title: Regulation of TRPP3 Channel Function by N-terminal Domain Palmitoylation and Phosphorylation.
abstract: Transient receptor potential polycystin-3 (TRPP3) is a cation channel activated by calcium and proton and is involved in hedgehog signaling, intestinal development, and sour tasting. How TRPP3 channel function is regulated remains poorly understood. By N-terminal truncation mutations, electrophysiology, and Xenopus oocyte expression, we first identified fragment Asp-21-Ser-42 to be functionally important. We then found that deletion mutant Δ1-36 (TRPP3 missing fragment Met-1-Arg-36) has a similar function as wild-type TRPP3, whereas Δ1-38 is functionally dead, suggesting the importance of Val-37 or Cys-38. Further studies found that Cys-38, but not Val-37, is functionally critical. Cys-38 is a predicted site of palmitoylation, and indeed TRPP3 channel activity was inhibited by palmitoylation inhibitor 2-bromopalmitate and rescued by palmitoylation substrate palmitic acid. The TRPP3 N terminus (TRPP3NT, Met-1-Leu-95) localized along the plasma membrane of HEK293 cells but stayed in the cytoplasm with 2-bromopalmitate treatment or C38A mutation, indicating that TRPP3NT anchors to the surface membrane through palmitoylation at Cys-38. By acyl-biotin exchange assays, we showed that TRPP3, but not mutant C38A, is indeed palmitoylated. When putative phosphorylation sites near Cys-38 were mutated to Asp or Glu to mimic phosphorylation, only T39D and T39E reduced TRPP3 function. Furthermore, TRPP3NT displayed double bands in which the upper band was abolished by λ phosphatase treatment or T39A mutation. However, palmitoylation at Cys-38 and phosphorylation at Thr-39 independently regulated TRPP3 channel function, in contrast to previous reports about correlated palmitoylation with a proximate phosphorylation. Palmitoylation at Cys-38 represents a novel mechanism of functional regulation for TRPP3.
authors.affiliation: From the Membrane Protein Disease Research Group, Departments of Physiology and.
authors.forename: Wang
authors.initials: W
authors.lastname: Zheng
authors.affiliation: From the Membrane Protein Disease Research Group, Departments of Physiology and.
authors.forename: JungWoo
authors.initials: J
authors.lastname: Yang
authors.affiliation: Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Erwan
authors.initials: E
authors.lastname: Beauchamp
authors.affiliation: From the Membrane Protein Disease Research Group, Departments of Physiology and.
authors.forename: Ruiqi
authors.initials: R
authors.lastname: Cai
authors.affiliation: From the Membrane Protein Disease Research Group, Departments of Physiology and.
authors.forename: Shaimaa
authors.initials: S
authors.lastname: Hussein
authors.affiliation: the Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, 66421 Homburg, Germany, and.
authors.forename: Laura
authors.initials: L
authors.lastname: Hofmann
authors.affiliation: From the Membrane Protein Disease Research Group, Departments of Physiology and.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.affiliation: the Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, 66421 Homburg, Germany, and.
authors.forename: Veit
authors.initials: V
authors.lastname: Flockerzi
authors.affiliation: Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Luc G
authors.initials: LG
authors.lastname: Berthiaume
authors.affiliation: the Institute of Biomedical and Pharmaceutical Sciences and Provincial Cooperative Innovation Center, College of Bioengineering, Hubei University of Technology, 430068 Wuhan, China jingfeng9930@163.com.
authors.forename: Jingfeng
authors.initials: J
authors.lastname: Tang
authors.affiliation: From the Membrane Protein Disease Research Group, Departments of Physiology and xzchen@ualberta.ca.|the Institute of Biomedical and Pharmaceutical Sciences and Provincial Cooperative Innovation Center, College of Bioengineering, Hubei University of Technology, 430068 Wuhan, China.
authors.forename: Xing-Zhen
authors.initials: XZ
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D054878:Lipoylation / Q000502:physiology*; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D000072417:Protein Domains; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014982:Xenopus laevis
chemical_list: D015220:Calcium Channels; C117185:PKD2L1 protein, human; D011956:Receptors, Cell Surface
keywords: Xenopus; acetylation; electrophysiology; phosphorylation; post-translational modification (PTM)