Evolution and desensitization of LGIC receptors.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J Dudel
Publications and source records attributed to J Dudel.
A patch-clamp study on a novel gamma-aminobutyric acid activated chloride channel of crayfish deep extensor abdominal muscle.
The patch-clamp technique was used to study the effect of rapid pulses of gamma-aminobutyric acid (GABA) on excised membrane-patches in the outside-out configuration of the deep extensor abdominal muscle (DEAM) of crayfish. Channel currents reversed at the equilibrium potential of Cl- and were blocked by picrotoxin. Rare channel openings were elicited by 0.1 mM GABA, and a saturating open probability of 0.9 was reached with 10 nM GABA. The investigated channel was only sensitive to GABA and is different from a previously described GABAergic channel in crayfish that is in addition sensitive to acetylcholine and glutamate and shows three subconductance states.
Abdominal Muscles↗
Reaction scheme for the glutamate-ergic, quisqualate type, completely desensitizing channels on crayfish muscle.
In analogy with a recently developed reaction scheme for a channel activated by acetylcholine (ACh; nicotinic), we propose a cyclical reaction scheme which agrees with all data published on the completely desensitizing channel type activated by glutamate (Glu) in crayfish muscle. For every high Glu concentrations, desensitization is mainly from the open state, while at lower Glu concentrations desensitization from the singly Glu liganded closed state contributes significantly. The latter pathway and the high Glu affinity of the desensitized channel states account for complete steady-state desensitization of the channel by Glu concentrations which occur without any channel opening.
Animals↗
Desensitization of embryonic nicotinic acetylcholine receptors expressed in Xenopus oocytes.
Xenopus oocytes where injected with mRNA coding for the embryonic type of the bovine nicotinic acetylcholine receptor (nAChR). Pulses of acetylcholine (ACh) were applied to outside-out patches of the injected oocytes with the liquid filament switch. With high concentrations of ACh (10(-3) M), the ACh response desensitized rapidly; the decay could be fitted with a single exponential with a time constant of about 50 ms. In double pulse experiments, recovery from desensitization was tested. Complete recovery was observed after 5 s. The affinity of ACh to the desensitized receptor was measured by preincubating the outside-out patch with different concentrations of ACh. Preincubation with 10(-6) M ACh led to complete desensitization of the nAChRs on the patch. The results can be fitted with the molecular scheme describing activation and desensitization of the nAChR which was developed recently for embryonic nAChRs of mouse muscle.
Acetylcholine↗
A molecular scheme for the reaction between acetylcholine and nicotinic channels.
In outside-out patches of mouse-muscle membrane, embryonic-like channels were activated by pulses of acetylcholine (ACh). On increasing the ACh concentration, the rate of desensitization, 1/tau d, increased linearly with the peak open probability, indicating desensitization from the open state. Desensitization had only one time constant tau d at each ACh concentration. Recovery from desensitization was only approximately 10 times slower than desensitization, whereas the probability of steady-state channel opening, declined to < 0.01 with > 10(-6) M ACh. The peak probability of opening in > 10(-4) M ACh pulse was close to 1. A linear reaction scheme was not compatible with these results. The scheme had to be expanded resulting in a circular scheme with two additional ACh binding steps to desensitized channel states. The approximate rate constants of all reaction steps in the circular scheme could be determined using computer simulations. The model predicted that clusters of channel opening had the average duration tau d at the respective ACh concentration. In cell-attached patches on intact muscle fibers, similar average cluster durations were observed at the respective ACh concentration. This indicates that tau d in the intact muscle fibers has similar values as in outside-out patches.
Acetylcholine↗
Spatial facilitation and depression within one motor nerve terminal of frogs.
