Functional expression of the epithelial Ca2+ channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex

Abstract

TRPV5 and TRPV6 constitute the Ca 2+ in¯ux pathway in a variety of epithelial cells. Here, we identi®ed S100A10 as the ®rst auxiliary protein of these epithelial Ca 2+ channels using yeast two-hybrid and GST pull-down assays. This S100 protein forms a heterotetrameric complex with annexin 2 and associates speci®cally with the conserved sequence VATTV located in the C-terminal tail of TRPV5 and TRPV6. Of these ®ve amino acids, the ®rst threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity. Using GST pull-down and co-immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5±S100A10 complex. Furthermore, the S100A10±annexin 2 pair colocalizes with the Ca 2+ channels in TRPV5-expressing renal tubules and TRPV6-expressing duodenal cells. Importantly, downregulation of annexin 2 using annexin 2-speci®c small interfering RNA inhibited TRPV5 and TRPV6-mediated currents in transfected HEK293 cells. In conclusion, the S100A10±annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.

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

“…Recently, we demonstrated a regulatory role for the S100A10-annexin 2 complex in the trafficking of TRPV5 and TRPV6 (van de Graaf et al 2003). S100A10 and annexin 2 were present along the apical membrane of TRPV5-expressing tubules and along the brush-border membrane of duodenum, which is in agreement with the TRPV6 localization.…”

“…Moreover, disruption of the S100A10-binding motif in TRPV5 or TRPV6 (Table 1) prevented the facilitation of Ca 2+ inward currents, which was accompanied by a major disturbance in their subcellular locali-zation (van de Graaf et al 2003). Importantly, downregulation of annexin 2 using annexin 2-specific siRNAs significantly inhibited the currents through TRPV5, indicating that annexin 2 in conjunction with S100A10 is crucial for TRPV5 activity (van de Graaf et al 2003). These results clearly show that the S100A10-annexin 2 complex is a significant component for the trafficking of the epithelial Ca 2+ channels towards the plasma membrane and, therefore, the functionality of these channels.…”

“…S100A10 and annexin 2 were present along the apical membrane of TRPV5-expressing tubules and along the brush-border membrane of duodenum, which is in agreement with the TRPV6 localization. Moreover, disruption of the S100A10-binding motif in TRPV5 or TRPV6 (Table 1) prevented the facilitation of Ca 2+ inward currents, which was accompanied by a major disturbance in their subcellular locali-zation (van de Graaf et al 2003). Importantly, downregulation of annexin 2 using annexin 2-specific siRNAs significantly inhibited the currents through TRPV5, indicating that annexin 2 in conjunction with S100A10 is crucial for TRPV5 activity (van de Graaf et al 2003).…”

Structure-function analysis of TRPV channels

“…Both TRPV5 and TRPV6 contain a conserved binding site for S100A10 binding localized to the intracellular C-terminal region shortly downstream of the last transmembrane domain (S6). Deletion of this PDZ binding motif "VATTV" or mutation of T601 within TRPV5 leads to greatly diminished surface expression and function (van de Graaf et al 2003). The S100A10-annexin 2 heterodimer is also important for the localization of the voltage gated sodium channel NA 1.8 and the potassium channel TASK1 (Girard et al 2002;Okuse et al 2002).…”

“…Deletion or mutation of the chaperone interaction site prevents surface expression (see van de Graaf et al 2003 for TRPV5). It is then also conceivable that overexpression of C-terminal fragments containing the chaperone interaction site could act as a dominant-negative regulator of wildtype channel expression by obstructing the transport pathway to the plasma membrane.…”