Effect of PCCP⁻ on recombinant α₁β₂γ₂ GABA_A receptors.

<p>A, GABA_A receptors were expressed in Xenopus oocytes. The electrical currents recorded by two-electrode voltage clamp were activated with a concentration of GABA eliciting 1% of the maximal current amplitude (EC₁) and inhibited with increasing concentrations of PCCP⁻. The lower bar indicates the time of GABA application, the upper bar the time of PCCP⁻ application. The numbers indicate the concentration of PCCP⁻ in µM. At concentrations >1 µM, induces an open-channel block, characterized by an apparent desensitization of the current and an off-current. B, Averaged concentration inhibition curve by PCCP⁻. Individual curves were fitted and standardized to the current elicited by GABA. Data are shown as mean ± SEM (n = 4). Open circle: peak current amplitudes at the beginning of the drug application. Filled squares: current amplitudes at the end of the drug application. Filled circles: current amplitudes at the end of the drug application corrected for the direct effect of PCCP⁻ on membranes. C) and D) same experiment carried out at a concentration of GABA eliciting 10% of the maximal current amplitude (EC₁₀).</p

Effect of the membrane potential on inhibition by PCCP⁻.

<p>A, GABA_A receptors were activated with a concentration of GABA eliciting 10% of the maximal current amplitude (EC₁₀) and inhibited with increasing concentrations of PCCP⁻. Averaged concentration inhibition curves by PCCP⁻ are shown for different membrane potentials. Individual curves were fitted and standardized to the current elicited by GABA. Data are shown as mean ± SD (n = 3).</p

PCCP⁻ prevents the increase in PCCP⁻ sensitivity of α₁V256β₂γ₂ mediated by MTSET⁺ + GABA.

<p>GABA (EC₁₀) was applied repetitively until a stable current response was observed followed by inhibition of the channel by PCCP⁻. Subsequently 5 mM MTSET was applied in the presence of GABA. After MTSET⁺ treatment GABA was applied twice followed by a combined application of GABA and the same concentration of PCCP⁻ used before. A, Wild type receptors were not affected by this treatment. B, The treatment leads to an enhanced inhibition in α₁V256Cβ₂γ₂. C, 5 mM MTSET⁺ was applied to α₁V256C mutant receptor in presence of GABA and 1 mM PCCP⁻. PCCP⁻ prevented enhanced inhibition and therefore covalent reaction. These experiments were repeated independently three times using different oocytes.</p

<b>Pharmacological evaluation of the expressed recombinant receptors.</b>

<p>EC₅₀ for GABA, IC₅₀ for PCCP⁻, and IC₅₀ for picrotoxin are given for wild type and mutant receptors.</p><p><b>Pharmacological evaluation of the expressed recombinant receptors.</b></p

Aligned sequences of the amino acid residues in the subunits α₁β₂γ₂ of the rat GABA_A receptor.

<p>A, Alignment of 2α, 2β and 1γ subunit contributing to the formation of a GABA_A pentamer. The residues in the α₁ subunit of the GABA_A mutated to Cys are shown in boldface letters. B, α-Helical wheel representation of the rat α₁ M2 membrane-spanning domain showing the mutated residues in boldface letters.</p

Molecular model of the interaction of PCCP⁻ with GABA_A receptors.

<p>A, The side view of the PCCP⁻ docking pose from the perspective of the γ₂ subunit. The ligand and the mutated residues of the α₁ subunit, which have an impact on the affinity of the ligand, are shown in space filling representation. The 2′ valines of the α₁ subunit are rendered grey; the 6′ threonines of the α₁ subunit in green. The GABA_A receptor is displayed in ribbon representation with α₁ subunits shown in yellow, β₂ subunits in red, γ₂ subunit in blue. The complete transmembrane domain (TMD) is shown only of the α₁ and the β₂ subunits in the back. Of the subunits in front, only a segment of the transmembrane domain 2 (TMD2) is depicted. The TMD2 of the γ₂ subunit is only partly displayed to provide a “window” through which the ligand is seen. B, Top view of the pose showing the symmetric molecular interactions between ligand and receptor. PCCP⁻ (space filling) forms H-bonds (blue dashed lines) to the –OH groups of the 6′ threonines (stick representation) of each of the five subunits.</p

High concentration of PCCP⁻ induce a current in non-injected Xenopus oocytes.

<p>A, Voltage jump of 25 ms duration from −80 mV to −30 mV. A µA sized transient current flows with each voltage step. B, A small transient outward current is induced after applications of 10, 30 and 100 µM PCCP⁻ of 30 s duration to an oocyte held at a membrane potential of −80 mV.</p

Symmetry-adapted anions, the chemical structure of PCCP⁻ and the X-ray structure of Na⁺PCCP⁻.

<p>A, Symmetry-adapted anions. B, X-ray structure of Na⁺PCCP⁻ (as the acetone solvate). The network of coordinative interactions between the partially negatively charged nitrogen atoms of PCCP⁻ and the Na⁺ cations is highlighted. The insert indicates the geometry of the molecule.</p

Concentration inhibition curves for rat and Drosophila GABA_A receptors.

<p>A, Concentration inhibition curve for α₁β₂γ₂ and α₁β₂δ GABA_A receptors. Increasing concentrations of PCCP⁻ were applied together with GABA EC₁₀. Individual inhibition curves were standardized and subsequently averaged (Mean ± SD, n = 4). Data are compared with those obtained from α₁β₂γ₂ receptors. Inhibition of the wild type and mutant RDL Drosophila GABA_A receptor by PCCP⁻ and Picrotoxin. Concentration inhibition curves of (B) PCCP⁻ and, (C) picrotoxin were determined in wild type (circles) and mutant (squares) receptors. Currents were activated with a concentration of GABA eliciting 10% of the maximal current amplitude (EC₁₀) and inhibited with increasing concentration of PCCP⁻ or picrotoxin. Individual curves were standardized to initial current amplitudes and subsequently averaged. Data are shown as mean ± SD (n = 3).</p

Hypothetical model for the mechanism of action of PCCP⁻.

<p>Mutation of residues α₁V256 and α₁T261 to cysteine alters strongly the apparent affinity for channel inhibition by PCCP⁻. The fact that MTSET⁺ can only react with cysteines introduced in M2 in the presence of GABA indicates that GABA widens the pore. For the mutations α₁V256C and α₁T260C the affinity for PCCP⁻ is strongly increased after MTSET⁺ treatment. MTSET⁺ reaction is prevented by PCCP⁻. This together implies these residues in PCCP⁻ binding. Introduction of two positive charges by reaction with MTSET⁺ further up in the channel leads to additional binding sites for PCCP⁻.</p