Intramolecular interactions and regulation of TRP channels

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Mammalian transient receptor potential (TRP) superfamily has 28 cation channels that are grouped into six subfamilies and implicated in sensory physiology by responding to various stimuli including light, force, temperature and chemicals. TRP proteins, though with modest sequence homology, share significant structural organizations, eg with six transmembrane domains and tetrameric assembly, mostly revealed using cryo-electron microscopy. However, little is known about how ligands, drugs and mutations (as inputs') induce changes in TRP intramolecular interactions and protein conformation through which they regulate TRP channel function (as output'), which hampers therapeutic interventions. For the past years my graduate/undergraduate students and postdoct fellows have made significant progress on understanding the function and regulation of several TRP members, mainly from the TRP polycystin (TRPP) and vanilloid (TRPV) subfamilies. Their preliminary experiments revealed novel and functionally important intramolecular interactions in TRPP3 and TRPV6, which is critical for formulating our hypotheses and initiating this program. The overall OBJECTIVE of this program is to study how intramolecular interactions in TRPP3 and TRPV6 and conformational changes mediate regulation of channel function by ligands and mutations, through which HQP will be trained. For this goal we will carry out experiments in three projects: 1) determining roles of TRPP3 intracellular Linker and its phosphorylation in regulating channel function and in mediating the effect of phospholipid, 2) characterizing interaction between TRPV6 pore-forming membrane helices and how it can mediate functional stimulation by phospholipid, and 3) examining interaction between TRPP3 pore-forming helices, which mediates functional inhibition by phospholipid. METHODOLOGY. Channel function will be assessed using electrophysiology in frog oocytes and mammalian cells. Interactions and conformational changes within a channel protein will be examined by protein-protein interaction, immunofluorescence and computer simulation (in collaboration with Dr J.B. Peng, Univ of Alabama at Birmingham), among others. Structure-function relationships of TRPP3 channel will be examined by electrophysiology and cryo-electron microscopy (in collaboration with Dr E. Cao, Univ of Utah). SIGNIFICANCE. By using state-of-the-art interdisciplinary approaches this program will gain novel insights into shared or distinct mechanisms underlying how TRPP3 and TRPV6 intramolecular interactions and conformational changes mediate regulation of channel function by ligands and mutations. Knowledge gained from this study will also help understanding other TRP channels and may provide novel targets for future therapeutic interventions. This program will be carried out by HQP trainees via local and national/international collaborations and will thus provide dynamic and productive training environments for them.