Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels.
Although PI(4,5)P2 is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ability of the TRPV1 ion channel to discriminate between PI(4,5)P2 and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC50 for activation by PI(4,5)P2, it decreased the EC50 for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P2 from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EGF receptor. Rather, conversion of a PI(4,5)P2-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5' position of the phosphoinositide.
University of Washington School of Medicine, Seattle, WA 98195.
Carmen A
CA
Ufret-Vincenty
University of Washington School of Medicine, Seattle, WA 98195.
Rebecca M
RM
Klein
University of Washington School of Medicine, Seattle, WA 98195.
Marcus D
MD
Collins
University of Washington School of Medicine, Seattle, WA 98195.
Mario G
MG
Rosasco
University of Washington School of Medicine, Seattle, WA 98195.
Gilbert Q
GQ
Martinez
University of Washington School of Medicine, Seattle, WA 98195 seg@uw.edu.
Sharona E
SE
Gordon
D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D050916:TRPV Cation Channels / Q000737:chemistry / Q000378:metabolism*
D019269:Phosphatidylinositol 4,5-Diphosphate; D050916:TRPV Cation Channels; C493922:Trpv1 protein, rat