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High-Resolution measurements of single-channel currents activated by glutamate in crayfish muscle
Neuroscience Letters, 59 (1985) 241-246
241
Elsevier Scientific Publishers Ireland Ltd. NSL 03480 HIGH-RESOLUTION MEASUREMENTS OF SINGLE-CHANNEL C U R R E N T S A C T I V A T E D BY G L U T A M A T E IN C R A Y F I S H M U S C L E
Ch. FRANKE and J. DUDEL Physiologisches Institut der Technischen Universitiit M,Tnchen, Biedersteinerstrasse 29, D-8000 Miinchen 40 (F.R.G.)
(Received April 17th, 1985;Accepted May 29th, 1985)
Key words: singlesynaptic channel - glutaminerglc synapse - kinetics of channel opening - sublevels of
channel current - crayfishneuromuscularjunction
Patch-clamp pipettes filled with 50-5000 #M glutamate were placed on crayfish muscle fibers treated with collagenase, formed Gf~ seals and elicited single-channel currents with a main amplitude of about - 8 pA at -70 mV membrane potential, representing a conductance of about 100 pS (19°C). Evaluation of the channel openingslonger than 1 ms yieldedthree sublevels of this conductance. The channels opened in bursts, the durations of which were distributed in two exponential components with time constants of about 0.1 and 0.3 ms at low glutamate concentrations, which rose to about 0.4 and 1.8 ms, respectively, at high glutamate concentrations. The distributions of closed times could be described by three time constants which also varied with glutamate concentration. Comparison of the burst durations with the decay time constants of natural synaptic currents indicates effective glutamate concentrations in the millimolar range during transmission.
Single-channel currents operated by glutamate have been recorded in muscles of locusts [2, 11], crayfish [5] and rock lobsters [10]. Especially in crustaceans, they showed very short open times and rapid flickering in bursts. The patch clamps had seals of some 100 Mfl, and the bandwidth of the recordings limited their interpretation seriously. Since the rapidly transmitting glutaminergic synapse is o f general interest also in vertebrates, we applied the improved patch-clamp techniques proposed by Hamill et al. [7] obtaining records with much improved resolution. The preparation was the lateral abdominal extensor muscle o f crayfish (Austropotamobius torrentium) [9], superfused with normal solution at 19°C [5]. The preparation was treated for 1.5-4 h with a 1 mg/ml collagenase (Sigma; type Ia) solution at 15-17°C. The patch-clamp amplifier contained a 50 Gf~ feed-back resistor and frequency compensation [13]. The patch-clamp pipettes had fire-polished tips with about l-#m openings which were coated with Syigard. They were filled with a bathing solution which usually contained glutamate. Touching the muscle surface in synaptic regions, Gfl seals were established in about 10~ of the trials. I f the pipette contained glutamate, single-channel currents appeared in 80~o of such patches. The recordings were stored on F M tape with a 10 kHz bandwidth. For evalution they were digitized at 100 kHz and displayed by a Motorola Exorset 30 computer which
242
provided an operator-controlled threshold crossing program for measurement of channel open times, and constructed histograms of distributions of open and closed times. Fig. 1 shows an example of bursts of openings triggered by 5 mM glutamate. At the resting potential of - 70 mV, the main amplitude of the current steps was about - 8 pA, which more than doubled on hyperpolarization to - 170 mV. The currentvoltage relation was linear between - 2 1 0 and + 70 mV, with a reversal potential at + 10 to + 2 0 mV and a slope of about 100 pS. Some of the current steps in Fig. 1 do not reach the full amplitude o f - 8 pA, and it is not clear whether these represent steps which are too short to be fully resolved or true 'sublevels' of incomplete channel openings or closings, as reported for some acetylcholine-, glycine- or ?-aminobutyric acid-activated synapses [1, 8]. Amplitudes of current steps were evaluted for the small subset lasting more than 1 ms with a noise level not larger than at rest, as shown in Fig. 2 for a recording elicited by 1 m M glutamate. In addition to the main open state of - 8 . 2 pA, three further sublevels are obvious at about - 6 . 2 , - 4 . 5 and - 2 . 5 pA. The absolute rates of occurrence of these substates cannot be given because only longer openings could be evaluated. The lower substates occurred at all glutamate concentrations but appeared to be relatively more frequent at lower concentrations (50-100/~M). They may represent spontaneous oscillations of the conformation of the channel, or they may reflect the binding of less than the optimal number of glutamate molecules to the receptor. The selected short recordings in Figs. 1 and 2 show channels open most o f the time, but this is misleading, as obvious in the longer recordings at 0.2 and 5 m M glutamate in Fig. 3. These results are representative for 8 experiments at the respective concentrations. For the evaluation of a burst length, the burst [12] was defined as an opening with gaps shorter than 0.4 ms, which comprise more than 95~o of the short closings
5pAI ~ c~
-,,o
m,
Fig. 1. Recordings of bursts of cha_n.nelopenings elicited by 5 mM glutamate, at resting potential of - 7 0
mV and hyperpolarizedto - 170 inV. Recordsplayed back from tape: band~th, 10 kHz; rise time, < 50 las; resting noise, < 1.5 pA r.m.s.
