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Function and Regulation of TRPP2 and TRPP3, and identification of pore gates of TRP channels

  • Author / Creator
    Zheng, Wang
  • The transient receptor potential (TRP) superfamily of cation channels is composed of eight subfamilies, TRPC/V/M/P/ML/A/N/Y, and play distinct sensory roles in response to various environmental stimuli. TRPP2, or polycystin-2, is mutated in autosomal dominant polycystic kidney disease (ADPKD). Recent studies have shown that protein dosage alterations play a critic role in cyst formation and disease progression, but mechanisms controlling TRPP2 expression remain incompletely characterized. TRPP3, or polycystin-L, is a homologue of TRPP2 but itself is not involved in ADPKD. It is a cation channel activated by calcium and protons. How TRPP3 channel function is regulated remains poorly understood. Despite structures of several TRP channels have recently been determined, how their pore gates, which control pore opening or closing, function and whether they share conserved gate residues(s) have remained elusive. In Chapter 2, we found that TRPP2 is translationally up-regulated by endoplasmic reticulum (ER) stress. Using cultured mammalian cells, we first showed that TRPP2 protein expression is up-regulated by ER stress which increases phosphorylation of eukaryotic initiation factor 2α (P-eIF2α). Increasing and reducing P-eIF2α was then found to increase TRPP2 expression and suppress ER stress-induced TRPP2 up-regulation, respectively. PCR and polysome-binding assays showed that ER stress does not affect the TRPP2 mRNA level but increases its binding to ribosomes. By mutation analyses, we found that an upstream open reading frame in the 5’-untranslated region (5’UTR) of TRPP2 mRNA represses TRPP2 translation and mediates ER stress-induced up-regulation. In Chapter 3, we identified far upstream element-binding protein 1 (FUBP1) that binds the 3’UTR of TRPP2 mRNA and suppresses its translation. Using dual-luciferase assays, we first identified nucleotides 691-1044 (called 3FI, located in the 3’UTR of TRPP2 mRNA) that represses the expression of luciferase. Using pull-down assays and mass spectrometry we identified FUBP1 as a 3FI-binding protein. In vitro over-expression of FUBP1 reduced the expression of TRPP2 protein but not mRNA. In embryonic zebrafish, FUBP1 knock-down by morpholino injection increased TRPP2 expression and alleviated tail curling caused by morpholino-mediated knock-down of TRPP2. Conversely, FUBP1 over-expression by mRNA injection significantly increased pronephric cyst occurrence and tail curling. Furthermore, FUBP1 binds directly to eIF4E-binding protein 1, indicating a link to the translation initiation complex. In Chapter 4, we identified a novel TRPP3 C-terminal domain critical for its trimerization and channel function. By SDS-PAGE, blue native PAGE and mutagenesis we first identified a novel C-terminal domain, called C1 (K575-T622), involved in stronger homo-trimerization of TRPP3 than the non-overlapping C-terminal coiled-coil 2 (CC2) domain which was reported to be important for TRPP3 trimerization. By electrophysiology and Xenopus oocyte expression, we found that C1, but not CC2, is critical for TRPP3 channel function. Co-immunoprecipitation and dynamic light scattering experiments further supported involvement of C1 in trimerization. Further, C1 acted as a blocking peptide that inhibits TRPP3 trimerization as well as TRPP3 and TRPP3/PKD1L3 channel function. In Chapter 5, we found that TRPP3 channel function is regulated by N-terminal domain palmitoylation and phosphorylation. By Xenopus oocyte electrophysiology, we first found that Cys-38 residue is functionally important. We then found that TRPP3 channel activity was inhibited by the palmitoylation inhibitor 2-bromopalmitate and rescued by the palmitoylation substrate palmitic acid. By acyl-biotin exchange assays, we showed that TRPP3, but not mutant C38A, is indeed palmitoylated. When Thr-39 was mutated to Asp or Glu to mimic phosphorylation, TRPP3 function was significantly reduced. Furthermore, TRPP3 N-terminus displayed double bands in which the upper band was abolished by λ phosphatase treatment or T39A mutation. In Chapter 6, we investigated gate residues within the distal fragment of helix S6 of TRPV/P/M/C channels based on Xenopus oocyte electrophysiology and hydrophobic gate theory. We found that channel activity drastically increases when TRPV6-Ala616, -Met617, TRPP3-Leu557 or -Ala558, but not any of their proximate residues, was changed to hydrophilic residues. Further mutation studies showed that channel activity strongly correlates with hydrophilicity and inversely with size of residues at these sites, suggesting that TRPV6-Ala616/-Met617 and TRPP3-Leu557/-Ala558 serve as gate residues. Similar studies only identified a single-residue gate in TRPP2(Leu677), TRPM8(Val976) and TRPC4(Iso617). Our identified double consecutive or single gate residues were all hydrophobic and were within motif "LIAM". In summary, our studies constitute valuable contributions to understanding the function and regulation of TRPP2 and TRPP3, and the nature of TRP channel gates.

  • Subjects / Keywords
  • Graduation date
    2017-06
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R30000C37
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Physiology
  • Supervisor / co-supervisor and their department(s)
    • Chen, Xing-Zhen (Physiology)
  • Examining committee members and their departments
    • Young, James (Physiology)
    • Chen, Xing-Zhen (Physiology)
    • Light, Peter (Pharmacology)
    • Altier, Christophe (Physiology and Pharmacology)
    • Cordat, Emmanuelle (Physiology)