Date of Graduation
2015
Document Type
Dissertation
Degree Type
PhD
College
School of Pharmacy
Department
Pharmaceutical Sciences
Committee Chair
Glenn H Dillon
Committee Co-Chair
Patrick S Callery
Committee Member
Gregory M Dick
Committee Member
Robert K Griffith
Committee Member
Jason D Huber
Abstract
Carisoprodol is a centrally acting skeletal muscle relaxant frequently prescribed for the treatment of acute musculoskeletal conditions. Recreational use of carisoprodol is an increasing problem. Recent reports highlight the dangers involved in carisoprodol abuse, including severe withdrawal leading to seizures and death. Indeed, carisoprodol has been placed into schedule IV drug at federal level effective January 11, 2012 considering its alarming abuse rate. Until recently, it was widely accepted that the sedative effects of carisoprodol were due predominantly to its metabolite, meprobamate. Our lab recently concluded, that carisoprodol itself, at low concentrations allosterically modulates and at higher concentrations directly activates gamma-aminobutyric acid, type A receptors (GABAARs), the predominant inhibitory neurotransmitter receptor in mammalian brain. This may underlie the capacity of carisoprodol to enhance the sedative effects of CNS depressants, contributing to its potential for abuse.;GABAA receptors are member of the cys-loop receptor family that are hetero-pentameric ligand gated chloride ion channels and play a critical role in mediating fast inhibition in the brain. A diverse number of GABA AR subunits as well as their isoforms have been identified including &agr; (1-6), beta (1-3), gamma (1-3), rho, delta, epsilon and theta. &agr;betagamma and &agr;betadelta receptor (2:2:1 stoichiometry) isoforms are the predominant GABAARs in the CNS. &agr;betagamma GABAARs generally located within the synapses mediating GABAergic phasic inhibition, whereas &agr;betadelta receptors are preferentially targeted to extrasynaptic membranes inducing tonic inhibition. Each GABAAR subunit is composed of a large extracellular N terminus, four transmembrane helices (TM1-TM4), one extracellular TM2-TM3 loop, two intracellular loops (TM1-TM2 and TM3-TM4), and an extracellular C terminus. TM2 from each subunit line the pore of the channel and gate. We hypothesized that carisoprodol modulates GABAARs in a subunit-dependent manner, possibly through a novel site of action, with different amino acid domains contributing to direct gating and allosteric modulatory actions of carisoprodol. We used HEK293 cells, stably and transiently expressing desired GABAA receptor and took advantage of whole cell patch clamp electrophysiology and site directed mutagenesis techniques to address following specific aims: 1) To identify subunit-dependent influence on direct gating and allosteric enhancement effects of carisoprodol on GABAARs; 2) To identify GABAA receptor subunit domains that confer carisoprodol sensitivity.;Our studies demonstrate direct gating and allosteric modulatory effects of carisoprodol are GABAA receptor isoforms dependent. Specifically, carisoprodol is most efficacious on &agr;1 containing receptors for allosteric modulatory action and least efficacious on &agr;3 containing receptors for direct gating action. Also, presence or absence of gamma subunit in GABA AR does not affect carisoprodol direct gating and allosteric modulatory efficacy. Regarding delta containing extrasynaptic receptors, carisoprodol is more efficacious than GABA and potentiated the maximal GABA-gated currents like barbiturates and general anesthetics. Mutagenesis studies showed, transmembrane 4 domains of &agr; subunit are involved in direct gating action of carisoprodol but not in allosteric modulatory action. Mutation of valine at 440 TM4 domain of &agr;3 subunit to corresponding &agr;1 subunit residue leucine 415 significantly increased the direct gating efficacy of carisoprodol without affecting allosteric modulation properties. In corresponding reverse mutation experiments, mutation of &agr;1 subunit residue leucine 415 to valine 440 residue of &agr;3 subunit significantly reduced direct gating efficacy of carisoprodol but not allosteric effects. In physicochemical analysis of TM4 415 residue, polarity and volume of amino acid influenced the direct gating efficacy of carisoprodol. Taken together, our data indicate that carisoprodol interacts with distinct sites to allosterically modulate and directly gate GABAA receptors. Also, the pharmacological profile of carisoprodol at GABAA receptors coincide with its therapeutic effects, and carisoprodol's subunit-dependence property may underlie its potential for abuse.
Recommended Citation
Kumar, Manoj, "Assessment of molecular action of direct gating and allosteric modulatory effects of carisoprodol (SomaRTM) on GABA A receptors" (2015). Graduate Theses, Dissertations, and Problem Reports. 6022.
https://researchrepository.wvu.edu/etd/6022