Pentobarbital Produces Activation and Block of α1β2γ2S GABAA Receptors in Rapidly Perfused Whole Cells and Membrane Patches: Divergent Results Can Be Explained by Pharmacokinetics

Abstract

Millimolar concentrations of the barbiturate pentobarbital (PB) activate γ-aminobutyric acid (GABA) type A receptors (GABARs) and cause blockade reported by a paradoxical current increase or “tail” upon washout. To explore the mechanism of blockade, we investigated PB-triggered currents of recombinant α1β2γ2S GABARs in whole cells and outside-out membrane patches using rapid perfusion. Whole cell currents showed characteristic bell-shaped concentration dependence where high concentrations triggered tail currents with peak amplitudes similar to those during PB application. Tail current time courses could not be described by multi-exponential functions at high concentrations (≥3,000 μM). Deactivation time course decayed over seconds and was slowed by increasing PB concentration and application time. In contrast, macropatch tail currents manifested eightfold greater relative amplitude, were described by multi-exponential functions, and had millisecond rise times; deactivation occurred over fractions of seconds and was insensitive to PB concentration and application time. A parsimonious gating model was constructed that accounts for macropatch results (“patch” model). Lipophilic drug molecules migrate slowly through cells due to avid partitioning into lipophilic subcellular compartments. Inclusion of such a pharmacokinetic compartment into the patch model introduced a slow kinetic component in the extracellular exchange time course, thereby providing recapitulation of divergent whole cell results. GABA co-application potentiated PB blockade. Overall, the results indicate that block is produced by PB concentrations sixfold lower than for activation involving at least three inhibitory PB binding sites, suggest a role of blocked channels in GABA-triggered activity at therapeutic PB concentrations, and raise an important technical question regarding the effective rate of exchange during rapid perfusion of whole cells with PB.

Barbiturates Bind in the GLIC Ion Channel Pore and Cause Inhibition by Stabilizing a Closed State

“…The micromolar to millimolar concentration range for the barbiturates to modulate GLIC channels is similar to that reported for these compounds to impart their effects at eukaryotic pLGICs (24). Moreover, it is notable that independent pharmacological screening studies of the related prokaryotic homolog ELIC (Erwinia chrysanthemi ligand-gated ion channel) revealed insensitivity to modulation by pentobarbital (25).…”

Synthetic Analogues of the Snail Toxin 6-Bromo-2-mercaptotryptamine Dimer (BrMT) Reveal That Lipid Bilayer Perturbation Does Not Underlie Its Modulation of Voltage-Gated Potassium Channels

“…(23) Third, the wash-in and wash-out kinetics of BrMT are multiphasic, suggesting slow accumulation of BrMT in the cell membrane during prolonged exposures. (23, 24) Together, these effects are consistent with BrMT partitioning in and out of cell membranes and acting through the membrane to alter channel function. Similar to many other amphiphilic molecules that act by bilayer perturbation, the biological effect of BrMT, with its two aminoethyl groups, depends on the side of the membrane to which it is applied.…”

Pentobarbital inhibition of human recombinant α1A P/Q‐type voltage‐gated calcium channels involves slow, open channel block

“…Transformed HEK‐293 cells, purchased from American Type Culture Collection (Bethesda, MD, USA), were plated on 12 × 12 mm glass coverslips in 60 × 15 mm Falcon dishes (Becton Dickinson, Lincoln Park, NJ, USA) and cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum and penicillin (100 IU·mL −1 ), streptomycin (0.1 mg·mL −1 ) and glutamine (2 mM) all from Invitrogen (Carlsbad, CA, USA). After incubation in 37°C, 5% CO2 for 48 h, cells were transiently transfected using a lipofection technique described previously (Gingrich et al ., 2009) with cDNAs encoding wild‐type human α 1A pore‐forming subunit (Soong et al ., 2002), rabbit β 2a (Perez‐Reyes et al ., 1992) and rabbit skeletal muscle α 2 δ‐1 (Tomlinson et al ., 1993) accessory subunits and the respiratory syncytial virus T antigen. The β 2a subunit also encoded a green fluorescent protein (GFP) sequence following an internal ribosomal entry site, so as to identify successfully transfected HEK‐293 cells by fluorescence (Chaudhuri et al ., 2007).…”