Molecular determinants in TRPV5 channel assembly.
The epithelial Ca(2+) channels TRPV5 and TRPV6 mediate the Ca(2+) influx in 1,25-dihydroxyvitamin D(3)-responsive epithelia and are therefore essential in the maintenance of the body Ca(2+) balance. These Ca(2+) channels assemble in (hetero)tetrameric channel complexes with different functional characteristics regarding Ca(2+)-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 (45)Ca(2+) uptake experiments in Xenopus laevis oocytes co-expressing TRPV5 wild-type and truncated proteins indicated that TRPV5 Delta N (deleted N-tail) and TRPV5 Delta C (deleted C-tail) decreased channel activity of wild-type TRPV5 in a dominant-negative manner, whereas TRPV5 Delta N Delta C (deleted N-tail/C-tail) did not affect TRPV5 activity. Oocytes co-expressing wild-type TRPV5 and TRPV5 Delta N or TRPV5 Delta C showed virtually no wild-type TRPV5 expression on the plasma membrane, whereas co-expression of wild-type TRPV5 and TRPV5 Delta N Delta C displayed normal channel surface expression. This indicates that TRPV5 trafficking toward the plasma membrane was disturbed by assembly with TRPV5 Delta N or TRPV5 Delta C but not with TRPV5 Delta N Delta 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.
Department of Physiology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center, NL-6500 HB Nijmegen, The Netherlands.
Qing
Q
Chang
Emmanouela
E
Gyftogianni
Stan F J
SF
van de Graaf
Susan
S
Hoefs
Freek A
FA
Weidema
René J M
RJ
Bindels
Joost G J
JG
Hoenderop
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane; D004594:Electrophysiology; D004622:Embryo, Mammalian; D004625:Embryo, Nonmammalian; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007163:Immunosorbent Techniques; D007668:Kidney; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D018075:RNA, Complementary / Q000235:genetics; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D050916:TRPV Cation Channels; D014162:Transfection; D014982:Xenopus laevis
D015220:Calcium Channels; D010446:Peptide Fragments; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; D005982:Glutathione Transferase; D002118:Calcium