Defense Date

2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Neuroscience

First Advisor

Ian Scott Ramsey

Second Advisor

Clive M. Baumgarten

Third Advisor

Michelle L. Block

Fourth Advisor

Pamela E. Knapp

Fifth Advisor

Roland N. Pittman

Abstract

Transient receptor potential vanilloid 2 channel (TRPV2) is a Ca2+-permeable ion channel that is highly expressed in leukocytes but is also present in skeletal and cardiac muscle and endocrine cells. The TRPV2 function is implicated in a number of physiological processes, including bacterial phagocytosis, pro-inflammatory cytokine production, cardiac hypertrophy, and cancer development. TRPV2 knockout mice exhibit a high incidence of perinatal mortality, arguing that the channel plays essential roles in physiology. Despite the importance of TRPV2 for normal homeostasis, the mechanisms that control TRPV2 gating in response to pharmacological agonists, heating, membrane stretch, bioactive lipids and reactive oxygen species (ROS) remain poorly understood. Here we demonstrate that TRPV2 is functionally expressed in microglia (i.e., ‘brain macrophages’) and the microglia-like BV-2 cell line, and demonstrate that the gating of an endogenous TRPV2-like conductance is positively modulated by the bacterial toxin lipopolysaccharide (LPS), which is known to cause pro-inflammatory (M1) activation and increase ROS production by NADPH oxidase. To determine how TRPV2 gating is modulated by ROS, we recorded single channel activity in inside-out patches excised from HEK-293 cells expressing GFP-rTRPV2. Unitary currents elicited by the TRPV2 agonist 2-aminophenyl borinate (2-APB) or cannabidiol (CBD) are linear in monovalent recording solutions and give rise to an estimated unitary conductance of ~100pS, which is similar to TRPV1 but significantly smaller than TRPV3. Intriguingly, we find that although TRPV2 is insensitive to ROS (in the form of exogenously applied H2O2) alone, apparent open probability is synergistically enhanced when H2O2 is applied together with CBD. We identify two intracellular Cys residues that are necessary for TRPV2 responses to H2O2 sensitivity and find that these residues are located close to one another, albeit in different subunits, in the TRPV2 structure, suggesting that ROS promote the formation of an inter-subunit disulfide bond that alters sensitivity to pharmacological agonists. We hypothesize that ROS-dependent modulation of TRPV2 activity may be an important contributor to pro-inflammatory activation of microglia underline central nervous system diseases and that TRPV2 antagonism could be a useful therapeutic strategy in the treatment of neuroinflammation.

Rights

Tuoxin Cao, Ian Scott Ramsey

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-10-2017

Available for download on Monday, May 09, 2022

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