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R L Ramsey

Publications and source records attributed to R L Ramsey.

At least 19 recordsLinked to original sources
PubMed1996

Potassium channels of adult locust (Schistocerca gregaria) muscle.

Two types of K+ channels have been identified in patches of plasma membrane of metathoracic extensor tibiae muscle fibres of adult locust, Schistocerca gregaria. One channel had a maximum conductance of 170 pS, fast open-closed kinetics, and a linear current/ voltage relationship. In inside-out patches it was activated by "internally applied" Ca2+, but at unexpectedly low levels (between 10(-10) and 10(-9)M). The other channel had a maximum conductance of 35 pS, slower open-closed kinetics, and was not activated by Ca2+. In cell-attached patches, its channel conductance measured in symmetrical salines was about three times greater for hyperpolarisations than for depolarisations. This inward rectification was proved to be due to block by intracellular Mg2+. For both channels, open probability (Po) and mean open time increased during depolarisations and decreased during hyperpolarisations, resulting in outward rectifications in terms of net current (I n, product of the single-channel current and Po). For both channels, the K+ conductance was 10 times greater than that for Na+. Internally applied tetraethylammonium or tetramethylammonium ions blocked both channels.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

Channel gating in the absence of agonist by a homo-oligomeric molluscan GABA receptor expressed in Xenopus oocytes from a cloned cDNA.

We have previously described the isolation of a complementary DNA (cDNA) from the freshwater mollusc Lymnaea stagnalis encoding a polypeptide that exhibits approximately 50% identity to the beta-subunits of vertebrate gamma-aminobutyric acid (GABA) type A (GABAA) receptor. When expressed in Xenopus laevis oocytes from in vitro-transcribed RNA, the snail subunit forms functional homo-oligomeric receptors possessing chloride-selective ion channels. In recordings from voltage-clamped oocytes held at -60 mV, GABA induced an inward current, whereas application of the chloride-channel blocker picrotoxin (in the absence of agonist) elicited an apparent outward current. Single channel recordings obtained from cell-attached patches have revealed a single population of approximately 20 pS channels, with an open probability greater than 90% (at a pipette potential of -100 mV) in the absence of GABA. The relationship between single channel current and pipette potential was linear over the studied range (-100 mV to +60 mV), but the open probability was less for hyperpolarizations than for depolarizations. The spontaneous channel openings were blocked by micromolar concentrations of picrotoxin. Functional hetero-oligomeric receptors were formed when the molluscan subunit was co-expressed in oocytes with the bovine GABAA receptor alpha 1-subunit, but the channels gated by these receptors did not open spontaneously.

Animals↗
PubMed1993

Effects of high pressure on the channel gated by the quisqualate-sensitive glutamate receptor of locust muscle and its blockade by ketamine; a single-channel analysis.

The effects of high pressure on the channel gating kinetics of the quisqualate-sensitive L-glutamate receptor (qGluR) of locust muscle have been investigated using a megaohm seal patch-clamp technique. Pressure was applied with helium gas and recordings were carried out at 20.5 degrees C with Rb+ as the main charge-carrying cation in the patch pipette. The mean open time of the qGluR channel was unaffected by 10 and 30 MPa, but it was significantly reduced at 50 MPa. A high proportion of brief openings (mean 0.808 ms) was seen at 50 MPa but not at lesser pressures. Also, in contrast to lesser pressures, 50 MPa prolonged the mean closed time and reduced both the frequency and probability of channel opening. 10(-6) M ketamine significantly reduced the mean channel open time, as previously reported. A pressure of 10 MPa which alone had no effect on the qGluR channel, restored the mean open time in the presence of 10(-6) M ketamine to the value obtained in the absence of the anaesthetic. This implies the shortening of qGluR channel open time by ketamine involves a large + delta V and, therefore, probably conformational changes in the channel. However 10 MPa did not restore the distribution of open times to normal.

Animals↗
PubMed1993

Gating kinetics of the quisqualate-sensitive glutamate receptor of locust muscle studied using agonist concentration jumps and computer simulations.

Outside-out patches excised from extrajunctional membrane of locust muscle were subjected to "concentration jumps" of L-glutamate, using the liquid filament switch technique, to study channel opening and closing rates, desensitization onset, and recovery from desensitization of a quisqualate-sensitive glutamate receptor (qGluR). Based on data obtained from these experimental studies, computer modeling techniques have been used in an attempt to simulate the behavior of qGluR during a concentration jump of L-glutamate. A linear model with three closed states (one unliganded, one monoliganded, and one biliganded), one open state (binding two molecules of L-glutamate), and two desensitization states (the one monoliganded, the other biliganded) leading from the unliganded closed state simulated all of the experimentally observed behavior. The results are discussed in the context of previous equilibrium studies in which desensitization was inhibited with concanavalin A and for which a ten-state model was required to simulate the behavior of qGluR.

Animals↗
PubMed1992

Single channel analysis of ketamine interaction with a quisqualate receptor.

The interactions of the general anaesthetic ketamine with the quisqualate-sensitive L-glutamate receptor (QUIS-R) of locust muscle have been investigated at the single channel level using a M omega seal patch clamp technique. Low concentrations (10(-10) to 10(-9) M) of ketamine did not significantly alter the kinetics of the QUIS-R channel. Higher concentrations of ketamine decreased the probability of the channel being open, the frequency of channel opening and the channel mean open time, and increased the channel mean closed time. Probability density functions of channel dwell times indicate that during application of greater than 10(-8) M ketamine the distribution of channel openings becomes restricted mainly to brief events. These results are consistent with the view that ketamine blocks the open, and possibly also the closed, channel of locust muscle QUIS-R and that this anaesthetic dissociates only slowly from its blocking site(s).

Animals↗
PubMed1990

Glutamatergic channels in locust muscle show a wide time range of desensitization and resensitization characteristics.

Outside-out patches of membrane were excised from locust muscle. 10 mM L-glutamate applied to such patches in short pulses elicited rapidly a peak of channel opening, followed by desensitization with time constants between 1 ms and 20 ms for different channel subtypes. Slowly rising glutamate concentrations could produce almost complete desensitization without any channel openings. Desensitization thus proceeds from a closed channel state, prior to channel opening. Resensitization, i.e. recovery from desensitization, was tested by applying pairs of glutamate pulses, varying the pulse interval. Resensitization has a biphasic time course. While in rapidly desensitizing channels more than 50% of resensitization was achieved within a few milliseconds, in slowly desensitizing channels resensitization took seconds.

Animals↗
PubMed1990

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.

Animals↗
PubMed1989

Curvature-electric effects in artificial and natural membranes studied using patch-clamp techniques.

Methods for applying sound pressure to membrane patches formed at the tips of patch-clamp pipettes have been developed. Artificial membrane patches were formed from diphytanoyl phosphatidylcholine using a pipette dipping technique. Natural membrane patches were excised (inside-out mode) from collagenase-treated locust muscle membrane. Curvature-electric signals were registered under both voltage clamp and current clamp conditions. The phenomenon of flexoelectricity in membranes has previously been attributed to curvature-induced polarization originating from the liquid crystalline properties of membranes. The estimated magnitude (2 x 10(-18) C) of the flexoelectric coefficient of the artificial lipid bilayers is consistent with previous findings while that of the muscle membrane was in certain cases several times larger. The present study is the first to report on flexoelectricity in a natural membrane and raises the question of the biological significance of this phenomenon.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

Enhancement of desensitization of quisqualate-type glutamate receptor by the dissociative anaesthetic ketamine.

Application of ketamine (10(-4)-10(-3)mol l-1) to locust retractor unguis muscle produced a reversible, dose-dependent reduction in neurally evoked twitches, and blocked agonist-induced contractions. With increasing ketamine concentration (5 x 10(-5)-10(-3) mol l-1), the amplitude of glutamate potentials was reduced and dose-response curves for ionophoresis of L-glutamate were shifted to the right, particularly after concanavalin A treatment. Ketamine (10(-4) mol l-1) enhanced the rate of desensitization to consecutive pulses of L-glutamate and this action was eliminated by concanavalin A. The amplitude of the excitatory postsynaptic current (EPSC) was reduced by ketamine (10(-5)-5 x 10(-4) mol l-1) in a dose-dependent manner but without a concomitant reduction in EPSC rise time. The decay phase of the EPSC was usually biphasic in the presence of ketamine (greater than 5 x 10(-5) mol l-1) but did not exhibit any voltage dependence. It is concluded that ketamine enhances desensitization and blocks the channel, particularly the closed form.

Action Potentials↗
PubMed1988

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.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

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.

Ion Channels↗
PubMed1987

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.

Animals↗
PubMed1985

5-Hydroxytryptamine-activated single-channel currents recorded from murine neuroblastoma cells.

Differentiated murine neuroblastoma cells (NIE-115) have receptors for 5-hydroxytryptamine (5-HT), the activation of which either depolarizes or hyperpolarizes these cells with associated increases in input conductance. Ionophoresis of 5-HT on to NIE-115 cells elicits brief depolarization, but the 5-HT receptors rapidly desensitize during repeated application of 5-HT. The reversal potential of 5-HT-induced depolarization is ca. 0 mV. With the use of the improved patch clamp technique it is possible to record single ion channel currents activated by 5-HT from intact cells as well as isolated membrane patches. These currents are invariably inward at the resting potential (approximately 50 mV). The channel has a conductance of approximately 140 pS and reversal potential of approximately 0 mV in normal saline. The channels recorded from the intact cells reveal more than one kinetically distinguishable open and closed states. The kinetic properties of 5-HT channels recorded from excised patches of membrane (as outside-out) are different from those recorded from the intact cell. Possible explanations for such differences are presented.

Action Potentials↗