1. Perfused macropatch electrodes were used to stimulate and simultaneously measure release from two sites on the same terminal of the frog cutaneous pectoris muscle. 2. It was found that release occurring at one site often affected release at an adjacent site 50 microns away, either enhancing it ('spatial facilitation') or depressing it ('spatial depression'). Spatial facilitation (or depression) was defined as the release produced by a test pulse at the second site (test electrode) when preceded by a pulse at the first site (prepulse electrode) divided by the release produced by the test pulse alone. 3. Spatial facilitation varied with the time interval between the prepulse and the test pulse. Peak spatial facilitation, which on the average was 2.14, occurred with an interval of 1-3 ms. With longer intervals spatial facilitation decayed with a time constant between 3-6 ms. When the time interval between the prepulse and the test pulse was zero (no delay), the release after the test pulse was always depressed. 4. When Ca2+ was omitted from the perfusate of the prepulse electrode, spatial facilitation was abolished. When a brief hyperpolarizing pulse followed the depolarizing prepulse with zero delay spatial facilitation was also abolished. 5. Electrotonic spread or Ca2+ diffusion within the axon terminal are excluded as coupling agents for spatial facilitation. It is suggested that the coupling agent may possibly be related to a hypothetical release-promoting factor.
Animals↗
Recovery from the rapid desensitization of nicotinic acetylcholine receptor channels on mouse muscle.
Pulses of acetylcholine (ACh) applied to outside-out patches of embryonic-like muscle membrane elicited channel currents which declined rapidly (tau d = 10-60 ms) due to desensitization. Recovery from desensitization was determined by pulse pairs, varying the pulse interval. When the pulse interval was about 300 ms, the response to the second pulse was about half that to the first pulse, i.e. about half of the channels had recovered from desensitization. The results are discussed in the frame of a cyclic reaction scheme. If this scheme includes high affinity binding of ACh to desensitized receptors, it can also explain the finding that low ACh concentrations (less than or equal to 1 microM) largely desensitize the receptors, but elicit very little channel opening.
Acetylcholine↗
Rapid activation and desensitization of transmitter-liganded receptor channels by pulses of agonists.
Explore the source record for details and available documents.
Animals↗
Contribution of Ca2+ inflow to quantal, phasic transmitter release from nerve terminals of frog muscle.
Evoked quantal release from sections of frog endplates contained in an extracellular electrode has been investigated with Ca2+ inflow prevented by superfusing the extracellular space with a Ringer's solution containing Cd2+e or with an "intracellular", EGTA-buffered solution containing less than 0.1 microM Ca2+e. Pulse application and recording were by a perfused macro-patch-clamp electrode. The muscle outside the electrode (bath) was superfused with Ringer's solutions containing Cd2+b to block Ca2+ inflow and normal (1.8 mM) or elevated (10 mM) Ca2+b. The depolarization level of the terminal during current pulses that generated maximal Ca2+ inflow was used as unit relative depolarization. Starting from a threshold above 0.5 relative depolarization, the average release increased by a factor of about 1000 with increasing depolarization, reaching a plateau above 1.2 relative depolarization. The high level of plateau release extended to at least a relative depolarization of 4, i.e. to about +200 mV. When Ca2+ inflow was prevented in the section of the terminal within the electrode, release was depressed strongly for relative depolarizations around 1, i.e. at potentials at which Ca2+ inflow is high. However, for large depolarizations (> 1.5 relative units), the depression of release by block of Ca2+ inflow was weak or absent. The time course of release, measured in distributions of the delays of quanta after the depolarizing pulse, was unaffected by block of Ca2+ inflow. If the extra-electrode superfusion of Ca2+b of the muscle was elevated to 10 mM and Cd2+b was 0.1 mM or 0.5 mM, perfusion of the electrode with solutions below 0.1 microM Ca2+e raised the average release paradoxically. With 0.5 mM Cd2+b this paradoxical increase of release was, on average, 4-fold at 6 degrees C, and 19-fold at 16 degrees C. Quantal endplate currents recorded in less than 0.1 microM Ca2+e had slightly increased amplitudes, and decay time constants were prolonged by about 50%. The results are interpreted to support the Ca2+/voltage theory of release, which proposes that evoked, phasic release is controlled by both intracellular Ca2+ concentration and another membrane-depolarization-related factor. If the resting intracellular Ca2+ concentration is sufficiently high, large depolarizations can elicit release independent of the presence or absence of Ca2+ inflow.
Animals↗
Activation and desensitization of embryonic-like receptor channels in mouse muscle by acetylcholine concentration steps.
1. Pulses of acetylcholine (ACh) in concentrations between 0.1 and 1000 microM were applied repetitively to outside-out patches of enzymatically denervated (14 days) mouse muscle with the liquid filament switch. Solutions superfusing the patch could be changed rapidly (within 0.2 ms). 2. Single-channel activity was studied under steady-state conditions in the outside-out and in the cell-attached mode. The single-channel conductance was 26 pS in outside-out patches, characteristic for embryonic-like channels. Apparent mean open time was about 2.5 ms, a shorter component of closed times was 800 microseconds and burst length was about 5 ms. 3. Channel currents elicited by pulses of ACh were averaged. The time-to-peak current was concentration dependent and decreased from a level of about 10 ms below 10 microM to about 400 microseconds at 100 microM-ACh. 4. For a typical experiment, the average peak current, imax, increased from -0.4 pA with 0.1 microM to -82 pA with 1000 microM-ACh, close to the value at saturation. The half-maximal response was at 60 microM-ACh. The dose-response curves for imax had double-logarithmic slopes of 1.1-1.3, consistent with two binding sites at the embryonic nicotinic acetylcholine receptor (nAChR). 5. The current elicited by ACh pulses decreased rapidly after the peak. The time constant of desensitization increased from 20-50 ms with 1000 microM-ACh to up to more than a second with 1 microM-ACh. 6. The current in steady state (fully desensitized) increased up to 10 microM-ACh, but decreased slightly to values of imax/100 to imax/500 when higher concentrations were applied. 7. In addition to the well-known differences between adult and embryonic nAChR concerning the apparent mean open time and burst length, we found differences in the slope of the dose-response curve for imax, in the ratio of peak to steady-state response, and in the rise time of the response.
Acetylcholine↗
Steep concentration dependence and fast desensitization of nicotinic channel currents elicited by acetylcholine pulses, studied in adult vertebrate muscle.
Skeletal muscles of adult mice and frogs were dissociated enzymatically and prepared for patch-clamping within less than 6 h. Outside-out patches were superfused with repetitive pulses of acetylcholine (ACh) with switching times of about 0.2 ms. Peak responses were reached within 1 ms. In mouse muscle the average channel conductance was 65 pS and the average open time 1 ms (20 degrees C). Between 1 and 10 microM ACh, the peak responses increased proportional to the second to third power of the ACh concentration, and less steeply between 10 and 1000 microM ACh. The apparent Km of the dose-response curve was about 100 microM. After the peak, channel opening probability declined with time constants decreasing from about 1 s with 1 microM ACh to 15-50 ms with 1 mM ACh. After 100 ms desensitization the channel opening had decreased to less than 1/300 peak value. The rate of desensitization increased with rising temperature, with Q10 values of 1.7-2.5 between 10 and 30 degrees C. The desensitization characteristics of channels from frog muscle were similar to that from mice. With pulses of 100 microM ACh the channels opened with a probability of 0.55, the open probability declining with a time constant of about 60 ms and dropping to less than 0.001 after 300 ms. The results support the view that three binding steps of ACh are necessary for opening of the channel. Desensitization in the presence of high ACh concentrations is slower than the decay of synaptic currents.
Acetylcholine↗
Evoked phasic release in frog nerve terminals obtained after block of Ca2+ entry by Cd2+.
