TRPV1 residues vital to protection against 4‐HNE modification, preservation of channel activity and microvascular function

Abstract

We previously demonstrated enhanced 4-hydroxynonenal (4-HNE) post-translational modification (PTM) of TRPV1 decreases TRPV1 functional expression and contributes to microvascular dysfunction in diabetes. Accordingly, we hypothesized that manipulation of residues associated with 4-HNE PTM would preserve TRPV1 function and restore vascular integrity. 4-HNE decreased capsaicin mediated increases in myocardial blood flow and capsaicin-mediated relaxation in isolated coronary microvessels. TRPV1 functional analysis using electrophysiology revealed blunted capsaicin-mediated currents in the presence of 4-HNE which were reversed by the reactive carbonyl species scavenger aminoguanidine (AGD). Using computational biology, we identified three probable residues, previously identified to be important for oxidative modification, as potential sites for 4-HNE modification. The corresponding residues, C616, C621 and C634, were mutated to alanine (individually and in combination) and subsequently we examined the effects of 4-HNE on TRPV1 currents induced by capsaicin via electrophysiology. The mutation of the three pore cysteine (individually or in combination) abrogated the effects of 4-HNE on capsaicin-mediated currents. These data suggest that TRPV1 is targeted by redox-active substances that directly modulate channel activity at numerous sites in diabetes to decrease TRPV1 functional expression and contribute to microvascular dysfunction. The results obtained demonstrate an optimal redox state is critical for a properly functioning TRPV1 channel.