title: Interplay between calmodulin and phosphatidylinositol 4,5-bisphosphate in Ca2+-induced inactivation of transient receptor potential vanilloid 6 channels.
abstract: The epithelial Ca(2+) channel transient receptor potential vanilloid 6 (TRPV6) undergoes Ca(2+)-induced inactivation that protects the cell from toxic Ca(2+) overload and may also limit intestinal Ca(2+) transport. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca(2+), calmodulin (CaM), and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in excised inside-out patches. The activity of TRPV6 strictly depended on the presence of PI(4,5)P(2), and Ca(2+)-CaM inhibited the channel at physiologically relevant concentrations. Ca(2+) alone also inhibited TRPV6 at high concentrations (IC(50) = ∼20 μM). A double mutation in the distal C-terminal CaM-binding site of TRPV6 (W695A/R699E) essentially eliminated inhibition by CaM in excised patches. In whole cell patch clamp experiments, this mutation reduced but did not eliminate Ca(2+)-induced inactivation. Providing excess PI(4,5)P(2) reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P(2) did not inhibit binding of CaM to the C terminus of the channel. Overall, our data show a complex interplay between CaM and PI(4,5)P(2) and show that Ca(2+), CaM, and the depletion of PI(4,5)P(2) all contribute to inactivation of TRPV6.
authors.affiliation: Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, USA.
authors.forename: Chike
authors.initials: C
authors.lastname: Cao
authors.forename: Eleonora
authors.initials: E
authors.lastname: Zakharian
authors.forename: Istvan
authors.initials: I
authors.lastname: Borbiro
authors.forename: Tibor
authors.initials: T
authors.lastname: Rohacs
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism*; D017124:Conserved Sequence; D004594:Electrophysiology / Q000379:methods; D005260:Female; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D050916:TRPV Cation Channels / Q000096:biosynthesis* / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D015220:Calcium Channels; D002147:Calmodulin; D007295:Inositol Phosphates; D011993:Recombinant Fusion Proteins; D050916:TRPV Cation Channels; C422827:TRPV6 protein, human; C066454:inositol 4,5-bisphosphate; D002118:Calcium