Molecular Determinants in TRPV5 Channel Assembly

Abstract

. Oocytes coexpressing wild-type TRPV5 and TRPV5⌬N or TRPV5⌬C showed virtually no wild-type TRPV5 expression on the plasma membrane, whereas co-expression of wild-type TRPV5 and TRPV5⌬N⌬C displayed normal channel surface expression. This indicates that TRPV5 trafficking toward the plasma membrane was disturbed by assembly with TRPV5⌬N or TRPV5⌬C but not with TRPV5⌬N⌬C. 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.TRPV5 and TRPV6 constitute the Ca 2ϩ influx pathway in 1,25-dihydroxyvitamin D 3 -responsive epithelia, including small intestine, kidney, and placenta, and play a vital role in the process of Ca 2ϩ (re)absorption (1-4). Both channels belong to a distinct subfamily (TRPV) within the superfamily of transient receptor potential channels (TRP). 1 The TRP family consists of a diverse group of non-voltage-gated cation channels, including TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies, which varies significantly in their selectivity and mode of activation (5). The understanding of the function, gating, regulation, and structure assembly of the TRP family is developing rapidly. Initially, it was demonstrated that the Drosophila TRP and TRPL members form heteromultimeric channels associated in a supramolecular signaling complex with specific receptors and regulators (6). Moreover, it has been identified that there are many channel compositions within the TRPC family, e.g. TRPC1/3, TRPC1/5, TRPC4/5, and TRPC3/ 6/7 (7-9). Within the TRPV family, the oligomeric structure of TRPV1 was studied by biochemical cross-linking, and the predominant existence of tetramers was suggested (10). More recently, it has been reported that TRPV5 and TRPV6 form homo-or heterotetramers in order to generate a pleiotropic set of functional channels with different Ca 2ϩ transport (11-13). TRPV5 and TRPV6 share 75% sequence homology at the amino acid level (11-13) and display several similar functional properties, including the permeation profile for monovalent and divalent cations (14) and regulation by calciotropic hormones (15-21). However, detailed sequence comparison of the N-and C-tails of the TRPV5 and TRPV6 channels reveals significant differences, which may account for the unique electrophysiological properties including differences of inactivation, kinetic properties, and affinity for the blocker ruthenium red between these two homologous channels (22).A considerable amount of information in channel subunit assembly has been accumulated by studies on voltage-gated K ϩ (K v ) channels that are structurally related to t...

The epithelial calcium channels TRPV5 and TRPV6: regulation and implications for disease

“…However, 80K-H was identified as a Ca 2+ sensor regulating TRPV5 activity at the plasma membrane rather than a role in routing towards the plasma membrane as discussed below. Importantly, these studies provide evidence that ankyrin repeats in the N-tail of TRPV5 and TRPV6 are essential for subunit assembly (Chang et al 2004;Erler et al 2004). In addition, it is likely that assembly also occurs in the N-tail and C-tail of TRPV5 and TRPV6, because they share more than 75% homology at the amino acid level, raising the possibility that both N-tail and C-tail assembled together in order to form functional heterotetrameric channel complexes of TRPV5 and TRPV6 (Hoenderop et al 2003b).…”

“…The membrane localization of 80K-H and TRPV5 was not altered, suggesting that 80K-H has a direct effect on TRPV5 activity (Gkika et al 2004). The 80K-H binding-site identified in the C-terminal tail of TRPV5 corresponds to the amino acid sequence MLERK, which is the same region where TRPV5 self-assembly can occur (Table 1; Chang et al 2004). Regulation of TRPV5 at this site could involve competition between the proteins, thereby differently regulating the channel.…”

“…The assembly domain in the N-tail of TRPV5 overlapped with the first ankyrin repeat. Deletion of these assembly domains abolished channel assembly and thereby the trafficking towards the plasma membrane and subsequent channel activity (Chang et al 2004). However, Erler et al indicated that tetramer formation is not essential for trafficking of TRPV6 to the cell surface since deletion of the N-terminal assembly domain did not affect plasma membrane staining (Erler et al 2004).…”

Structure-function analysis of TRPV channels

“…Garcia-Sanz et al (2004), Erler et al (2004) and Chang et al (2004) describe the identification of tetramerization domains of TRPV channels with partially divergent results. Garcia-Sanz et al identify a tetramerization domain in the C-terminus of TRPV1.…”

“…One asterisk indicates that tetramerization requires ANK 3 as the assembly anchor and ANK 5 as one potential stabilizing region (from Erler et al 2004). Two asterisks mark the region around ANK 1 as a critical assembly domain for TRPV5 identified by Chang et al (2004). Intracellular C-terminal regions, which are not implicated in TRPV6 assembly (Erler et al 2004), but appear important for TRPV5 assembly , have been omitted for clarity.…”