Functional expression of the epithelial Ca(2+) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex.
TRPV5 and TRPV6 constitute the Ca(2+) influx pathway in a variety of epithelial cells. Here, we identified S100A10 as the first 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 specifically with the conserved sequence VATTV located in the C-terminal tail of TRPV5 and TRPV6. Of these five amino acids, the first 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-specific 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.
van de Graaf
Stan F J
SF
Department of Cell Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands.
Hoenderop
Joost G J
JG
Gkika
Dimitra
D
Lamers
Dennis
D
Prenen
Jean
J
Rescher
Ursula
U
Gerke
Volker
V
Staub
Olivier
O
Nilius
Bernd
B
Bindels
René J M
RJ
D000818:Animals; D017306:Annexin A2 / Q000378:metabolism; D001483:Base Sequence; D002117:Calcitriol / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D017931:DNA Primers; D004848:Epithelium / Q000378:metabolism; D020868:Gene Silencing; D007150:Immunohistochemistry; D051379:Mice; D011485:Protein Binding; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D009418:S100 Proteins; D020798:Two-Hybrid System Techniques; D000099248:S100 Calcium Binding Protein A10
D017306:Annexin A2; D002117:Calcitriol; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D009418:S100 Proteins; D000099248:S100 Calcium Binding Protein A10