243 1 mM GLUTAMATE
i
60
20 ms
=
N = 473
1
z_ 40 rn cF
CO
~: 20u.J
!
-2
-4
-6
CURRENT AMPLITUDE
-8
-10
(oA)
Fig. 2. Amplitude distribution o f current levels of more than 1 m s duration, obtained in a recording filtered at 2.5 kHz. n = 4 7 3 events evaluated. G l u t a m a t e concentration 1 m M . Inset: sample record filtered at 2.5 kHz.
at 5 mM glutamate (Fig. 3, middle diagram). These bursts with short gaps (1.2 gaps/ burst) are spaced at about 100-ms intervals on average (lowest diagram). At the lower glutamate concentration of 0.2 mM short gaps are relatively rare (0.13 gaps/burst), and the mostly single openings are separated by closed times distributed around the time constants of 1.6 and 22 ms (Fig. 3, lowest diagram). The distributions of burst durations (Fig. 3, upper diagrams) each show two time constants: 0.12 and 0.44 ms at 0.2 mM glutamate, and 0.36 and 1.75 ms at 5 mM glutamate. The openings thus were almost four times longer at 5 mM glutamate than at 0.2 mM. However, 70 burst/s were seen at 0.2 mM glutamate compared to 10 bursts/s at 5 mM, and consequently total open time was shorter at 5 mM glutamate than at 0.2 mM. It should be noticed that the macroscopic synaptic current elicited by superfusion of 0.2 mM glutamate for 1 s is already in the saturation range [3], and desensitizes in longer applications with a time constant of about 1 s [3, 4, 14]. All recordings shown here, therefore, are from deeply desensitized receptors, and it is astonishing that any responses were seen at all. However, at 50/zM glutamate there is usually no desensitization and synaptic current reaches about 10% of the saturation level [3]. Gf~ seal recordings of single channels at 50 #M glutamate (not illustrated) yield very short openings distributed with time constants of 0.35 ms and 0.1 ms, occurring at a rate of about 5/s. This agrees with the previous results at lower resolution [5], which only overestimated the contribution of the shortest openings (probably misinterpreting lower open states). The prolongation of single openings or bursts with increasing glutamate concentration thus starts from a range without desensitization and should be a true effect of glutamate concentration on the open state. An even stronger depen-
244
0.2 mM GLUTAMATE
5 mM GLUTAMATE
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Fig. 3. Recordings and distributions of burst durations and closed times of single channels for 0.2 mM (left-hand column) and 5 m M glutamate (right=hand column). 19°C. At 0.2 m M g l ~ a m a t e , 5306 bursts were evaluated in a 74-s recording, while at 5 mM glutamate 960 bursts were evaluated in a 89-s recording. The distributions show the evaluated number of events (logarithmic scales) per bin, i.e. per time intervals of 0 . 1 . 0 . 0 4 or 5 ms, respectively, as indicated in the different distributions. The respective distributions were fitted by one or two exponentials (continuous curves) characterized by theh- time constants which are indicated next to the curves. In the distributions of burst durations, the longer component of 0.44 or 1.75 ms, respectively, accounted for more than 80°,/0 of the sample.
