Modulation of single-channel properties of TRPV1 by phosphorylation.
Activation of TRPV1, the heat and capsaicin receptor, is known to be promoted by phosphorylation, but the molecular details are unclear. In the present study we recorded from single TRPV1 ion channels using the cell-attached patch clamp technique. The influence of capsaicin concentration on the time constants of open and closed states demonstrates the existence of at least four closed and three open states, and shows that channel opening can occur from partially liganded states. Activation of protein kinase C (PKC) promotes channel opening in some channels but not others, consistent with some channels being inaccessible to the kinase. The changes in open and closed state time constants following activation of PKC are equivalent to an increased affinity of capsaicin binding, but other arguments suggest that channel opening must be potentiated by downstream changes in channel activation rather than by a direct action of phosphorylation on the capsaicin binding site. Mutation of functionally important PKC phosphorylation sites on TRPV1, or application of staurosporine, a broad-spectrum kinase inhibitor, completely inhibited the effect of PKC in enhancing channel open time. Staurosporine also inhibited channel activity in the absence of overt PKC activation, showing that TRPV1 is partially phosphorylated at rest. This study elucidates the mechanism by which phosphorylation by PKC potentiates the activation of single TRPV1 ion channels.
Studer
Milena
M
Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK.
McNaughton
Peter A
PA
D000465:Algorithms; D002211:Capsaicin / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D055724:Electrophysiological Phenomena; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D020932:Nerve Growth Factor / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D047428:Protein Kinase Inhibitors; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D020932:Nerve Growth Factor; D011493:Protein Kinase C; D019311:Staurosporine; C071423:capsazepine; D013755:Tetradecanoylphorbol Acetate; D002211:Capsaicin