title: Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking.
abstract: The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.
authors.affiliation: Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands.
authors.forename: Theun
authors.initials: T
authors.lastname: de Groot
authors.forename: Eline A E
authors.initials: EA
authors.lastname: van der Hagen
authors.forename: Sjoerd
authors.initials: S
authors.lastname: Verkaart
authors.forename: Veronika A M
authors.initials: VA
authors.lastname: te Boekhorst
authors.forename: René J M
authors.initials: RJ
authors.lastname: Bindels
authors.forename: Joost G J
authors.initials: JG
authors.lastname: Hoenderop
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D011817:Rabbits; D017384:Sequence Deletion; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D050916:TRPV Cation Channels; D002118:Calcium