245
dence of duration of bursts on the glutamate concentration has been reported for locust muscle [6]. The rate of bursts rises when the glutamate concentration increases from low levels (up to 0.2 mM), but at higher concentrations desensitization depresses the rate of channel openings. This effect of desensitization may be even more dramatic than apparent in Fig. 3, since more than one channel probably is present in the patch (although no superpositions were observed), and different channels may become active from the desensitized state, as concluded also for acetylcholine receptors [ 12]. The observed dependence of burst duration on glutamate concentration may be used for an interpretation of synaptic currents elicited by quanta of transmitter. Occasionally, spontaneous synaptic currents appeared in patch-clamp recordings which showed amplitudes of up to - 1.5 nA and decayed with time constants of 0.8-2 ms, and similar values were also obtained in macro-patch-clamp ( ~ 50/#m opening) measurements (Franke and Dudel, unpublished). These time constants are much longer than the 0.4 ms observed at 0.2 mM glutamate (Fig. 3) but agree with burst durations found for millimolar concentrations of glutamate in the pipette. A quantum of transmitter thus may present a glutamate concentration around 1 mM at the receptors and open 100-200 channels almost simultaneously. The authors wish to thank Mr. W. Zeitz for programming of the computer, Miss I. Horstmann for technical assistance and Mrs. M. Griessl for secretarial help. This investigation was supported by the Deutsche Forschungsgemeinschaft. 1 Auerbach, A. and Sachs, F., Flickering of a nicotinic ion channel to a subconductance state, Biophys. J., 42 (1983) 1-10. 2 Cull-Candy, S.G. and Parker, I., Rapid kinetics of single glutamate-receptor channels, Nature (Lond.), 295 (1982) 410-412. 3 Dudel, J., Dose-response curve of glutamate applied by superfusion to crayfish muscle synapses, Pfliigers Arch., 368 (1977) 49-54. 4 Dudel, J., Aspartate and other inhibitors of excitatory synaptic transmission in crayfish muscle, Pfliigers Arch., 369 (1977) 7--16. 5 Franke, C., Dudel, J. and Finger, W., Single synaptic channels recorded at glutamate sensitive patches on a crayfish muscle, Neurosci. Lett., 42 (1983) 7-12. 6 Gration, K.A.F., Lambert, J.J., Ramsey, R. and Usherwood, P.N.R., Non-random openings and concentration-dependent lifetimes of glutamate-gated channels in muscle membrane, Nature, (Lond.), 291 (1981) 423-425. 7 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Nature (Lond.), 305 (1981) 85-100. 8 Hamill, O.P., Bormann, J. and Sakmann, B., Activation of multiple-conductance state chloride channels in spinal neurones by glycine and GABA, Nature (Lond.), 305 (1983) 805-808. 9 Parnas, I. and Atwood, H.L., Phasic and tonic neuromuscular systems in the abdominal extensor muscles of the crayfish and rock lobster, Comp. Biochem. Physiol., ! 8 (1966) 701-723. 10 Parnas, I., Dudel, J., Cohen, I. and Franke, Ch., Strengthening of synaptic contacts of an excitatory axon on elimination of a second excitatory axon innervating the same target, J. Neurosci., 4 (1984) 1912-1923. I 1 Patlak, J.B., Gration, K.A.F. and Usherwood, P.N.R., Single glutamate-activated channels in locust muscle, Nature (Lond.), 278 (1979) 643~J45.
246 12 Sakmann, B., Patlak, J. and Neher, E., Single acetylcholine-activated channels show burst-kinetics in presence of desensitizing concentrations of agonist, Nature (Lond.), 286 (1980) 71-73. 13 Sigworth, F.J., Electronic design of the patch clamp. In B. Sakmann and E. Neher (Eds.), Single-Channel Recording, Plenum Press, New York, London, 1983, Chapt. 1, pp. 3-35. 14 Stettmeier, H., Finger, W. and Dudel, J., Glutamate activated postsynaptic channels in crayfish muscle investigated by noise analysis, Pfl/igers Arch., 397 (1983) 13-19.
