Null method to estimate the maximal PA at subsaturating concentrations of agonist.

The maximal probability of being in an active state (PA,max) is a measure of gating efficacy for a given agonist acting on a given receptor channel. In macroscopic electrophysiological recordings, PA,max is typically estimated by comparing the amplitude of the current response to a saturating concentration of a test agonist to that of a reference agonist with known PA. Here, we describe an approach to estimate the PA,max for low-efficacy agonists at subsaturating concentrations.

Stereospecific Properties and Intracellular Transport of Novel Intrinsically Fluorescent Neurosteroids.

Allopregnanolone (AlloP) is an example of neuroactive steroids (NAS), which is a potent allosteric activator of the γ-aminobutyric acid A (GABA) receptor. The mechanisms underlying the biological activity of AlloP and other NAS are only partially understood. Here, we present intrinsically fluorescent analogs of AlloP (MQ-323) and its 3β-epimer, epi-allopregnanolone (E-AlloP) (YX-11), and show, by a combination of spectroscopic and computational studies, that these analogs mimic the membrane properties of AlloP and E-AlloP very well.

Three classes of propofol binding sites on GABA receptors.

Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator of gamma-aminobutyric acid type A (GABA) receptors. Several potential propofol binding sites that may mediate this effect have been identified using propofol-analogue photoaffinity labeling. Ortho-propofol diazirine (o-PD) labels β-H267, a pore-lining residue, whereas AziPm labels residues β-M286, β-M227, and α-I239 in the two membrane-facing interfaces [β(+)/α(-) and α(+)/β(-)] between α and β subunits.

Inhibitory Actions of Potentiating Neuroactive Steroids in the Human α1β3γ2L γ-Aminobutyric Acid Type A Receptor.

The γ-aminobutyric acid type A (GABA) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3-hydroxy steroids inhibit, while 3-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the 13γ2L GABA receptor by the endogenous neurosteroid 3-hydroxy-5-pregnan-20-one (35P) and the synthetic neuroactive steroid 3-hydroxy-5-androstane-17-carbonitrile (ACN).

Direct measurements of neurosteroid binding to specific sites on GABA receptors.

Background And Purpose: Neurosteroids are allosteric modulators of GABA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABA receptor.

Experimental Approach: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites.