The action of philanthotoxin-343 and photolabile analogues on locust (Schistocerca gregaria) muscle.

The effects of philanthotoxin-343 (PhTX-343; tyrosyl-butanoyl-spermine) and photolabile analogues of this synthetic toxin on locust (Schistocerca gregaria) skeletal muscle have been investigated using whole muscle preparations (twitch contractions), single muscle fibres (excitatory postsynaptic currents (EPSCs)) and muscle membrane patches containing single quisqualate-sensitive glutamate receptors (qGluR). Analogues containing an azido group attached to either the butanoyl side-chain of PhTX-343 or as a substitute for the hydroxyl moiety of the tyrosyl residue were about 6 fold more potent antagonists than PhTX-343; those with an azido group located at the distal end of the toxin molecule were generally 2-3 fold less potent than PhTX-343. When these compounds were tested in subdued light, they were reversible antagonists of the muscle twitch, EPSC and qGluR. When a muscle was irradiated with U.V. during application of photolabile toxin combined with either neural stimulation of the muscle or L-glutamate application, antagonism of the twitch, EPSC and qGluR was complete and irreversible.

Reconstitution of glutamate receptor proteins purified from Xenopus central nervous system into artificial bilayers.

Excitatory amino acid (EAA) receptor (EAAR) proteins purified from Xenopus central nervous system using a domoate affinity column and then separated into fractions using sucrose density gradient centrifugation were reconstituted, first into liposomes and then into planar lipid bilayers, using pipette-dipping and black lipid membrane techniques. Although the protein was eluted from the column with either alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) or kainate and could not be eluted with N-methyl-D-aspartate (NMDA), channel openings were obtained after exposure of the bilayers to kainate, AMPA, or NMDA (usually only in the presence of glycine). In bilayers exhibiting a single open channel conductance level this was approximately 6 pS with AMPA, approximately 9 pS with kainate, and approximately 50 pS with NMDA. However, with a few batches of protein unitary channel openings of up to 400 pS were observed, suggesting that reconstituted EAAR may sometimes form functional aggregates. The protein eluted from the domoate column was divided into two fractions on a sucrose density gradient. After reconstitution, one fraction responded to all three EAAs, whereas the other responded only to the non-NMDA receptor agonists. An explanation for these results is that some of the EAAR eluted from the column contain NMDA receptor subunits in addition to non-NMDA receptor subunits.

Quisqualate-sensitive glutamate receptors of the locust Schistocerca gregaria are antagonised by intracellularly applied philanthotoxin and spermine.

The effects of intracellularly and extracellularly applied synthetic analogues of delta-philanthotoxin (PhTX-433) and the polyamine spermine on the excitatory postsynaptic current (EPSC) of glutamatergic synapses and single channel currents gated by quisqualate-sensitive glutamate receptors (QUIS-R) on locust leg muscle have been compared. When applied extracellularly all 3 compounds reversibly antagonised the EPSC and the single channel currents. Antagonism was voltage independent, but use (agonist) dependent. Antagonism also occurred when they were injected into muscle fibres, but in this case it was not use dependent. It is proposed that spermine and the two toxins bind to the closed and open channel conformations of QUIS-R at a site near the intracellular opening of the channel gated by this receptor.

Markov, fractal, diffusion, and related models of ion channel gating. A comparison with experimental data from two ion channels.

The gating kinetics of single-ion channels are generally modeled in terms of Markov processes with relatively small numbers of channel states. More recently, fractal (Liebovitch et al. 1987. Math. Biosci. 84:37-68) and diffusion (Millhauser et al. 1988. Proc. Natl. Acad. Sci. USA. 85:1502-1507) models of channel gating have been proposed. These models propose the existence of many similar conformational substrates of the channel protein, all of which contribute to the observed gating kinetics. It is important to determine whether or not Markov models provide the most accurate description of channel kinetics if progress is to be made in understanding the molecular events of channel gating. In this study six alternative classes of gating model are tested against experimental single-channel data. The single-channel data employed are from (a) delayed rectifier K+ channels of NG 108-15 cells and (b) locust muscle glutamate receptor channels. The models tested are (a) Markov, (b) fractal, (c) one-dimensional diffusion, (d) three-dimensional diffusion, (e) stretched exponential, and (f) expo-exponential. The models are compared by fitting the predicted distributions of channel open and closed times to those observed experimentally. The models are ranked in order of goodness-of-fit using a boot-strap resampling procedure. The results suggest that Markov models provide a markedly better description of the observed open and closed time distributions for both types of channel. This provides justification for the continued use of Markov models to explore channel gating mechanisms.

Single channel studies of non-competitive antagonism of a quisqualate-sensitive glutamate receptor by argiotoxin636--a fraction isolated from orb-web spider venom.

The effects of purified spider toxin (argiotoxin636) on single glutamate-activated channels in voltage-clamped locust muscle fibres have been examined using a megaohm seal, patch-clamp technique. Four experimental protocols were employed in which the composition of the patch pipette and bathing solutions were varied. Three types of channel behaviour were broadly defined when argiotoxin636 was present either in the patch pipette or in the muscle bath; the type of channel behaviour being dependent upon the concentration of argiotoxin636 and/or the duration of its application. Type I behaviour was characterized by reductions in channel open probability (Po) and channel event frequency (f), by an increase in mean channel closed time (mc) and either no change in mean channel open time (mo) or, infrequently, an increase in this parameter; Type II behaviour was characterized by apparent absence of channel openings. For example, with 10(-12) M argiotoxin636 in the patch pipette Type I behaviour changed to Type II behaviour after approximately 60 s and from Type II behaviour to Type III behaviour after approximately 120 s. The results of this study are consistent with the idea that argiotoxin636 blocks the cation-selective channel gated by excitatory glutamate receptors in insect muscle at the level of the open channel although there remains the possibility that it is also either a closed channel blocker and/or a competitive antagonist. The increase in mo seen in a few recordings during the initial stage of argiotoxin636 antagonism raises the possibility that the toxin interacts allosterically with the glutamate binding sites on the excitatory glutamate receptor.

The use of dwell time cross-correlation functions to study single-ion channel gating kinetics.

The derivation of cross-correlation functions from single-channel dwell (open and closed) times is described. Simulation of single-channel data for simple gating models, alongside theoretical treatment, is used to demonstrate the relationship of cross-correlation functions to underlying gating mechanisms. It is shown that time irreversibility of gating kinetics may be revealed in cross-correlation functions. Application of cross-correlation function analysis to data derived from the locust muscle glutamate receptor-channel provides evidence for multiple gateway states and time reversibility of gating. A model for the gating of this channel is used to show the effect of omission of brief channel events on cross-correlation functions.

Glutamate receptor channel kinetics: the effect of glutamate concentration.

Single channel recordings from the locust muscle D-glutamate receptor channel were obtained using glutamate concentrations ranging from 10(-6) to 10(-2) M. Channel kinetics were analyzed to aid in the development of a model for the gating mechanism. Analysis of channel dwell time histograms demonstrated that the channel possessed multiple open and closed states at concentrations of glutamate between 10(-5) and 10(-2) M. Correlations between successive dwell times showed that the gating mechanism was nonlinear (i.e., branched or cyclic) over the same glutamate concentration range. The glutamate concentration dependence of the channel open probability, and of the event frequency, was used to explore two possible allosteric gating mechanisms in more detail.

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the presence of 10(-4) M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.