Intramolecular Interactions in TRP Channels, and Gate and Proton Activation of PAC Channel

• Author / Creator

  Cai, Ruiqi

• The mammalian transient receptor potential (TRP) channels, composed of six subfamilies and 28 members, play crucial roles in sensory physiology and malfunctions of TRP channels cause channelopathies. High-resolution structures of TRP channels indicate conformational arrangements showing physical proximity of the TRP or TRP-like domain to the pre-S1 helix and S4-S5 linker, and between the pore-forming region S5/S6 helix. Since the structures, resolved under unnatural conditions, are representative states showing snapshots of highly dynamic conformations of TRP channels, whether these domains do physically interact with each other and their functional importance have not yet been determined in living cells. Inner membrane located phosphatidylinositol 4,5-bisphosphate (PIP2) is well known to modulate the majority of the TRP channels but the underlying mechanism is not well understood. TRPV6 channel is one of the most calcium-selective TRP channels implicated in breast cancer progression with largely undetermined mechanisms. A novel proton-activated chloride channel (PAC) was recently identified as an acid-induced outward rectified anion channel, without sequence similarity to any known ion channels. How the PAC pore gate is composed and proton sensing remain unclear.
  In Chapter 2, by two-electrode voltage clamping in Xenopus oocytes, immunofluorescence and patch clamping on mammalian cells, we identified interactions between a conserved aromatic residue in the N-terminal pre-S1 helix and a cationic residue in the C-terminal TRP (-like) helix of TRPP3 (Trp81:K568), -P2 (Trp201:K688), -V1 (Trp426:Arg701), -M8 (Trp682:Arg998) and -C4 (Trp314:Arg639), and found that these N/C interactions are required for the channel function. The PIP2 distinctly affected the N/C binding to regulate the channel function. This study describes a PIP2-modulated interaction between N/C domains that is functionally critical and likely shared among TRP channels.
  In Chapter 3, by electrophysiology in Xenopus oocytes, calcium imaging in mammalian cells and biotinylation. we functionally characterized residues W593 and I597 in the C-terminal TRP, R470 in the S4-S5 linker and W321 in the N-terminal pre-S1 helix of TRPV6 channel, and found that the Linker to C-terminal TRP helix (L/C) and the N-terminal pre-S1 helix to C-terminal TRP helix (N/C) interactions are autoinhibitory for the channel function. The stimulatory effect of PIP2 on the TRPV6 function can be accounted for by its ability to disrupt the autoinhibitory L/C and N/C bindings. This study revealed PIP2-regulated autoinhibitory interactions within TRPV6, which represents a novel mechanism of regulation that may be shared by other TRP channels.
  In Chapter 4, by electrophysiology, in-vitro pull down, disulfide bond formation assays and molecular dynamic simulations, we found that in TRPV6 a highly conserved residue R532 in the S5 helix presumably forms a salt bridge with D620 in the S6 helix, which acts as an autoinhibitory intramolecular (S5/S6 helix) interaction. Neutralized mutant R532Q is known to be gain-of-function in breast cancer cells. We found that both WT and R532Q mutant TRPV6 interact with subunit p85 of PI-3 kinase thereby promoting breast cancer progression through activating a PI3K/Akt pathway to promote epithelial-mesenchymal transition and inhibit apoptosis. However, mutant R532Q showed stronger effects than WT TRPV6. This study uncovered novel mechanistic insights into the intramolecular regulation of the TRPV6 channel function and roles of TRPV6 in breast cancer development, which may contribute to drug discovery for TRPV6-related diseases such as breast cancer.
  In Chapter 5, by means of two-electrode voltage clamp, immunofluorescence, biotinylation and flow cytometry we characterized PAC channel gate and the mechanism of proton-induced channel activation. We found that hydrophobic residue W304 acts as the closed gate while I307 as the open gate in the pore-lining S2 helix, controlling the Cl- permeation at high (e.g., pH 7.5) and low extracellular pH (e.g., pH 4.5) conditions, respectively. Four protonatable residues located in the extracellular loop were found to be involved in acid-sensing. Hydrophilic substitutions of the closed gate residue W304 constitutively opened the channel at high extracellular pH for WT PAC and even when PAC was rendered proton-insensitive. This study revealed that PAC adopts a mechanism of alternate pore gates formed by hydrophobic residues W304 and I307 and identified four protonatable extracellular residues involved in proton binding or sensing.
  In summary, this thesis consists of investigations on functionally important intramolecular interactions in TRP channels that are differently regulated by PIP2, and on gates and proton activation of the PAC channel.

• Subjects / Keywords

  • TRPV6
  • TRP channel
  • TRPP3
  • Intramolecular interaction
  • PAC channel
  • Breast cancer
  • Hydrophobic gate

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-vdqj-w708

• License

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