Expression of the polycystin-1 C-terminal cytoplasmic tail increases Cl- channel activity in Xenopus oocytes

Abstract

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 36Cl -, 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 36Cl- flux was not blocked by injected ethyleneglycol tetraacetate (EGTA) or by the cation channel inhibitor SKF96365, and was stimulated by the cation channel inhibitor La3+, suggesting that CD16.7-PKD1 (115-226)-associated cation conductance was not required for 36CI- 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 Ca2+-permeable cation channels. © 2005 by the International Society of Nephrology.