti-BK-beta 4 antibody on wild-type tissue and anti-GFP on GFP-replaced BK-beta 4 mouse (BK-beta 4(-/-)) tissue. Both antibodies (anti-BK-beta 4 and anti-GFP) localized BK-beta 4 to the thick ascending limb (TAL), distal convoluted tubule (DCT), and ICs of the distal nephron. It is concluded that BK-beta 1 is narrowly confined to the apical membrane of CNTs in the mouse, whereas BK-beta 4 is expressed in the TAL, DCT, and ICs.