Graduation Year

2021

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Thomas Taylor-Clark, Ph.D.

Committee Member

Jahanshah Amin, Ph.D.

Committee Member

Javier Cuevas, Ph.D.

Committee Member

Robert Deschenes, Ph.D.

Committee Member

Sami Noujaim, Ph.D.

Keywords

glutathione, NEM, nociceptor, patch-clamp, reactive

Abstract

A subset of sensory nerves called nociceptors are stimulated by noxious stimuli and evoke nocifensive responses in different organ systems. Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, nonselective, cation channel that initiates the generator potential that evokes afferent signaling in nociceptive nerves. TRPA1 is activated many harmful irritants, such as food chemicals, environmental pollutants, reactive oxygen species and other endogenous mediators. Most TRPA1 agonists have electrophilic properties, which covalently modify cysteine residues on the cytosolic side of the channel. Biochemical studies in our lab have identified four cysteines (C273, C621, C665, C1085) that rapidly bound iodoacetamide (irreversible electrophile); C621 bound at a rate that was 6,000-fold greater than nonreactive cysteines. Electrophilic adduction of cysteines can result in TRPA1 activation, but the detailed mechanism underlying this process is poorly understood. We hypothesize that electrophilic modification of C621 and C665 independently contributes to TRPA1 activation. In this study, we have utilized live-cell calcium imaging, whole-cell patch clamp and single-channel patch clamp to determine the role of highly reactive cysteine residues in TRPA1 activation via electrophiles. We determined that the highly reactive C621 is critical for TRPA1 and the local environment surrounding C621 contributes to its reactivity. C665 is not required to initiate TRPA1 activation but could contribute to the transition from partial to full activation. We determined that depletion of cytosolic glutathione in whole-cell recordings decreases electrophile-evoked currents. Lastly, in single-channel recordings we identified two sequential activation profiles for TRPA1 which were differentially regulated by C621 and C665, suggesting that TRPA1 has multiple open states. Our data will provide novel functional insights consistent with recently published structural data of TRPA1 activation.

Download

Share

COinS