GABAergic inhibition of crayfish deep extensor abdominal muscle exhibits a steep dose-response relationship and a high degree of cooperativity

Abstract

A patch-clamp study was done to characterize the recently found GABAergic (i.e. gamma-aminobutyric acid) inhibitory synaptic channels of crayfish deep extensor abdominal muscle. Outside-out patches were rapidly activated by GABA to measure the dose/response curves for the open probability of the channels, Po, and the rise time, tr, (time from Po = 0.1 to Po = 0.9). In some of the patches the GABA-activated currents decayed due to desensitization and such patches were not studied further. Rare channel openings were elicited with 0.1 mM GABA. The Po at this low concentration of GABA was 0.0005 to 0.01. Application of 10 mM GABA was necessary to reach the maximal Po of 0.9. The slope of the dose/response relationship in the double logarithmic plot was 5.4 +/- 1.1 (mean +/- SD; n = 9) between 0.1 mM and 0.2 mM GABA. The plot of tr versus GABA concentration had a peculiar shape, recently found to be characteristic for positive cooperativity of the binding sites. tr increased from a minimum at 10 mM GABA with declining concentrations of GABA and reached a peak at 0.4 mM GABA. Below 0.4 mM GABA, tr decreased again. With 0.2 mM GABA tr was 0.40 +/- 0.1 (mean +/- SD; n = 4) of the peak value measured at 0.4 mM GABA. Simulations were compared with the experimental results and a linear reaction scheme with five binding sites for GABA was established to describe the dose/response curves for Po and tr.

Characterization and molecular reaction scheme of a chloride channel expressed after axotomy in crayfish

“…Unlike control muscles, in which 5% of about 120 patches containing chloride channels showed desensitization [6], desensitization was never observed to occur in axotomized muscles in more than 200 channel-containing outside-out patches, even when GABA was applied for more than 1 s.…”

“…Upon decreasing the GABA concentrations the rise times increased uniformly up to values of about 300 ms. A sharp peak of the rise times and a decrease of the rise times at very low GABA concentrations, as characteristic for the control channel, were not observed to occur in the concentration range tested but cannot be excluded for GABA concentrations below 0.2 mM. Consequently there is no reason to assume cooperativity for the binding of the agonist to the receptor [6,17].…”

“…The one on distal fibres of the first walking leg reacts relatively slowly, with three binding steps of GABA, and is probably able to mediate a gradual control of inhibition of the muscle fibres (H. Adelsberger et al unpublished results). On the DEAM of the same animal we have shown the presence of a completely different GABA-activated chloride channel with five binding steps for the agonist and strong positive cooperativity [3,6]. This channel is able to react very fast in an all-or-none fashion mediating inhibition of the DEAM.…”

“…Some of them are activated by yaminobutyric acid (GABA) as well as by other agonists, for instance glutamate [15,27]. Others are activated by GABA only, and different types on distal fibres of the opener muscle (H. Adelsberger, unpublished results) and on the DEAM [2,3,6] have been characterized. The chloride channel of the opener muscle is probably the synaptic receptor, transmitting a strong inhibitory action; it needs three binding steps of GABA to the receptor prior to opening.…”

“…2Bfor the range of 0.05-10 mM GABA. For comparison, an average dose/response curve for the non-operated muscle[6] is shown (dashed line, open circles). In the double-logarithmic plot, the steepest part of the dose/response relationship is in the concentration range of between 0.05 and 0.15 mM GABA.…”