Robert L. Ramsey

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.

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.

Rapid activation and desensitization by glutamate of excitatory, cation-selective channels in locust muscle

Outside-out patches of membrane were excised from extensor tibiae muscles of locusts. L-Glutamate or its agonists were applied to such patches in short pulses by means of a lipid filament switch. Cationselective, excitatory channels were activated by quisqualate, L-glutamate and aspartate (in decreasing order of effectivity), but not by ibotenate, kainate, N-methyl-D-aspartate and glycine. At high agonist concentrations, channel activation reached a peak within 1 ms. Two kinetic types of channels have been identified: L-channels with on average relatively long and S-channels with short openings. Both types of channel openings showed surprisingly high rates of desensitization, channel activity declining after the initial surge to zero with time constants of about 25 and 3 ms, respectively. The L-channels exhibit open times close to those of channels recorded in M omega-seal studies. The S-channel has not been reported previously.

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.

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.

The effects of nitrous oxide on a glutamate-gated ion channel and their reversal by high pressure; a single channel analysis

Nitrous oxide reversibly affects the kinetics, but not the conductance, of the qGluR channel of locust muscle. 0.5 atm N2O at 20.5 degrees C was without effect but both 1.5 and 2.7 atm significantly reduced the probability of the channel opening, the frequency of opening and the mean open time, and prolonged the mean closed time. 100 atm helium was without effect on these parameters, but when 98.5 atm He was combined with 1.5 atm N2O they, and the associated dwell time distributions, were restored to normal. 100 atm similarly combined with 2.7 atm N2O exerted a comparable trend which fell short of significance. The results are consistent with nitrous oxide binding to the channel with a significant molar volume increase, which pressure opposes. This suggests that nitrous oxide may cause conformational changes in the channel, and that the pressure reversal of nitrous oxide anaesthesia in animals could be caused by molecular antagonism.

Agonist potency determination by patch clamp analysis of single glutamate receptors

Agonists (L-quisqualate, L-glutamate and L-cysteine sulphinate) of the locust muscle glutamate receptor differ in potency. Patch clamp analysis of single glutamate receptors reveals that the relative potencies of agonists are determined both by the life-times of the channels that they gate and their probability of activating the glutamate receptor-ionophore complex. Furthermore, agonists which most readily activate the receptor channel complex gate channels with longer life-times than agonists which are less efficient in this respect.

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 gating : maximum likelihood analysis of gigaohm seal recordings from locust muscle

Gigaohm recordings have been made from glutamate receptor channels in excised, outside-out patches of collagenase-treated locust muscle membrane. The channels in the excised patches exhibit the kinetic state switching first seen in megaohm recordings from intact muscle fibers. Analysis of channel dwell time distributions reveals that the gating mechanism contains at least four open states and at least four closed states. Dwell time autocorrelation function analysis shows that there are at least three gateways linking the open states of the channel with the closed states. A maximum likelihood procedure has been used to fit six different gating models to the single channel data. Of these models, a cooperative model yields the best fit, and accurately predicts most features of the observed channel gating kinetics.