Abstract
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.
Highlights
Calcium (Ca2ϩ) plays a critical role in many cellular and physiological processes in the human body
TRPV5 belongs to the superfamily of TRP channels, which share the mutual composition of six transmembrane domains and permeability to cations [6]
A functional TRPV5 channel consists of four identical subunits, forming a single central pore located between transmembrane domains five and six [7]
Summary
Calcium (Ca2ϩ) plays a critical role in many cellular and physiological processes in the human body. Expression of the TRPV5 channel resulted in an elevated [Ca2ϩ]i compared with mock-transfected cells (Fig. 1, A and C). Fura-2 analysis of TRPV5– 698X-expressing HEK293 cells revealed an elevated basal [Ca2ϩ]i compared with TRPV5-WT.
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