Robins Gerard G, Sandle Geoffrey I
Leeds Institute of Biomedical and Clinical Sciences, St James's University Teaching Hospital, Level 7, Clinical Sciences Building, Beckett Street, Leeds, LS9 7TF, UK.
J Membr Biol. 2014 Aug;247(8):729-37. doi: 10.1007/s00232-014-9698-0. Epub 2014 Jul 1.
Increases in intracellular calcium (Ca(2+)) inhibit renal sodium (Na(+)) absorption in cortical collecting ducts, but the precise mechanism is unclear. We, therefore, studied the effects of raising intracellular Ca(2+) (using 10 µmol/L A23187, a Ca(2+) ionophore) on wild-type and Liddle-mutated human epithelial Na(+) channels (hENaC) expressed in Xenopus oocytes, using the dual-electrode voltage clamp technique. A23187 decreased amiloride-sensitive Na(+) current by 55% in oocytes expressing wild-type hENaC, an effect prevented by co-exposure to 50 μmol/L W-7 (to inhibit the Ca(2+)/calmodulin complex). By contrast, co-exposure to 50 μmol/L calphostin (to inhibit protein kinase C) or 5 μmol/L KN-62 (to inhibit Ca(2+)/calmodulin-dependent protein kinase II) had no effect on the decrease in amiloride-sensitive Na(+) current elicited by A23187 alone. Whereas A23187 reduced amiloride-sensitive Na(+) current in oocytes expressing wild-type hENaC, it had no similar effect in those expressing Liddle-mutated hENaCs, suggesting that the activity of individual Na(+) channels in situ was unchanged by the rise in intracellular Ca(2+). These data suggest that the A23187-induced rise in intracellular Ca(2+) inhibited wild-type hENaC through a W-7-sensitive mechanism, which likely reflected enhanced removal of Na(+) channels from the cell membrane by endocytosis. We, therefore, propose that Na(+) absorption in cortical collecting duct cells is inhibited by Ca(2+), possibly when complexed with calmodulin.
细胞内钙（Ca(2+)）水平升高会抑制皮质集合管对肾钠（Na(+)）的重吸收，但其确切机制尚不清楚。因此，我们使用双电极电压钳技术，研究了提高细胞内Ca(2+)水平（使用10 µmol/L A23187，一种Ca(2+)离子载体）对非洲爪蟾卵母细胞中表达的野生型和利德尔突变型人上皮钠通道（hENaC）的影响。A23187使表达野生型hENaC的卵母细胞中amiloride敏感的Na(+)电流降低了55%，而同时暴露于50 μmol/L W-7（以抑制Ca(2+)/钙调蛋白复合物）可阻止这种效应。相比之下，同时暴露于50 μmol/L钙泊三醇（以抑制蛋白激酶C）或5 μmol/L KN-62（以抑制Ca(2+)/钙调蛋白依赖性蛋白激酶II）对A23187单独引起的amiloride敏感的Na(+)电流降低没有影响。虽然A23187降低了表达野生型hENaC的卵母细胞中amiloride敏感的Na(+)电流，但在表达利德尔突变型hENaC的卵母细胞中没有类似作用，这表明细胞内Ca(2+)升高并未改变原位单个Na(+)通道的活性。这些数据表明，A23187诱导的细胞内Ca(2+)升高通过一种W-7敏感机制抑制野生型hENaC，这可能反映了通过内吞作用增强了Na(+)通道从细胞膜的移除。因此，我们提出皮质集合管细胞对Na(+)的重吸收受到Ca(2+)的抑制，可能是在与钙调蛋白结合时。
