Lachheb Sahran, Cluzeaud Françoise, Bens Marcelle, Genete Mathieu, Hibino Hiroshi, Lourdel Stéphane, Kurachi Yoshihisa, Vandewalle Alain, Teulon Jacques, Paulais Marc
Université Pierre et Marie Curie, 75720 Paris Cedex 06, France.
Am J Physiol Renal Physiol. 2008 Jun;294(6):F1398-407. doi: 10.1152/ajprenal.00288.2007. Epub 2008 Mar 26.
K(+) channels in the basolateral membrane of mouse cortical collecting duct (CCD) principal cells were identified with patch-clamp technique, real-time PCR, and immunohistochemistry. In cell-attached membrane patches, three K(+) channels with conductances of approximately 75, 40, and 20 pS were observed, but the K(+) channel with the intermediate conductance (40 pS) predominated. In inside-out membrane patches exposed to an Mg(2+)-free medium, the current-voltage relationship of the intermediate-conductance channel was linear with a conductance of 38 pS. Addition of 1.3 mM internal Mg(2+) had no influence on the inward conductance (G(in) = 35 pS) but reduced outward conductance (G(out)) to 13 pS, yielding a G(in)/G(out) of 3.2. The polycation spermine (6 x 10(-7) M) reduced its activity on inside-out membrane patches by 50% at a clamp potential of 60 mV. Channel activity was also dependent on intracellular pH (pH(i)): a sigmoid relationship between pH(i) and channel normalized current (NP(o)) was observed with a pK of 7.24 and a Hill coefficient of 1.7. By real-time PCR on CCD extracts, inwardly rectifying K(+) (Kir)4.1 and Kir5.1, but not Kir4.2, mRNAs were detected. Kir4.1 and Kir5.1 proteins cellularly colocalized with aquaporin 2 (AQP2), a specific marker of CCD principal cells, while AQP2-negative cells (i.e., intercalated cells) showed no staining. Dietary K(+) had no influence on the properties of the intermediate-conductance channel, but a Na(+)-depleted diet increased its open probability by approximately 25%. We conclude that the Kir4.1/Kir5.1 channel is a major component of the K(+) conductance in the basolateral membrane of mouse CCD principal cells.
采用膜片钳技术、实时聚合酶链反应（PCR）和免疫组织化学方法，对小鼠皮质集合管（CCD）主细胞基底外侧膜中的钾离子（K(+)）通道进行了鉴定。在细胞贴附式膜片上，观察到三种电导分别约为75、40和20皮西门子（pS）的K(+)通道，但中间电导（40 pS）的K(+)通道占主导地位。在暴露于无镁（Mg(2+)）培养基的内向外膜片中，中间电导通道的电流-电压关系呈线性，电导为38 pS。加入1.3 mM的细胞内Mg(2+)对内向电导（G(in) = 35 pS）无影响，但将外向电导（G(out)）降低至13 pS，使G(in)/G(out)为3.2。在60 mV的钳制电位下，多价阳离子精胺（6×10(-7) M）使内向外膜片上的通道活性降低50%。通道活性还依赖于细胞内pH值（pH(i)）：观察到pH(i)与通道标准化电流（NP(o)）之间呈S形关系，pK为7.24，希尔系数为1.7。通过对CCD提取物进行实时PCR，检测到内向整流钾离子通道（Kir）4.1和Kir5.1的信使核糖核酸（mRNA），但未检测到Kir4.2的mRNA。Kir4.1和Kir5.1蛋白在细胞水平上与水通道蛋白2（AQP2）共定位，AQP2是CCD主细胞的特异性标志物，而AQP2阴性细胞（即闰细胞）无染色。饮食中的钾离子对中间电导通道的特性无影响，但低钠饮食使其开放概率增加约25%。我们得出结论，Kir4.1/Kir5.1通道是小鼠CCD主细胞基底外侧膜中钾离子电导的主要成分。