241
Elsevier Scientific Publishers Ireland Ltd. NSL 03480 HIGH-RESOLUTION MEASUREMENTS OF SINGLE-CHANNEL C U R R E N T S A C T I V A T E D BY G L U T A M A T E IN C R A Y F I S H M U S C L E
Ch. FRANKE and J. DUDEL Physiologisches Institut der Technischen Universitiit M,Tnchen, Biedersteinerstrasse 29, D-8000 Miinchen 40 (F.R.G.)
(Received April 17th, 1985;Accepted May 29th, 1985)
Key words: singlesynaptic channel - glutaminerglc synapse - kinetics of channel opening - sublevels of
channel current - crayfishneuromuscularjunction
Patch-clamp pipettes filled with 50-5000 #M glutamate were placed on crayfish muscle fibers treated with collagenase, formed Gf~ seals and elicited single-channel currents with a main amplitude of about - 8 pA at -70 mV membrane potential, representing a conductance of about 100 pS (19°C). Evaluation of the channel openingslonger than 1 ms yieldedthree sublevels of this conductance. The channels opened in bursts, the durations of which were distributed in two exponential components with time constants of about 0.1 and 0.3 ms at low glutamate concentrations, which rose to about 0.4 and 1.8 ms, respectively, at high glutamate concentrations. The distributions of closed times could be described by three time constants which also varied with glutamate concentration. Comparison of the burst durations with the decay time constants of natural synaptic currents indicates effective glutamate concentrations in the millimolar range during transmission.
Single-channel currents operated by glutamate have been recorded in muscles of locusts [2, 11], crayfish [5] and rock lobsters [10]. Especially in crustaceans, they showed very short open times and rapid flickering in bursts. The patch clamps had seals of some 100 Mfl, and the bandwidth of the recordings limited their interpretation seriously. Since the rapidly transmitting glutaminergic synapse is o f general interest also in vertebrates, we applied the improved patch-clamp techniques proposed by Hamill et al. [7] obtaining records with much improved resolution. The preparation was the lateral abdominal extensor muscle o f crayfish (Austropotamobius torrentium) [9], superfused with normal solution at 19°C [5]. The preparation was treated for 1.5-4 h with a 1 mg/ml collagenase (Sigma; type Ia) solution at 15-17°C. The patch-clamp amplifier contained a 50 Gf~ feed-back resistor and frequency compensation [13]. The patch-clamp pipettes had fire-polished tips with about l-#m openings which were coated with Syigard. They were filled with a bathing solution which usually contained glutamate. Touching the muscle surface in synaptic regions, Gfl seals were established in about 10~ of the trials. I f the pipette contained glutamate, single-channel currents appeared in 80~o of such patches. The recordings were stored on F M tape with a 10 kHz bandwidth. For evalution they were digitized at 100 kHz and displayed by a Motorola Exorset 30 computer which
242
provided an operator-controlled threshold crossing program for measurement of channel open times, and constructed histograms of distributions of open and closed times. Fig. 1 shows an example of bursts of openings triggered by 5 mM glutamate. At the resting potential of - 70 mV, the main amplitude of the current steps was about - 8 pA, which more than doubled on hyperpolarization to - 170 mV. The currentvoltage relation was linear between - 2 1 0 and + 70 mV, with a reversal potential at + 10 to + 2 0 mV and a slope of about 100 pS. Some of the current steps in Fig. 1 do not reach the full amplitude o f - 8 pA, and it is not clear whether these represent steps which are too short to be fully resolved or true 'sublevels' of incomplete channel openings or closings, as reported for some acetylcholine-, glycine- or ?-aminobutyric acid-activated synapses [1, 8]. Amplitudes of current steps were evaluted for the small subset lasting more than 1 ms with a noise level not larger than at rest, as shown in Fig. 2 for a recording elicited by 1 m M glutamate. In addition to the main open state of - 8 . 2 pA, three further sublevels are obvious at about - 6 . 2 , - 4 . 5 and - 2 . 5 pA. The absolute rates of occurrence of these substates cannot be given because only longer openings could be evaluated. The lower substates occurred at all glutamate concentrations but appeared to be relatively more frequent at lower concentrations (50-100/~M). They may represent spontaneous oscillations of the conformation of the channel, or they may reflect the binding of less than the optimal number of glutamate molecules to the receptor. The selected short recordings in Figs. 1 and 2 show channels open most o f the time, but this is misleading, as obvious in the longer recordings at 0.2 and 5 m M glutamate in Fig. 3. These results are representative for 8 experiments at the respective concentrations. For the evaluation of a burst length, the burst [12] was defined as an opening with gaps shorter than 0.4 ms, which comprise more than 95~o of the short closings
5pAI ~ c~
-,,o
m,
Fig. 1. Recordings of bursts of cha_n.nelopenings elicited by 5 mM glutamate, at resting potential of - 7 0
mV and hyperpolarizedto - 170 inV. Recordsplayed back from tape: band~th, 10 kHz; rise time, < 50 las; resting noise, < 1.5 pA r.m.s.