Cutaneous pectoris muscles of frogs were isolated, mounted in a chamber and superfused with Ringer's solution. With a macro-patch-clamp electrode placed on a section of a motor nerve terminal, quantal synaptic currents were elicited by depolarizing pulses and recorded. The electrode tip and the section of the terminal recorded from were perfused rapidly by Ringer's solution alone or containing 20-500 microM Cd2+ to block Ca2+ inflow. Separate superfusion of the muscle and the rest of the terminal with normal or elevated Ca2+ Ringer's solution provided a sufficiently high resting Ca2+ concentration in the terminal even when Ca2+ was blocked by Cd2+. The depolarization level of maximal Ca2+ inflow into the terminal was found by measuring maximal test pulse facilitation, Fc. In control solution as well as in the case of Cd2+ block, the rate of phasic release after depolarizing pulses rose further when depolarization was increased past the level of Fc, and reached a saturation level which was maintained at estimated depolarizations up to +200 mV. Block of Ca2+ inflow by Cd2+ decreased release substantially, but did not suppress it. The depression of release was greater in the range of large Ca2+ inflow (around Fc) than for very large depolarizations. The time course of phasic release was unaltered by blockage of Ca2+ inflow. It is concluded that Ca2+ inflow contributes to the promotion of evoked release only in the depolarization range in which Ca2+ inward current is large.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Kinetic constants of the acetylcholine (ACh) receptor reaction deduced from the rise in open probability after steps in ACh concentration.
Outside-out patches of enzymatically dissociated adult and denervated mouse muscle fibers were superfused repetitively by pulses of acetylcholine (ACh) containing solution. Up to 300 channels opened simultaneously 300 microseconds after the beginning of a 1,000 microM ACh pulse corresponding to a peak current i of almost -1 nA. Single responses to ACh were averaged and the concentration dependence of i and of the rise time tr from 0.1 i to 0.9 i was measured. In adult receptors, i increased proportional to the second to third power of ACh concentration, whereas in embryonic-type receptors it was proportional to the first to the second power. tr increased from approximately 0.3 ms at 1,000 microM ACh to a plateau value of approximately 5 ms for adult and of approximately 10 ms for embryoniclike receptors at concentrations less than 10 microM ACh. The concentration dependence of i and tr was simulated using the standard model of ACh binding with different combinations of rate constants and two and three binding sites for ACh. The calculated curves were compared to the measurements and a set of well fitting rate constants was determined for adult and embryoniclike receptors. Three binding sites for ACh were necessary to fit the dose response for i for adult receptors. A method for deriving rate constants in a model of ACh-receptor interaction is described that avoids analysis of open-closed kinetics of single channels, which in rapid systems, as the ones studied here, are at the limit of the frequency response of the current measurement.
Acetylcholine↗
Synaptic transmission in decentralized axons of rock lobster.
Axons of the lobster deep abdominal extensor muscles were cut, and the resulting effects on their synaptic properties were observed. Decentralized axons continued to conduct action potentials and to release transmitter for at least a year after the cut. In controls, the single quanta were brief, and their decay phase could be fit by a single exponent, with a time constant of about 2 msec. Quanta of "cut axons" were slower, and their decay phase could not be fit by a single exponent. At midamplitude, the duration of the cut-axon quanta varied between 1.6 and 5.8 msec, as opposed to 0.6-2.8 msec in controls. Synaptic delay histograms were taken as a measure of time course of evoked release. In controls, evoked release lasted less than 10 msec at 14 degrees C. In cut axons, release lasted up to 10 times longer. The duration of release was not affected by tetrodotoxin, membrane depolarization, or hyperpolarization. It appears that the basic mechanism that controls the time course of evoked release is altered in degenerating terminals.
Action Potentials↗
Depolarizing pulses to neuromuscular terminals of frogs can elicit graded, phasic transmitter release in the absence of Ca influx.
