Ca²⁺ transport via TRPV6 is regulated by rapid internalization of the channel

Amongst the superfamily of transient receptor potential (TRP) channels, TRPV5 and TRPV6 are specialized members that mediate Ca²⁺-selective transport across epithelial membranes. Intriguingly, fluorescent fusion proteins of TRPV5 or TRPV6 are hardly discernible within the plasma membrane of living cells. Instead, TRPV6 is mostly found in vesicular membrane compartments, indicating either a rapid degradation or cycling of channel-bearing vesicles between endomembrane compartments and the plasma membrane. In TRPV6-expressing cells, brefeldin A, a toxin that blocks the transit between the endoplasmic reticulum and the Golgi apparatus, caused a drop in [Ca²⁺]_i with a half time in the range of 0.5-1 h. Upon wash-out of the toxin, the [Ca²⁺]_i rose to a steady-state level within 2-3 h. Consistently, the synchronized forward trafficking of TRPV6_VL-eGFP after brefeldin A wash-out led to a visible accumulation of the protein within the plasma membrane, as shown by confocal and total internal reflection microscopy. Analysis of the internalization route and differentiation of vesicle populations provided evidence for a clathrin-dependent internalization pathway. Most TRPV6_VL-bearing vesicles co-stained with Rab5a, a marker protein for early endosomes. Fewer vesicles were co-localized with Rab7a (late endosomes) or with Rab11 (recycling endosomes). From these data, we propose that the lack of plasma membrane visibility of the channel results from a rapid internalization, which in addition to transcriptional regulation, adds a layer of functional channel regulation to modulate transepithelial Ca²⁺ transport.