The identification of histidine 712 as a critical residue for constitutive TRPV5 internalization

Abstract

The epithelial Ca2+ channel TRPV5 constitutes the apical entry gate for Ca2+ transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca2+ reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca2+ concentration ([Ca2+] i) using fura-2 analysis. Removal of amino acid His712 elevated the [Ca2+]i, indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His712 for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His712 was confirmed by patch clamp analysis, which demonstrates increased Na+ and Ca2+ currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca 2+-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca2+]i. Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His712 plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.