Received for publication March 1, 2006.
Revised June 8, 2006.
Accepted for publication June 8, 2006.

FUNCTIONAL MAPPING OF THE TRPV1 INTRACELLULAR BINDING SITE

Abstract

Capsaicin (vanilloid) sensitivity has long served as the functional signature of a subset of nociceptive sensory neurons. Mutagenesis studies have revealed seemingly distinct regions involved in mediating ligand binding and channel activation at the capsaicin binding site. Residue 547 (TM4) mediates significant species differences in RTX sensitivity and the Ser⁵¹² residue is critical in discriminating between pH and capsaicin gating. In the present study, the pharmacological profiles of a variety of ligands were studied to investigate cross-talk between these two regions. Exchange of residue 547 between species mediated a difference in capsaicin and RTX-dependent gating. Likewise, the potency of I-RTX, and a novel TRPV1 antagonist were also altered. Previously, experiments using the S512Y mutant channel have confirmed the importance of residue 512 for functional interaction of capsaicin and our novel antagonist. Surprisingly, in this study, the mutation S512Y converted the activity of the antagonist, I-RTX, into an intrinsic agonist, albeit with a lower potency than its parent compound, RTX. Recent studies have proposed a novel model for the receptor, based on the X-ray crystal structure of the Kvap channel where both the 512 and 547 amino acid residues are in close proximity. Our data support the model whereby intracellular ligand interaction occurs within a S3-S4 'sensor' domain, enabling binding of ligands to be transduced to functional gating of the channel. The binding pocket also appears to be exquisitely sensitive to residue-specific interaction with ligands, as subtle changes in either ligand or channel structure can have profound effects on channel activity.

Key words: Capsaicin/vanilloid, Ion channel regulation, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches, Receptor binding studies

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