Subject:
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Radboudumc 11: Renal disorders RIMLS: Radboud Institute for Molecular Life Sciences |
Abstract:
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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 beta-galactosidase (beta-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 beta-gal, showing that the stimulation is dependent on the presence of the TRPV5N-glycan. In addition, beta-gal was found to stimulate transcellular Ca(2+)transport in isolated mouse primary DCT2/CNT cells. beta-gal expression was detected in the apical membrane of the proximal tubules, and the protein was found in mouse urine. In summary, beta-gal is present in the pro-urine from where it is thought to stimulate TRPV5 activity.
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