Manish Kumar

Date of Graduation

2016

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Glenn H Dillon

Committee Co-Chair

Patric S Callery

Committee Member

Robert K Griffith

Committee Member

Rae Matsumoto

Committee Member

Hanting Zhang

Abstract

Meprobamate (tradenames Miltown, Equanil) is a schedule IV anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (gamma-aminobutyric acid type A) receptors, and has been described as having barbiturate-like activity. A thorough understanding of its mechanism remains ill-defined, however. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In alphaxbetazgamma2 GABAA receptors (where x = 1-6 and z = 1-3), the ability to enhance GABA-mediated current was evident for all alpha subunit isoforms, with the largest effect observed in alpha5-expressing receptors. Direct gating was comparable with all alpha subunits, although the effect was attenuated in alpha3-expressing receptors. Allosteric potentiating and direct gating effects were comparable in alpha1beta1gamma2 and alpha1beta2gamma2 receptors, whereas allosteric effects were enhanced in alpha1beta2 compared to alpha1beta2gamma2 receptors. In "extrasynaptic" GABA A receptors (alpha1beta3delta and alpha4beta3delta receptors), meprobamate allosterically enhanced both EC20 and saturating GABA currents, and directly activated these receptors with an efficacy comparable to that of GABA. Bemegride, which antagonizes effects of pentobarbital, attenuated direct gating effects of meprobamate. Whereas pentobarbital directly gated homomeric beta3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric rho1 receptors, both pentobarbital and meprobamate were ineffective in direct gating; a mutation (W328M) previously shown to confer sensitivity to pentobarbital, did not confer sensitivity to meprobamate. Our results provide additional insight into the actions of meprobamate and parent therapeutic agents such as Carisoprodol as well as possible domains involved in the direct gating actions of the ligands including transmembrane domains TM4. Further, using site-directed-mutagenesis and whole cell patch clamp electrophysiology in transiently transfected HEK293 cells, we examined the role of GABAA receptor alpha-subunit transmembrane domain 4 (TM4) and transmembrane domain 2 (TM2) amino acids in direct gating, and inhibitory actions of meprobamate. Mutation of alpha3 valine at position 440 to leucine (present in the equivalent position in the alpha1 subunit) increased the direct gating potency and efficacy of meprobamate. In R-alpha1beta2 heteromeric GABAA receptor, both pentobarbital and carisoprodol had inhibitory effect for allosteric modulatory action at higher concentrations. Meprobamate did not have an inhibitory effect at the concentrations tested. T6'F mutation of the beta2-subunit abolished the inhibition of carisoprodol but not of pentobarbital. Thus, our studies provide further insight into actions of meprobamate, its parent drug carisoprodol and their comparison to barbiturate pentobarbital.

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