Quantal synaptic currents were recorded by means of a macro-patch-clamp electrode, through which the terminal could be also depolarized by current pulses. The tip of the electrode was perfused rapidly, applying either Ringer's solution or an EGTA-buffered less than 0.1 microM Ca Ringer's. Muscle and nerve outside the electrode were superfused with normal or 10 mM Ca Ringer which served to keep the resting intracellular Ca concentration, Cair, in the terminal below the electrode relatively high. When Ca inflow was prevented by decreasing the Ca concentration to less than 0.1 microM, release was depressed, but still measurable, for low depolarizations, and much less or not at all depressed for large depolarizations to positive membrane potentials. The time course of the depressed release without Ca-inflow was the same as that in the controls with Ca inflow. It appears that the voltage dependent activator proposed in the Ca-voltage theory of release can elicit maximal release in the absence of Ca inflow, provided Cair is sufficiently high. The voltage dependencies of this activator as well as that of Ca inflow can be estimated from the results.
Animals↗
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↗
Inhibition of Ca2+ inflow at nerve terminals of frog muscle blocks facilitation while phasic transmitter release is still considerable.
Action potentials were triggered in the motor nerve by a suction electrode and calcium currents (iCa) in the nerve terminals were measured by means of a perfused macro-patch-clamp electrode on the distal portion of the end-plates. Postsynaptic currents were blocked by adding d-tubocurarine, whereas presynaptic Na+ (iNa) and K+ (iK) currents were blocked by adding tetrodotoxin (TTX), tetraethylammonium and 3,4-diaminopyridine, respectively, to the perfusate of the electrode. The current components which could be suppressed by addition of Cd2+ to the perfusate were taken as presynaptic iCa. The observed effects on the presynaptic current components were very similar to those reported previously. If the electrode was perfused with Ringer's solution containing the blockers for iNa and iK, the same, obviously complete block of iCa was obtained by 50 and 100 microM Cd2+, an average of 96% block by 20 microM Cd2+ and 50% block by about 5 microM Cd2+. Using the same type of electrode and similar locations on motor nerve terminals, postsynaptic quantal currents and twin-pulse facilitation (Fd) were elicited by variable-duration (0.5-3 ms) depolarizing pulses. When the electrode was perfused with Ringer's solution containing TTX, 20 microM Cd2+ added to the perfusate reduced the rate of phasic release of quanta insignificantly for short depolarizing pulses and by a factor of about 10 for longer pulses. Fd was blocked almost completely. Addition of 50 microM Cd2+ to the perfusate had a greater depressive effect on release after short depolarizing pulses and reduced release after longer pulses by a factor of about 100.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Rapid activation, desensitization, and resensitization of synaptic channels of crayfish muscle after glutamate pulses.
Completely desensitizing excitatory channels were activated in outside-out patches of crayfish muscle membrane by applying glutamate pulses with switching times of approximately 0.2 ms for concentration changes. Channels were almost completely activated with 10 mM glutamate. Maximum activation was reached within 0.4 ms with greater than or equal to 1 mM glutamate. Channel open probability decayed with a time constant of desensitization of 2 ms with 10 mM glutamate and more rapidly at lower glutamate concentrations. The rate of beginnings of bursts (average number of beginnings of bursts per time bin) decayed even faster but approximately in proportion to the glutamate concentration. The dose-response curve for the channel open probability and for the rate of bursts had a maximum double-logarithmic slope of 5.1 and 4.2, respectively. Channels desensitized completely without opening at very low or slowly rising glutamate concentrations. Desensitization thus originates from a closed channel state. Resensitization was tested by pairs of completely desensitizing glutamate pulses. Sensitivity to the second pulse returned rapidly at pulse intervals between 1 and 2 ms and was almost complete with an interval of 3 ms. Schemes of channel activation by up to five glutamate binding steps, with desensitization by glutamate binding from closed states, are discussed. At high agonist concentrations bursts are predominantly terminated by desensitization. Quantal currents are generated by pulses of greater than 1 mM glutamate, and their decay is determined by the duration of presence of glutamate and possibly by desensitization.
Animals↗