K.A.F. Gration

Block of locust muscle glutamate receptors by δ-philathotoxin occurs after receptor activations

Abstract One component (δ-philathottoxin (δ-PTX)) of the venom from the wasp ( Philanthus triangulum blocks transmission postsynaptically at excitatory synapses on locust muscle. δ-PTX depresses both the iontophoretic glutamate potential and the excitatory junctional current (e.j.c.) in a glutamate receptor activation-dependent manner. The rate of recovery from the effects of the toxin is reduced following either prolonged application of l -glutamate or repetitive iontophoretic application of this amino acid or high frequency neural stimulation of the muscle in the presence of δ-PTX. The decay phase of the e.j.c. is shortened by δ-PTX. The effects of δ-PTX on the e.j.c. are not voltage dependent. The open-close kinetics of glutamate channels in extrajunctional muscle membrane are modified by δ-PTX as shown by patch clamp analysis. The mean life time of the glutamate channel is reduced, whilst the mean interval between single opening events is increased with the events often occurring in bursts. These data are consistent with glutamate channel blocking by this toxin. It is proposed that the toxin blocks open channels gated by both junctional and extrajunctional glutamate receptors on locust muscle. It is further proposed that δ-PTX enters a compartment of the muscle through the glutamate open channels and that it can also block the open channels from this site.

RESPONSES TO dl‐IBOTENIC ACID AT LOCUST GLUTAMATERGIC NEUROMUSCULAR JUNCTIONS

1 The responses of excitatory junctions on locust skeletal muscle fibres to iontophoretically applied l‐glutamic acid and dl‐ibotenic acid, a rigidly extended analogue of glutamate, were recorded by means of intracellular microelectrodes. 2 Iontophoresis of l‐glutamate to junctional sites produced transient depolarizations. Ibotenate applied iontophoretically to these sites usually evoked small hyperpolarizations which probably resulted from the activation of glutamate H‐receptors on the extrajunctional membrane surrounding the junctions. However, at a minority (∼20%) of junctions, ibotenate iontophoresis evoked transient depolarizations. 3 Iontophoretically applied glutamate desensitized the ibotenate receptors, and vice versa. In experiments performed at junctional sites at which ibotenate depolarizations were absent, ibotenate had no effect on the responses to glutamate. 4 Glutamate and ibotenate junctional currents had similar reversal potentials, measured under voltage‐clamp, suggesting that the ionic bases for these currents are identical. 5 It is proposed that the excitation caused by ibotenate results from the activation of receptors for extended l‐glutamate and that these receptors co‐exist on the post‐junctional membranes of locust excitatory nerve‐muscle synapses with ibotenate‐insensitive glutamate receptors activated by glutamate in partially folded conformation.

Influence of glutamate and aspartate on time course of decay of excitatory synaptic currents at locust neuromuscular junctions

The influence of glutamate and aspartate on the time course of decay of excitatory currents at neuromuscular junctions of locust skeletal muscle was examined. Both aspartate (up to 10 mM) and glutamate (up to 0.3 mM) had little influence on the decay time of the synaptic currents, whether measured intracellularly by voltage-clamp or extracellularly with a focal electrode. These results do not exclude a role for an active uptake system in sequestering neurally released transmitter at locust neuromuscular junctions, but they suggest that it has a negligible influence on the time course of the synaptic currents.

Denervation of insect muscle: A comparative study of the changes in L-Glutamate sensitivity on locust retractor unguis and extensor tibiae muscle

Abstract The sensitivity of the extrajunctional membrane of the locust retractor unguis (R.U.) muscle to microiontophoretic application of l -glutamate was compared with that of the extensor tibiae (E.T.) muscle. The distribution of extrajunctional D-receptors for l -glutamate on R.U. muscle was more uniform than on E.T. muscle. Chronic denervation (at 30°C) caused an increase in the mean extrajunctional D-sensitivity of both muscles, complete development of which occurred 12–14 days post-denervation for R.U. muscle and 10–12 days post-denervation for E.T. muscle. The maximum mean extrajunctional glutamate D-sensitivity of denervated R.U. fibres was about 20 times greater than the sensitivity of innervated control fibres, while the increase for denervated E.T. fibres was about 6-fold. For both muscles the increase in glutamate sensitivity was only partially accounted for by an increase in the input resistance of the fibres after denervation. The extrajunctional D-receptors on denervated R.U. muscle were uniformly distributed, but on E.T. fibres there was considerable variation in local glutamate sensitivity. Between 20–28 days post-denervation the extrajunctional sensitivity of both muscles declined at a time when muscle atrophy was evident. The reversal potential of extrajunctional glutamate responses of denervated muscle, determined using voltage-clamp, was +3.5 ± 2.6 mV (mean ± S.D., n = 8), similar to the reversal potential of junctional glutamate responses of both innervated and denervated fibres.

GLUTAMATE-ACTIVATED CHANNELS IN LOCUST MUSCLE

Publisher Summary This chapter discusses glutamate-activated channels in locust muscle. The patch clamp technique has been used to measure the currents passing through individual d-receptor gated channels in the extrajunctional membrane of locust muscle. Patch clamp recordings with electrodes containing low (10−4 M) concentrations of l-glutamate from different, sometimes even close sites, on a muscle fiber exhibit various levels of activity, ranging from a single channel showing random openings to the simultaneous openings of two or more channels. When two channels open simultaneously, the total current is approximately twice that of a single channel. In most cases, the activity appears to be of a single channel. In a study, single channels in the absence of Con. A were recorded and it was established that at the resting potential, channel lifetimes are similar to those recorded in Con.A-treated muscles. There is a close correspondence between the properties of glutamate channels recorded using the patch clamp technique and those seen using noise analysis, which leads to suggest that extrajunctional d-receptors on locust muscle gate similar channels to their junctional counterparts.

Influence of sodium and calcium ions and membrane potential on glutamate receptor desensitization

1. The effects of Na, Ca and membrane potential on desensitization of postjunctional glutamate receptors on locust muscle were investigated. 2. The kinetics of desensitization were measured ionophoretically. 3. Replacement of Na by equimolar concentrations of the permeant cations Li, NH4 and guanidine and the impermeant cation choline accelerated desensitization onset, increased the steady-state leve of desensitization and reduced the rate of recovery from desensitization. 4. Desensitization onset rates and steady-state levels of desensitization were not significantly altered either by changing the extracellular Ca concentration or by changing the membrane potential of voltage clamped muscle fibres.

Analysis of Single-Channel Data from Glutamate Receptor-Channel Complexes on Locust Muscle

In view of the wealth of information available on the physiology and pharmacology of cholinergic synapses, particularly those on vertebrate excitable cells, it is not surprising that the cholinergic nicotinic receptor has been greatly influential in shaping current views of receptor pharmacology, receptor-channel kinetics, and synaptic function. But it would be unwise to assume that these views are generally applicable to other receptor systems. For many years, our laboratory has studied the noncholinergic, glutamatergic synapses found on locust leg muscle (Usherwood, 1978) and has recently demonstrated that this system offers considerable advantages for patch clamp analyses of single receptor-channel complexes. Of significance is the discovery that the kinetics of glutamate receptors on locust muscle may differ in some significant respects from their nicotinic acetylcholine receptor counterparts on vertebrate skeletal muscle.