243 1 mM GLUTAMATE
i
60
20 ms
=
N = 473
1
z_ 40 rn cF
CO
~: 20u.J
!
-2
-4
-6
CURRENT AMPLITUDE
-8
-10
(oA)
Fig. 2. Amplitude distribution o f current levels of more than 1 m s duration, obtained in a recording filtered at 2.5 kHz. n = 4 7 3 events evaluated. G l u t a m a t e concentration 1 m M . Inset: sample record filtered at 2.5 kHz.
at 5 mM glutamate (Fig. 3, middle diagram). These bursts with short gaps (1.2 gaps/ burst) are spaced at about 100-ms intervals on average (lowest diagram). At the lower glutamate concentration of 0.2 mM short gaps are relatively rare (0.13 gaps/burst), and the mostly single openings are separated by closed times distributed around the time constants of 1.6 and 22 ms (Fig. 3, lowest diagram). The distributions of burst durations (Fig. 3, upper diagrams) each show two time constants: 0.12 and 0.44 ms at 0.2 mM glutamate, and 0.36 and 1.75 ms at 5 mM glutamate. The openings thus were almost four times longer at 5 mM glutamate than at 0.2 mM. However, 70 burst/s were seen at 0.2 mM glutamate compared to 10 bursts/s at 5 mM, and consequently total open time was shorter at 5 mM glutamate than at 0.2 mM. It should be noticed that the macroscopic synaptic current elicited by superfusion of 0.2 mM glutamate for 1 s is already in the saturation range [3], and desensitizes in longer applications with a time constant of about 1 s [3, 4, 14]. All recordings shown here, therefore, are from deeply desensitized receptors, and it is astonishing that any responses were seen at all. However, at 50/zM glutamate there is usually no desensitization and synaptic current reaches about 10% of the saturation level [3]. Gf~ seal recordings of single channels at 50 #M glutamate (not illustrated) yield very short openings distributed with time constants of 0.35 ms and 0.1 ms, occurring at a rate of about 5/s. This agrees with the previous results at lower resolution [5], which only overestimated the contribution of the shortest openings (probably misinterpreting lower open states). The prolongation of single openings or bursts with increasing glutamate concentration thus starts from a range without desensitization and should be a true effect of glutamate concentration on the open state. An even stronger depen-
244
0.2 mM GLUTAMATE
5 mM GLUTAMATE
50 ms
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Fig. 3. Recordings and distributions of burst durations and closed times of single channels for 0.2 mM (left-hand column) and 5 m M glutamate (right=hand column). 19°C. At 0.2 m M g l ~ a m a t e , 5306 bursts were evaluated in a 74-s recording, while at 5 mM glutamate 960 bursts were evaluated in a 89-s recording. The distributions show the evaluated number of events (logarithmic scales) per bin, i.e. per time intervals of 0 . 1 . 0 . 0 4 or 5 ms, respectively, as indicated in the different distributions. The respective distributions were fitted by one or two exponentials (continuous curves) characterized by theh- time constants which are indicated next to the curves. In the distributions of burst durations, the longer component of 0.44 or 1.75 ms, respectively, accounted for more than 80°,/0 of the sample.
