Comparison of biophysical properties of α1ß2 and α3ß2 GABAA receptors in whole-cell patch-clamp electrophysiological recordings.

In the present study we have characterized the biophysical properties of wild-type (WT) α1ß2 and α3ß2 GABAA receptors and probed the molecular basis for the observed differences. The activation and desensitization behavior and the residual currents of the receptors expressed in HEK293 cells were determined in whole-cell patch clamp recordings. Kinetic parameters of α1ß2 and α3ß2 activation differed significantly, with α1ß2 and α3ß2 exhibiting rise times (10-90%) of 24 ± 2 ms and 51 ± 7 ms, respectively. In contrast, the two receptors exhibited largely comparable desensitization behavior with decay currents that could be fitted to exponential functions with two or three components. Most notably, the two receptor compositions displayed different degrees of desentization, with the residual currents of α1ß2 and α3ß2 constituting 34 ± 2% and 21 ± 2% of the peak current, respectively. The respective contributions of the extracellular domains and the transmembrane/intracellular domains of the α-subunit to these physiological profiles were next assessed in recordings from cells expressing αß2 receptors comprising chimeric α-subunits. The rise times displayed by α1ECD/α3TMDß2 and α3ECD/α1TMDß2 receptors were intermediate to those of WT α1ß2 and WT α3ß2, and the distribution of the different components of the current decays exhibited by the two chimeric receptors followed the same pattern as the two WT receptors. The residual current exhibited by α1ECD/α3TMDß2 (23 ± 3%) was similar to that of α3ß2 but significantly different from that of α1ß2, whereas the residual current displayed by α3ECD/α1TMDß2 (27 ± 2%) was intermediate to and did not differ significantly from either of the WT receptors. This points to molecular differences in the transmembrane/intracellular domains of the α-subunit as the main determinants of the observed differences in receptor physiology between α1ß2 and α3ß2 receptors.

Delineation of the functional properties and the mechanism of action of AA29504, an allosteric agonist and positive allosteric modulator of GABAA receptors.

The retigabine analog 2-amino-4-[(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504) is a positive allosteric modulator (PAM) of γ-aminobutyric acidA receptors (GABAARs), and the modulator has been used in ex vivo/in vivo studies to probe the physiological roles of native δ-containing GABAARs. In this study, the functional properties and mode of action of AA29504 were investigated at human GABAARs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. AA29504 was found to be an allosteric GABAAR agonist displaying low intrinsic activities at 3-30 µM. AA29504 was essentially equipotent as a PAM at the 13 GABAAR subtypes tested (EC50: 0.45-5.2 µM), however GABA EC5-evoked currents through αßδ subtypes were modulated to substantially higher levels than those through αßγ2S subtypes (relative to GABA Imax). While the δ/γ2S-difference clearly was key for this differential GABA efficacy modulation, studies of the AA29504-mediated modulation of different α4,5,6-containing αß, αßγ2S and αßδ GABAARs revealed the α-subunit identity to be another important determinant. Based on its functional properties at numerous mutant GABAARs and on in silico analysis of its low-energy conformations, AA29504 is proposed to act through an allosteric site in the transmembrane ß(+)/α(-) interface in the GABAAR also targeted by etomidate and several other modulators. In contrast to these modulators, however, AA29504 did not display substantial ß2/ß3-over-ß1 GABAAR preference, which challenges the notion of ligands targeting this site always possessing this subtype-selectivity profile. Hence, the detailed pharmacological profiling of AA29504 both highlights the complexity of allosteric GABAAR modulation and provides valuable information about this modulator as a pharmacological tool.

Functional properties and mechanism of action of PPTQ, an allosteric agonist and low nanomolar positive allosteric modulator at GABAA receptors.

The former sedative-hypnotic and recreational drug methaqualone (Quaalude) is a moderately potent, non-selective positive allosteric modulator (PAM) at GABAA receptors (GABAARs) (Hammer et al., 2015). In the present study, we have identified a novel methaqualone analog, 2-phenyl-3-(p-tolyl)quinazolin-4(3H)-one (PPTQ), in a screening of 67 analogs at five αß2γ2S GABAAR subtypes and delineated its functional properties and mechanism of action at wild-type and mutant GABAARs expressed in Xenopus laevis oocytes by two-electrode voltage clamp electrophysiology. PPTQ was found to be an allosteric agonist and a PAM (ago-PAM) at human α1ß2γ2S and α4ß2δ GABAARs, exhibiting intrinsic activity at micromolar concentrations and potentiating the GABA-evoked signaling through the receptors at concentrations down to the low-nanomolar range. Whereas PPTQ exclusively increased the potency of GABA at the α1ß2γ2S receptor, it increased both GABA potency and efficacy at α4ß2δ and displayed modest potency-based preference for α4ß2δ over α1ß2γ2S. In elaborate mutagenesis and competition experiments PPTQ was found to act through the same or an overlapping site as etomidate in the transmembrane ß(+)/α(-) subunit interfaces, whereas it did not seem to target the other three transmembrane interfaces in the GABAAR. Finally, the PPTQ site was shown to be allosterically linked with sites targeted by neurosteroids and barbiturates but not with the high-affinity benzodiazepine site in the α1ß2γ2S receptor. In conclusion, the development of a highly potent, bioavailable GABAAR ago-PAM by subtle modifications to the methaqualone scaffold demonstrates that derivatization of this infamous drug from the past can lead to modulators with distinct functional characteristics at the receptors.

Molecular Cloning and Functional Expression of the Equine K+ Channel KV11.1 (Ether à Go-Go-Related/KCNH2 Gene) and the Regulatory Subunit KCNE2 from Equine Myocardium.

The KCNH2 and KCNE2 genes encode the cardiac voltage-gated K+ channel KV11.1 and its auxiliary ß subunit KCNE2. KV11.1 is critical for repolarization of the cardiac action potential. In humans, mutations or drug therapy affecting the KV11.1 channel are associated with prolongation of the QT intervals on the ECG and increased risk of ventricular tachyarrhythmia and sudden cardiac death--conditions known as congenital or acquired Long QT syndrome (LQTS), respectively. In horses, sudden, unexplained deaths are a well-known problem. We sequenced the cDNA of the KCNH2 and KCNE2 genes using RACE and conventional PCR on mRNA purified from equine myocardial tissue. Equine KV11.1 and KCNE2 cDNA had a high homology to human genes (93 and 88%, respectively). Equine and human KV11.1 and KV11.1/KCNE2 were expressed in Xenopus laevis oocytes and investigated by two-electrode voltage-clamp. Equine KV11.1 currents were larger compared to human KV11.1, and the voltage dependence of activation was shifted to more negative values with V1/2 = -14.2±1.1 mV and -17.3±0.7, respectively. The onset of inactivation was slower for equine KV11.1 compared to the human homolog. These differences in kinetics may account for the larger amplitude of the equine current. Furthermore, the equine KV11.1 channel was susceptible to pharmacological block with terfenadine. The physiological importance of KV11.1 was investigated in equine right ventricular wedge preparations. Terfenadine prolonged action potential duration and the effect was most pronounced at slow pacing. In conclusion, these findings indicate that horses could be disposed to both congenital and acquired LQTS.