Block by picrotoxin of a GABAergic chloride channel expressed on crayfish muscle after axotomy

Abstract

Outside-out patches containing a gamma-aminobutyric acid (GABA)-activated chloride channel expressed after axotomy on crayfish deep extensor abdominal muscle were excised. GABA and the blocker picrotoxin (PTX) were applied to the patches using a liquid filament switch to study the effects of picrotoxin on the GABA-elicited currents. Coapplication of GABA and PTX resulted in a reduction of the current amplitude compared with that elicited by the same GABA concentration alone. This reduction of the amplitude was dependent on both the GABA and PTX concentrations. The rise time of the current decreased after coapplication of GABA and PTX. Evaluation of the single channel currents and off-currents in the presence of GABA and PTX showed a dramatic shortening of the burst duration of the channel. The open time distributions were not altered, whereas in the closed time distributions a new closed time was apparent in presence of PTX. Preincubation with PTX prior to the GABA pulse resulted in an increase of the rise time. This effect was dependent on the PTX concentration only. Possible mechanisms are discussed to explain the effects of PTX and are implemented into the existing molecular reaction scheme of the channel.

Mechanisms for Picrotoxinin and Picrotin Blocks of α2 Homomeric Glycine Receptors

“…The picrotin kinetic scheme differs from the picrotoxinin kinetic scheme in that it was not necessary to add desensitization states (A 2 ϩAPD, AϩA 2 PD, and A 3 PD) linked to the glycine-bound closed states plus picrotin (A 2 ϩAPC, AϩA 2 PC, and A 3 PC) to predict our experimental data. Rather, such a kinetic model can be interpreted as picrotin promoting desensitization-like glycine-bound closed states, as suggested previously for picrotoxinin-evoked inhibition of homomeric ␣ 1 GlyRs and GABA C receptors (16,17,29). According to models 4 and 5, it was necessary to allow these desensitization-like closed states to interconvert when glycine dissociates (A 3 PC to AϩA 2 PC and AϩA 2 PC to A 2 ϩAPC) to fit our experimental data.…”

“…Recovery from Picrotoxinin or Picrotin Block Requires Channel Reopening-The lengthening of the rise time of glycineevoked outside-out current was also observed in the presence of PTX on ␣ 2 homomeric GlyR, and it was proposed to reflect recovery from channel block (9) rather than being the consequence of PTX binding to the unliganded receptor as proposed for GABA A R in the crayfish muscle (29). To determine whether the lengthening of the rise time of glycine-evoked outside-out current evoked by picrotoxinin and picrotin can also reflect recovery from open channel block, we first estimated for comparison the recovery time constant of picrotoxinin and picrotin inhibition by a transient application of 10 M picrotoxinin or 100 M picrotin during glycine-evoked outside-out current.…”

“…12B, this procedure gave a poor fit of the experimental data; it overestimated the speed of the activation time course of the glycine response and underestimated the decrease in the deactivation time course of the glycineevoked current when picrotin was applied during the relaxation phase of the glycine response. Discarding the links between the glycine-liganded closed states with and without picrotin in Another hypothesis already proposed for PTX block on homomeric ␣ 1 GlyR (17) and on GABAergic chloride channels expressed on crayfish muscle (29) proposed that PTX can promote GlyR desensitization-like closed states. We tested this hypothesis using the kinetic models shown in Fig.…”

“…All other parameters were set as free parameters. In these two models, it was also necessary to constrain reactions depending on the reaction cycles (one reaction cycle in model 1 and three reaction cycles in model 2) to satisfy the principle of microscopic reversibility (29). As performed previously for picrotoxin, the association and dissociation rate constants for picrotoxinin linking the glycine-liganded closed states with and without picrotoxinin binding (A 2 ϩAC to A 2 ϩAPC, AϩA 2 C to AϩA 2 PC, and A 3 C to A 3 PC) were set as equivalent (9).…”

Mechanisms for Picrotoxin Block of α2 Homomeric Glycine Receptors

“…Recovery from PTX Block Requires Channel Reopening-A lengthening in the rise time of responses evoked by PTX preincubation has been described for GABA-evoked outside-out currents in crayfish muscle (35). This was interpreted as the consequence of PTX binding to the unliganded receptor (35).…”