245
dence of duration of bursts on the glutamate concentration has been reported for locust muscle [6]. The rate of bursts rises when the glutamate concentration increases from low levels (up to 0.2 mM), but at higher concentrations desensitization depresses the rate of channel openings. This effect of desensitization may be even more dramatic than apparent in Fig. 3, since more than one channel probably is present in the patch (although no superpositions were observed), and different channels may become active from the desensitized state, as concluded also for acetylcholine receptors [ 12]. The observed dependence of burst duration on glutamate concentration may be used for an interpretation of synaptic currents elicited by quanta of transmitter. Occasionally, spontaneous synaptic currents appeared in patch-clamp recordings which showed amplitudes of up to - 1.5 nA and decayed with time constants of 0.8-2 ms, and similar values were also obtained in macro-patch-clamp ( ~ 50/#m opening) measurements (Franke and Dudel, unpublished). These time constants are much longer than the 0.4 ms observed at 0.2 mM glutamate (Fig. 3) but agree with burst durations found for millimolar concentrations of glutamate in the pipette. A quantum of transmitter thus may present a glutamate concentration around 1 mM at the receptors and open 100-200 channels almost simultaneously. The authors wish to thank Mr. W. Zeitz for programming of the computer, Miss I. Horstmann for technical assistance and Mrs. M. Griessl for secretarial help. This investigation was supported by the Deutsche Forschungsgemeinschaft. 1 Auerbach, A. and Sachs, F., Flickering of a nicotinic ion channel to a subconductance state, Biophys. J., 42 (1983) 1-10. 2 Cull-Candy, S.G. and Parker, I., Rapid kinetics of single glutamate-receptor channels, Nature (Lond.), 295 (1982) 410-412. 3 Dudel, J., Dose-response curve of glutamate applied by superfusion to crayfish muscle synapses, Pfliigers Arch., 368 (1977) 49-54. 4 Dudel, J., Aspartate and other inhibitors of excitatory synaptic transmission in crayfish muscle, Pfliigers Arch., 369 (1977) 7--16. 5 Franke, C., Dudel, J. and Finger, W., Single synaptic channels recorded at glutamate sensitive patches on a crayfish muscle, Neurosci. Lett., 42 (1983) 7-12. 6 Gration, K.A.F., Lambert, J.J., Ramsey, R. and Usherwood, P.N.R., Non-random openings and concentration-dependent lifetimes of glutamate-gated channels in muscle membrane, Nature, (Lond.), 291 (1981) 423-425. 7 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Nature (Lond.), 305 (1981) 85-100. 8 Hamill, O.P., Bormann, J. and Sakmann, B., Activation of multiple-conductance state chloride channels in spinal neurones by glycine and GABA, Nature (Lond.), 305 (1983) 805-808. 9 Parnas, I. and Atwood, H.L., Phasic and tonic neuromuscular systems in the abdominal extensor muscles of the crayfish and rock lobster, Comp. Biochem. Physiol., ! 8 (1966) 701-723. 10 Parnas, I., Dudel, J., Cohen, I. and Franke, Ch., Strengthening of synaptic contacts of an excitatory axon on elimination of a second excitatory axon innervating the same target, J. Neurosci., 4 (1984) 1912-1923. I 1 Patlak, J.B., Gration, K.A.F. and Usherwood, P.N.R., Single glutamate-activated channels in locust muscle, Nature (Lond.), 278 (1979) 643~J45.
246 12 Sakmann, B., Patlak, J. and Neher, E., Single acetylcholine-activated channels show burst-kinetics in presence of desensitizing concentrations of agonist, Nature (Lond.), 286 (1980) 71-73. 13 Sigworth, F.J., Electronic design of the patch clamp. In B. Sakmann and E. Neher (Eds.), Single-Channel Recording, Plenum Press, New York, London, 1983, Chapt. 1, pp. 3-35. 14 Stettmeier, H., Finger, W. and Dudel, J., Glutamate activated postsynaptic channels in crayfish muscle investigated by noise analysis, Pfl/igers Arch., 397 (1983) 13-19.