Elucidation of molecular impediments in the α6 subunit for in vitro expression of functional α6β4* nicotinic acetylcholine receptors

Explorations into the α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric α6/α3 and mutant α3 and α6 subunits and functional characterization of these co-expressed with β4 or β4β3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe(223) residue in transmembrane helix 1 of α6 with the corresponding α3 segment or residue was found to enhance α6β4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both α3 elements into α6 resulted in assembly of α6β4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by α6β4β3 receptors comprising one of these novel α6/α3 chimeras in oocytes were found to be in good agreement with those from previous studies of α6* nAChRs formed from other surrogate α6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this α6/α3 chimera with β2 or β2β3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in α6 could be specific impediments for the expression of functional α6β4* nAChRs

Kavain, the Major Constituent of the Anxiolytic Kava Extract, Potentiates GABAA Receptors:Functional Characteristics and Molecular Mechanism

Extracts of the pepper plant kava (Piper methysticum) are effective in alleviating anxiety in clinical trials. Despite the long-standing therapeutic interest in kava, the molecular target(s) of the pharmacologically active constituents, kavalactones have not been established. γ-Aminobutyric acid type A receptors (GABAARs) are assumed to be the in vivo molecular target of kavalactones based on data from binding assays, but evidence in support of a direct interaction between kavalactones and GABAARs is scarce and equivocal. In this study, we characterised the functional properties of the major anxiolytic kavalactone, kavain at human recombinant α1β2, β2γ2L, αxβ2γ2L (x = 1, 2, 3 and 5), α1βxγ2L (x = 1, 2 and 3) and α4β2δ GABAARs expressed in Xenopus oocytes using the two-electrode voltage clamp technique. We found that kavain positively modulated all receptors regardless of the subunit composition, but the degree of enhancement was greater at α4β2δ than at α1β2γ2L GABAARs. The modulatory effect of kavain was unaffected by flumazenil, indicating that kavain did not enhance GABAARs via the classical benzodiazepine binding site. The β3N265M point mutation which has been previously shown to profoundly decrease anaesthetic sensitivity, also diminished kavain-mediated potentiation. To our knowledge, this study is the first report of the functional characteristics of a single kavalactone at distinct GABAAR subtypes, and presents the first experimental evidence in support of a direct interaction between a kavalactone and GABAARs

A multifaceted GABAA receptor modulator: Functional properties and mechanism of action of the sedative-hypnotic and recreational drug methaqualone (Quaalude)

In the present study, we have elucidated the functional characteristics and mechanism of action of methaqualone (2-methyl-3-o-tolyl-4(3H)-quinazolinone, Quaalude), an infamous sedative-hypnotic and recreational drug from the 1960s– 1970s. Methaqualone was demonstrated to be a positive allosteric modulator at human a1,2,3,5b2,3g2S GABAA receptors (GABAARs) expressed in Xenopus oocytes, whereas it dis-played highly diverse functionalities at the a4,6b1,2,3d GABAAR subtypes, ranging from inactivity (a4b1d), through negative (a6b1d) or positive allosteric modulation (a4b2d, a6b2,3d), to superagonism (a4b3d). Methaqualone did not interact with the benzodiazepine, barbiturate, or neurosteroid binding sites in the GABAAR. Instead, the compound is proposed to act through the transmembrane b(1)/a(–) subunit interface of th

Kavain did not affect propofol potentiation, but modestly reduced propofol activation at α1β2γ2L GABA_ARs.

<p><i>Top</i>, Representative traces of current responses to 10 mM GABA (control); 10 μM GABA; 10 μM GABA and 300 μM kavain; 10 μM GABA and 10 μM propofol; 10 μM GABA, 300 μM kavain and 10 μM propofol. <i>Middle</i>, Continuous traces demonstrating two consecutive applications of control (100 μM propofol) followed by the co-application of 300 μM kavain with control; and control. <i>Bottom</i>, Receptor modulation produced by propofol alone (G2 + P) was not significantly different from the combination of kavain and propofol (G2 + K + P; <i>n</i> = 5; <i>p</i> > 0.05; paired <i>t</i> test). The agonist effect of propofol (P2) was significantly reduced in the presence of kavain (P2 + K; <i>n</i> = 5; <i>p</i> < 0.01; paired <i>t</i> test). Data are presented as mean ± SEM.</p