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

The epithelial Ca(2+) channel TRPV5 constitutes the apical entry gate for Ca(2+) transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca(2+) 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 Ca(2+) concentration ([Ca(2+)](i)) using fura-2 analysis. Removal of amino acid His(712) elevated the [Ca(2+)](i), indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His(712) for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His(712) was confirmed by patch clamp analysis, which demonstrates increased Na(+) and Ca(2+) 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 [Ca(2+)](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 His(712) plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.