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.
Chang
Qing
Q
Department of Physiology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center, NL-6500 HB Nijmegen, The Netherlands.
Gyftogianni
Emmanouela
E
van de Graaf
Stan F J
SF
Hoefs
Susan
S
Weidema
Freek A
FA
Bindels
René J M
RJ
Hoenderop
Joost G J
JG
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