Chernova Marina N, Vandorpe David H, Clark Jeffrey S, Alper Seth L
Molecular and Vascular Medicine Unit and Renal Division, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
Kidney Int. 2005 Aug;68(2):632-41. doi: 10.1111/j.1523-1755.2005.00441.x.
Expression of the polycystin-1 C-terminal cytoplasmic tail increases Cl(-) channel activity in Xenopus oocytes. Background. Cyst expansion in autosomal-dominant polycystic kidney disease (ADPKD) is characterized by active Cl(-) secretion in excess of solute reabsorption. However, the connections between elevated epithelial Cl(-) secretion and loss-of-function or dysregulation of either ADPKD gene polycystin-1 (PC1) or polycystin-2 (PC2) remain little understood. Methods. Cl(-) transport in Xenopus oocytes expressing the CD16.7-PKD1 (115-226) fusion protein containing the final 112 amino acid (aa) of the PC1 C-terminal cytoplasmic tail, or in oocytes expressing related PC1 fusion protein mutants, was studied by isotopic flux, two-electrode voltage clamp, and outside-out patch clamp recording. Results. Expression in oocytes of CD16.7-PKD1 (115-226) increased rates of both influx and efflux of (36)Cl(-), whereas CD16.7-PKD1 (1-92) containing the initial 92 aa of the PC1 C-terminal cytoplasmic tail was inactive. The increased Cl(-) transport resembled CD16.7-PKD1 (115-226)-stimulated cation current in its sensitivity to ADPKD-associated missense mutations, to mutations in phosphorylation sites, and to mutations within or encroaching upon the PC1 coiled-coil domain, as well as in its partial suppression by coexpressed PC2. The NS3623- and 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive (36)Cl(-) flux was not blocked by injected ethyleneglycol tetraacetate (EGTA) or by the cation channel inhibitor SKF96365, and was stimulated by the cation channel inhibitor La(3+), suggesting that CD16.7-PKD1 (115-226)-associated cation conductance was not required for (36)CI(-) flux activation. Outside-out patches from oocytes expressing CD16.7-PKD1 (115-226) also exhibited increased NS3623-sensitive Cl(-) current. Conclusion. These data show that CD16.7-PKD1 (115-226) activates Cl(-) channels in the Xenopus oocyte plasma membrane in parallel with, but not secondary to, activation of Ca(2+)-permeable cation channels.
多囊蛋白-1 C末端胞质尾的表达增加非洲爪蟾卵母细胞中的Cl(-)通道活性。背景。常染色体显性多囊肾病（ADPKD）中的囊肿扩张特征为Cl(-)的主动分泌超过溶质重吸收。然而，上皮细胞Cl(-)分泌增加与ADPKD基因多囊蛋白-1（PC1）或多囊蛋白-2（PC2）功能丧失或调节异常之间的联系仍知之甚少。方法。通过同位素通量、双电极电压钳和外向膜片钳记录，研究了表达包含PC1 C末端胞质尾最后112个氨基酸（aa）的CD16.7-PKD1（115-226）融合蛋白的非洲爪蟾卵母细胞中的Cl(-)转运，或表达相关PC1融合蛋白突变体的卵母细胞中的Cl(-)转运。结果。CD16.7-PKD1（115-226）在卵母细胞中的表达增加了(36)Cl(-)的流入和流出速率，而包含PC1 C末端胞质尾最初92个aa的CD16.7-PKD1（1-92）则无活性。增加的Cl(-)转运在对ADPKD相关错义突变、磷酸化位点突变以及PC1卷曲螺旋结构域内或其附近突变的敏感性方面，以及在共表达PC2对其的部分抑制方面，类似于CD16.7-PKD1（115-226）刺激的阳离子电流。NS3623和4,4'-二异硫氰酸根合芪-2,2'-二磺酸（DIDS）敏感的(36)Cl(-)通量未被注射的乙二醇四乙酸（EGTA）或阳离子通道抑制剂SKF96365阻断，且被阳离子通道抑制剂La(3+)刺激，这表明CD16.7-PKD1（115-226）相关的阳离子电导对于(36)Cl(-)通量激活并非必需。表达CD16.7-PKD1（115-226）的卵母细胞的外向膜片也表现出NS3623敏感的Cl(-)电流增加。结论。这些数据表明，CD16.7-PKD1（115-226）与Ca(2+)可渗透阳离子通道的激活并行但非继发于此，激活了非洲爪蟾卵母细胞质膜中的Cl(-)通道。
