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Molecular determinants in TRPV5 channel assembly Chang Q; Gyftogianni E; van de Graaf SF; Hoefs S; Weidema FA; Bindels RJ; Hoenderop JGJ Biol Chem 2004[Dec]; 279 (52): 54304-11The 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.|Amino Acid Sequence[MESH]|Animals[MESH]|Calcium Channels/*chemistry/*genetics/physiology[MESH]|Calcium/metabolism[MESH]|Cell Line[MESH]|Cell Membrane[MESH]|Electrophysiology[MESH]|Embryo, Mammalian[MESH]|Embryo, Nonmammalian[MESH]|Escherichia coli/genetics[MESH]|Gene Expression[MESH]|Glutathione Transferase/genetics[MESH]|Humans[MESH]|Immunosorbent Techniques[MESH]|Kidney[MESH]|Oocytes/metabolism[MESH]|Patch-Clamp Techniques[MESH]|Peptide Fragments/chemistry/genetics/physiology[MESH]|RNA, Complementary/genetics[MESH]|Recombinant Fusion Proteins[MESH]|Sequence Alignment[MESH]|Structure-Activity Relationship[MESH]|TRPV Cation Channels[MESH]|Transfection[MESH]|Xenopus laevis[MESH] |
