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"MODULATION OF TRPV6 ACTIVITY BY PHOSPHATIDYLINOSITOL(4,5)-BISPHOSPHATE, CYTOSOLIC ATP AND CALMODULIN"

by
Chike Cao
Interdisciplinary Biomedical Sciences Program
M.S., University of Arkansas at Fayetteville, 2006
B.S., Central China Normal University, 2000



Thesis Advisor: Tibor Rohacs, M.D., Ph.D.
Associate Professor
Department of Pharmacology and Physiology

Tuesday, January 22, 2013
1:00 P.M., MSB H-609b


Abstract

Transient receptor potential vanilloid 6 (TRPV6) is an epithelial Ca2+ channel responsible for active Ca2+ absorption in the intestines. TRPV6 is constitutively active and both phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and cytoplasmic ATP have been shown to be important for TRPV6 activity. TRPV6 undergoes Ca2+ -induced inactivation, which has been proposed to be mediated by Ca2+-calmodulin (CaM) and depletion of PtdIns(4,5)P2 via phospholipase C (PLC) activation. However, how these factors are coordinated with each other in regulating TRPV6 activity is not clear.
We showed that in excised inside-out macropatches, ATP reactivated the human TRPV6 channels after current rundown only in the presence of Mg2+. The stimulatory effect of MgATP was blocked by three structurally different compounds that inhibit type III phosphatidylinositol 4-kinases (PI4Ks) and by PI-PLC which selectivity hydrolyzes phosphatidylinositol (PtdIns), the substrate for PI4Ks. PtdIns(4,5)P2 stimulated TRPV6 activity in excised patches, whereas PtdIns(4)P had only minimal effect. These data demonstrate that MgATP supports TRPV6 activity by providing substrate for lipid kinases thus allowing resynthesis of PtdIns(4,5)P2.
We also showed that in excised macropatches, Ca2+-CaM significantly inhibited TRPV6 both during current rundown and when the channels were re-activated by either PtdIns(4,5)P2 or MgATP. Ca2+ blocked TRPV6 monovalent currents in a concentration-dependent manner. By using both biochemical binding assays and excised patch measurements of wild-type and mutant channels, we showed that CaM binds to TRPV6 via a distal C-terminal region in a Ca2+-dependent manner. Two highly conserved amino acid residues in this region (W695, R699) were responsible for interacting with CaM. W695A/R699E double mutant of TRPV6 channel was essentially not inhibited by Ca2+-CaM. The inhibitory effect of Ca2+-CaM on wild-type TRPV6 was alleviated to some extent by higher PtdIns(4,5)P2 concentrations in excised patches. However, we did not observe direct competition between PtdIns(4,5)P2 and Ca2+-CaM in biochemical binding experiments. Our data show complex regulation of TRPV6 activity by the interplay between PtdIns(4,5)P2 and Ca2+-CaM.


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