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Intramolecular interactions in TRPV6, and functional regulation of TRPP channels
- Author / Creator
- Liu, Xiong
There are 28 members in the mammalian transient receptor potential (TRP) channel superfamily, which are divided into six subfamilies, TRPA/C/M/ML/P/V, and play distinct sensory roles in response to various environmental stimuli. High-resolution structures of various TRP channels show a number of potential contacts between different domains, indicating potential interactions that may be functionally important. In TRPV6, a highly Ca2+ selective ion channel, whether these domains physically interact with each other and whether they are of functional importance have not been determined. TRPP3 is a cation channel regulated by Ca2+ and activated by calmodulin (CaM) antagonist, calmidazolium (CMZ). How TRPP3 is regulated by CaM is poorly understood. TACAN was recently reported to be a mechano-sensitive ion channel with diverse tissue distribution and was indicated to interact with TRPP2 by a proteomic screening. Whether they physically or functionally interact with each other has yet to be explored.
In Chapter 2, we found that residue R470 in the S4-S5 Linker and W593 in the C-terminal TRP-like domain mediate an intramolecular interaction between the S4-S5 Linker and C-terminus (called L/C interaction). We also found that residue W321 in the N-terminal pre-S1 domain and I597 in the TRP-like domain mediate an intramolecular interaction between the N- and C-terminus (called N/C interaction). These interactions were autoinhibitory for the TRPV6 channel function. We also found that phosphatidylinositol 4,5-bisphosphate (PIP2) interacts with R470 to disrupt these interactions, which activates TRPV6. This study revealed a mechanism of how PIP2 regulates TRPV6, which could represent a shared mechanism by other TRP channels.
In Chapter 3, we found that CaM inhibits TRPP3 in Xenopus oocytes and that the two proteins interact with each other through the calcified CaM N-lobe and the TRPP3 C-terminal fragment E566-F621. Furthermore, our data also showed that CaM inhibits TRPP3 mainly through enhancing phosphorylation of TRPP3 at T591 by Ca2+/CaM-dependent protein kinase II (CaMK2). Ca2+ entry through TRPP3 increased the formation of the CaM/Ca2+ complex that binds to the TRPP3 C-terminus to enhance the TRPP3/CaMK2 interaction thereby promoting the T591 phosphorylation, which inhibits channel function.
In Chapter 4, we found that TACAN is in complex with TRPP2 in different kidney cell lines and that they are co-localized in primary cilia. Using Xenopus oocyte expression, we found that TACAN inhibits the channel activity of the TRPP2 gain-of-function (GOF) mutant F604P. Using Chinese hamster ovary (CHO) cell expression, we found that TACAN inhibits both wild-type (WT) TRPP2 and mutant F604P by reducing their single-channel conductance and open probability. We also found that co-expression of TACAN with TRPP2 enhances the cell sensitivity to stretch. Furthermore, our data showed that while both the first and last transmembrane (TM) segments (S1 and S6, respectively) of TACAN are involved in the interaction with the TM domains of TRPP2, only the TACAN S6-involved interaction is functionally relevant. Of note, we also found that the TACAN S6 interacts with the peripheral domain (S1-S4) of TRPP2 and that the TACAN S1 interacts with the pore domain (S5-S6) of TRPP2. This study demonstrated that TACAN is a regulator of TRPP2 channel.
In summary, this thesis is composed of functional characterization of critical PIP2-regulated intramolecular interactions in TRPV6 and studies of how TRPP3 is regulated by CaM and how TRPP2 is regulated by TACAN. Our work constitutes valuable contributions to understanding the function and regulation of TRPV6, TRPP3 and TRPP2.
- Graduation date
- Fall 2021
- Type of Item
- Doctor of Philosophy
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