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

Kidney Int. 2005 Aug;68(2):632-41. doi: 10.1111/j.1523-1755.2005.00441.x.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Calcium / metabolism
  • Cations / metabolism
  • Cell Membrane / metabolism
  • Chloride Channels / metabolism*
  • Chlorides / metabolism
  • Cytoplasm / metabolism
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mutation
  • Oocytes / cytology
  • Oocytes / physiology
  • Patch-Clamp Techniques
  • Phosphorylation
  • Polycystic Kidney, Autosomal Dominant / genetics
  • Polycystic Kidney, Autosomal Dominant / metabolism*
  • Polycystic Kidney, Autosomal Dominant / physiopathology
  • Protein Structure, Tertiary
  • Proteins / chemistry
  • Proteins / genetics*
  • Proteins / metabolism*
  • RNA, Complementary
  • Radioisotopes
  • Serine / metabolism
  • TRPP Cation Channels
  • Tyrosine / metabolism
  • Xenopus

Substances

  • Cations
  • Chloride Channels
  • Chlorides
  • Membrane Proteins
  • Proteins
  • RNA, Complementary
  • Radioisotopes
  • TRPP Cation Channels
  • polycystic kidney disease 1 protein
  • polycystic kidney disease 2 protein
  • Tyrosine
  • Serine
  • Calcium