Molecular determinants in TRPV5 channel assembly

The epithelial Ca2+ channels TRPV5 and TRPV6 mediate the Ca2+ influx in 1,25-dihydroxyvitamin D-3-responsive epithelia and are therefore essential in the maintenance of the body Ca2+ balance. These Ca2+ channels assemble in (hetero) tetrameric channel complexes with different functional characteristics regarding Ca2+-dependent inactivation, ion selectivity, and pharmacological block. Glutathione S-transferase pull-downs and co-immunoprecipitations demonstrated an essential role of the intracellular N- and C-tails in TRPV5 channel assembly by physical interactions between N-N tails, C-C tails, and N-C-tails. Patch clamp analysis in human embryonic kidney (HEK293) cells and Ca-45(2+) uptake experiments in Xenopus laevis oocytes co-expressing TRPV5 wild-type and truncated proteins indicated that TRPV5DeltaN (deleted N-tail) and TRPV5DeltaC (deleted C-tail) decreased channel activity of wild-type TRPV5 in a dominant-negative manner, whereas TRPV5DeltaNDeltaC (deleted N-tail/C-tail) did not affect TRPV5 activity. Oocytes co-expressing wild-type TRPV5 and TRPV5DeltaN or TRPV5DeltaC showed virtually no wild-type TRPV5 expression on the plasma membrane, whereas co-expression of wild-type TRPV5 and TRPV5DeltaNDeltaC displayed normal channel surface expression. This indicates that TRPV5 trafficking toward the plasma membrane was disturbed by assembly with TRPV5DeltaN or TRPV5DeltaC but not with TRPV5DeltaNDeltaC. TRPV5 channel assembly signals were refined between amino acid positions 64-77 and 596-601 in the N- tail and C-tail, respectively. Pull-down assays and co-immunoprecipitations demonstrated that N- or C-tail mutants lacking these critical assembly domains were unable to interact with tails of TRPV5. In conclusion, two domains in the N- tail (residues 64-77) and C-tail (residues 596-601) of TRPV5 are important for channel subunit assembly, subsequent trafficking of the TRPV5 channel complex to the plasma membrane, and channel activity.