Urinary β-galactosidase Stimulates Ca2+ Transport by Stabilizing TRPV5 at the Plasma Membrane

Glycobiology. 2016 May;26(5):472-81. doi: 10.1093/glycob/cwv172. Epub 2016 Jan 7.

Abstract

Transcellular Ca(2+)transport in the late distal convoluted tubule and connecting tubule (DCT2/CNT) of the kidney is a finely controlled process mediated by the transient receptor potential vanilloid type 5 (TRPV5) channel. A complex-type-N-glycan bound at the extracellular residue Asn358 of TRPV5 through post-translational glycosylation has been postulated to regulate the activity of TRPV5 channels. Using in vitro Ca(2+)transport assays, immunoblot analysis, immunohistochemistry, patch clamp electrophysiology and total internal reflection fluorescence microscopy, it is demonstrated that the glycosidase β-galactosidase (β-gal), an enzyme that hydrolyzes galactose, stimulates TRPV5 channel activity. However, the activity of the non-glycosylated TRPV(N358Q)mutant was not altered in the presence of β-gal, showing that the stimulation is dependent on the presence of the TRPV5N-glycan. In addition, β-gal was found to stimulate transcellular Ca(2+)transport in isolated mouse primary DCT2/CNT cells. β-gal expression was detected in the apical membrane of the proximal tubules, and the protein was found in mouse urine. In summary, β-gal is present in the pro-urine from where it is thought to stimulate TRPV5 activity.

Keywords: TRPV5; Urine; calcium channel; glycoprotein; post-transcriptional regulation; β-galactosidase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Calcium Channels / genetics
  • Calcium Channels / metabolism*
  • Cell Membrane / genetics
  • Cell Membrane / metabolism*
  • Humans
  • Ion Transport / genetics
  • Kidney Tubules, Distal / metabolism*
  • Mice
  • Mice, Transgenic
  • Protein Stability
  • TRPV Cation Channels / genetics
  • TRPV Cation Channels / metabolism*
  • beta-Galactosidase / genetics
  • beta-Galactosidase / metabolism*
  • beta-Galactosidase / urine

Substances

  • Calcium Channels
  • TRPV Cation Channels
  • TRPV5 protein, human
  • Trpv5 protein, mouse
  • beta-Galactosidase
  • Calcium