A Single Amino Acid Residue at Transmembrane Domain 4 of the α Subunit Influences Carisoprodol Direct Gating Efficacy at GABAA Receptors

The muscle relaxant carisoprodol has recently been controlled at the federal level as a Schedule IV drug due to its high abuse potential and consequences of misuse, such as withdrawal syndrome, delusions, seizures, and even death. Recent work has shown that carisoprodol can directly gate and allosterically modulate the type A GABA (GABA(A)) receptor. These actions are subunit-dependent; compared with other GABA(A) receptors, carisoprodol has nominal direct gating effects in alpha 3 beta 2 gamma 2 receptors. Here, using site-directed mutagenesis and whole-cell patch-clamp electrophysiology in transiently transfected human embryonic kidney 293 cells, we examined the role of GABAA receptor a subunit transmembrane domain 4 (TM4) amino acids in direct gating and allosteric modulatory actions of carisoprodol. Mutation of alpha 3 valine at position 440 to leucine (present in the equivalent position in the alpha 1 subunit) significantly increased the direct gating effects of carisoprodol without affecting its allosteric modulatory effects. The corresponding reverse mutation, alpha 1( L415V), decreased carisoprodol direct gating potency and efficacy. Analysis of a series of amino acid mutations at the 415 position demonstrated that amino acid volume correlated positively with carisoprodol efficacy, whereas polarity inversely correlated with carisoprodol efficacy. We conclude that alpha 1(415) of TM4 is involved in the direct gating, but not allosteric modulatory, actions of carisoprodol. In addition, the orientation of alkyl or hydroxyl groups at this position influences direct gating effects. These findings support the likelihood that the direct gating and allosteric modulatory effects of carisoprodol are mediated via distinct binding sites.