Dialysis of an Unknown Cytosolic Cofactor Results in Partial, Delayed Activation of TRPA1

Abstract

In many organ systems, a subset of sensory nerve fibers termed nociceptors are stimulated by noxious stimuli and evoke defensive sensations, behaviors and reflexes. Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel that is expressed in sensory neurons and evokes action potentials in nociceptive fibers when activated. Agonists of TRPA1 are electrophilic compounds that covalently modify reactive cysteine residues on TRPA1 subunits, which result in activation. Electrophiles can modify cysteines through reversible or irreversible reactions based on the structure of the compound. Whole‐cell patch clamp and live‐cell Ca2+ imaging have been used to characterize human TRPA1 (hTRPA1) channel activity (expressed in HEK293 cells) in the presence of electrophiles. Ca2+ imaging experiments show that irreversible electrophiles like N‐ethylmaleimide (NEM) rapidly increase Ca2+ influx, which suggests TRPA1 activation. However, whole‐cell patch clamp studies show a delayed, partial activation of TRPA1 with NEM treatment, but display rapid activation by reversible electrophiles. Furthermore, inside‐out single channel patch clamp experiments exhibit a reduction in open probability with NEM treatment that is comparable to the whole‐cell patch clamp experiments. To determine the mechanism of TRPA1 activation, the cause for the disparity of TRPA1 activation between whole‐cell patch clamp and Ca2+ imaging assays must be identified. We hypothesized that dialysis of cellular components in whole‐cell patch clamp that promote rapid, robust TRPA1 activation with NEM. To test our hypothesis, I performed whole‐cell patch clamp and gramicidin‐perforated patch clamp, a non‐invasive patch clamp method, to observe and compare the rate‐of‐change of NEM‐induced TRPA1 responses. Both methods contain 5mM Na‐polyphosphates in the pipette solution to prevent rundown of the TRPA1 channel. Studies using gramicidin‐perforated patch clamp exhibit a significant increase in the rate‐of‐change of NEM‐induced TRPA1 responses compared to activation in whole‐cell experiments. We also hypothesize that glutathione is the unidentified cofactor that is dialyzed out of the cell in whole‐cell experiments. Future whole‐cell experiments with glutathione (5mM) in the pipette solution will be performed to determine if rapid TRPA1 activation can be rescued. Identification of the unknown cytosolic cofactor will promote a complete understanding of the electrophilic cysteine modification events that underlie TRPA1 activation.Support or Funding InformationR01 from the NHLBIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.