ANKYRIN-REPEAT-DOMAIN AFFECTS TRPV1 CHANNEL GATING: A STRUCTURE-FUNCTION STUDY

TRPV1 is a polymodal channel that is natively expressed in nociceptor neurons. It can be activated directly by heat (>42°C) and by several molecules such as endovaniloids, protons (pH < 5.9) and different toxins, such as capsaicin (the pungent ingredient of chili pepper), resinferatoxin (RTX) or the tarantula toxin (DkTx). At the structural level, TRPV1 consists of six ankyrin-repeat-domains (ARD) located at the N-terminus, followed by six transmembrane segments with a pore-forming region and a highly conserved segment at the C-terminus called the TRP-domain. The ARD was shown to bind chemicals that modulate channel activity, however, the mechanism by which these chemicals modulate channel activity is unknown. In an attempt to understand the role of the ARD of TRPV1, we performed multiple sequence alignment among members of the TRPV and TRPC channel subfamilies, searching for conserved amino acids. Using the inducible T-REX- HEK293 expression system and Ca²⁺-imaging experiments, we found two mutations in Asparagine residues located at the ARD that render the expressed TRPV1 channel inactive. This mutation-induced loss-of-function was not due to the instability or mislocalization of the protein. Moreover, the mutated channels were not rescued by mutations causing constitutive activity of the channel. Using the solved atomic structure of the ARD, we found that these mutations interrupt a network of hydrogen bonds, which are probably essential for channel activation. Our results raise the question of how single point mutation located in a remote region of the protein, the ARD, affects TRPV1 channel gating.