Transient receptor potential (TRP) channels are nonselective cation channels that act as sensors of heat and noxious chemicals and thus are important in pain perception. One member of this family, TRPV1, responds to such stimuli as heat, low pH, and capsaicin, the ingredient of chili peppers that makes them "hot." Two mechanisms are known to reduce the activity of TRP channels. The first, desensitization, occurs after prolonged exposure to a single stimulus. The second, tachyphylaxis, occurs after sequential exposures to the same stimulus. Increased intracellular Ca2+ causes the desensitization of TRPV1 currents, and this may be mediated by the calcium-binding protein calmodulin (CaM). Lishko et al. solved the crystal structure of the ankyrin repeat domain (ARD) found in the N-terminal region of TRPV1. Ankyrin repeats are motifs containing 33 amino acid residues, and they are important for protein-protein interactions. As well as determining the protein structure of the ARD, the authors also discovered that ATP, which was present in the crystallization solution, was bound to the ARD. This interaction was not an artifact of the crystallization process as ATP-agarose formed a complex with purified TRPV1-ARD recombinant protein, and free ATP inhibited this interaction. The addition of either Mg2+ or Ca2+ reduced the binding of TRPV1-ARD to ATP-agarose. As discussed in the accompanying commentary by Myers and Julius, these findings are interesting because the N terminus of TRPV1 does not contain the "Walker box" motif often found in other proteins that bind ATP, and divalent cations usually stabilize the association between binding proteins and ATP, rather than disrupt them. Whole-cell patch clamp assays were performed on TRPV1-expressing insect cells to assess the effect of ATP on capsaicin-stimulated changes in TRPV1 current. The authors found (consistent with previous studies) that the addition of ATP sensitized TRPV1 and reduced tachyphylaxis after repeated exposure to capsaicin. Surprisingly, mutation of residues in the ATP-binding site resulted in mutant TRPV1 channels that showed a reduction in tachyphylaxis, even in the absence of ATP. The authors suggested that another factor that promotes tachyphylaxis must bind to the same site on TRPV1, and so mutations of this site block the binding of both ATP and this other factor, resulting in a net decrease in tachyphylaxis. Exclusion chromatography analysis showed that CaM could form a complex with TRPV1-ARD that was Ca2+-dependent and inhibitable by ATP. Patch clamp experiments with an antibody that sequesters CaM and blocks its activity showed that CaM promoted tachyphylaxis. ATP-binding site mutants of TRPV1-ARD did not bind CaM. Together, these data not only increase our understanding of the mechanisms that regulate TRPV1 channel activity but also present a new role for ankyrin repeats that may have consequences for the function of other ankyrin repeat-containing proteins.
P. V. Lishko, E. Procko, X. Jin, C. B. Phelps, R. Gaudet, The ankyrin repeats of TRPV1 bind multiple ligands and modulate channel sensitivity. Neuron 54, 905-918 (2007). [PubMed]
B. R. Myers, D. Julius, TRP channel structural biology: New roles for an old fold. Neuron 54, 847-850 (2007). [PubMed]