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A patch-clamp study was done to characterize the recently found GABAergic (i.e. gamma-aminobutyric acid) inhibitory synaptic channels of crayfish deep extensor abdominal muscle. Outside-out patches were rapidly activated by GABA to measure the dose/response curves for the open probability of the channels, Po, and the rise time, tr, (time from Po = 0.1 to Po = 0.9). In some of the patches the GABA-activated currents decayed due to desensitization and such patches were not studied further. Rare channel openings were elicited with 0.1 mM GABA. The Po at this low concentration of GABA was 0.0005 to 0.01. Application of 10 mM GABA was necessary to reach the maximal Po of 0.9. The slope of the dose/response relationship in the double logarithmic plot was 5.4 +/- 1.1 (mean +/- SD; n = 9) between 0.1 mM and 0.2 mM GABA. The plot of tr versus GABA concentration had a peculiar shape, recently found to be characteristic for positive cooperativity of the binding sites. tr increased from a minimum at 10 mM GABA with declining concentrations of GABA and reached a peak at 0.4 mM GABA. Below 0.4 mM GABA, tr decreased again. With 0.2 mM GABA tr was 0.40 +/- 0.1 (mean +/- SD; n = 4) of the peak value measured at 0.4 mM GABA. Simulations were compared with the experimental results and a linear reaction scheme with five binding sites for GABA was established to describe the dose/response curves for Po and tr.

Electrophysiological studies on muscle fibres from patients with hyperkalemic periodic paralysis with myotonia have shown that the episodes of weakness are caused by a sustained depolarization of the sarcolemma to potentials between -40 and -60 mV. In muscle fibre segments from three such patients this sustained depolarization was caused by noninactivating Na+ channels with reduced single-channel conductance blocked by TTX and procainamide. As the chloride conductance was normal, myotonia may be best explained with the abnormal reopenings of the Na+ channels. The recently described genetic linkage between hyperkalemic periodic paralysis with myotonia and the gene coding for the TTX-sensitive Na+ channel suggests an altered primary structure of this channel causing its abnormal function.

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.

The patch-clamp technique was used to study Na+ channels of human skeletal muscle. Preparations were from biopsies of quadriceps muscle from adults who were not suffering from neuromuscular diseases. Activity of Na+ channels was recorded from inside-out patches when the membrane potential was stepped from a holding potential of -110 mV to potential above a threshold of about -65 mV. Single channel activity increased within minutes after hyperpolarizing the patch due to recovery from ultra-slow inactivation. Up to ten Na+ channels were active in individual patches. Macroscopic currents were reconstructed by averaging single channel currents. The time-to-peak current declined from 1.6 ms at -60 mV to 0.5 ms at + 10 mV. The currents decayed mono-exponentially with time constants between 12.1 ms at -60 mV and 0.4 ms at + 10 mV (21 C). The conductance of single Na+ channels was 1.65 pS and the mean open time was voltage-dependent. At -50 mV, the mean open time was 0.4 ms, while positive to -10 mV it increased to values above 1 ms. In the threshold potential range, the number of openings per depolarizing pulse was larger than the number of channels under the patch-clamp pipette, indicating reopening of Na+ channels at this potential. Openings could be observed only rarely 10 ms after onset of depolarization and the macroscopic current produced by late openings was less than 0.1% of the peak current. Human skeletal muscle is thus suitable for investigation with the patch-clamp technique and the determination of properties of Na+ channels with this technique could be the basis for an assessment of possible defects of these channels in diseased muscle.

Single, glutamate activated ionic channel currents were recorded from crayfish muscle in the cell attached mode. Different concentrations of glutamate were present in the patch clamp pipette. Bursts of openings were observed with a concentration dependent number of short gaps per burst. Also the mean burst length was concentration dependent and varied between 0.3 ms (100 microM) and 1.3 ms (20 mM). Even with the highest concentrations of glutamate the channel activations were well separated and the beginning and the end of a burst could be defined. The distributions of open times and of burst lengths could be fitted well with a single exponential component for all studied concentrations of glutamate. The distributions of closed times were composed of two or three exponential components (with possibly more than one channel contributing). The mean burst length was compared with the time constants of decay of synaptic currents (0.8-3.0 ms at 19 degrees C) which were measured either with the same pipette as the single channel currents or with a macro patch technique. An estimation of the glutamate concentration at the receptors during synaptic transmission gave values in the millimolar range. The most simple model of glutamate-receptor interaction contains two binding sites for glutamate but no singly liganded open states. Rate constants were estimated for this model.

In the deep abdominal L1-extensor muscle of the spiny lobster (Panulirus penicillatus) quantal excitatory postsynaptic currents (EPSCs) were recorded through macro-patch-clamp electrodes. Release of transmitter quanta from terminals was also elicited by depolarizing current pulses given through the recording electrode. The majority of terminals were excitable: on increasing the depolarization pulses, release was triggered at a threshold in an all-or-nothing manner. If excitation was blocked by tetrodotoxin (TTX), release was graded with depolarization reaching the amplitude of the all-or-nothing response at pulse amplitudes several times higher than the former threshold level. Some inexcitable terminals were also found: in these, release was graded for increasing depolarization pulses, and TTX did not alter the depolarization-release relation. Among the other types of terminals studied with the same technique, the proportion of excitable terminals in this lobster tail muscle is higher than in the crayfish opener and lower than in the frog's cutaneous pectoris muscle. The contribution of the increase in intraterminal Ca concentration to the control of release was estimated using facilitation of a test EPSC as an indicator of Ca inflow during a preceding depolarization pulse. This facilitation was found to have a maximum at a certain pulse amplitude, PF, and to decline for larger depolarizations. Release, however, rose considerably for depolarizations larger than those effected at PF. It is concluded that, like in crayfish and frog motor terminals, release is controlled directly by depolarization in addition to the control by Ca-inflow.

Excitatory, glutamate-activated single channel currents were measured in outside-out patches of crayfish muscle. The open times of single channel openings, and the durations and rates of bursts were evaluated. These kinetic parameters were not appreciably affected by replacement of extracellular Na+ by Li+ or choline. Changes in extracellular Ca2+ concentration Cao also did not influence the duration of single openings. However the mean burst duration decreased for Cao less than 13.5 mM and the rate of bursts declined with a power of almost 2 in low Cao. At Cao less than 1 mM practically no channel openings were observed in presence of glutamate. In order to exclude more rapid desensitization of the glutamate receptors in low Cao as the cause of disappearance of channel openings, glutamate was applied in short pulses with a liquid-filament switch. In 0 Cao also a glutamate pulse did not trigger channel openings. In presence of 13.5mM Cao, the inorganic Ca-channel blockers La3+ and Cd2+ diminished the duration and rate of bursts of channel openings in a similar manner as low Cao. The effects of low Cao and of Cd2+ were tested also on quantal postsynaptic currents, EPSCs, which were recorded through a perfused macro-patch-clamp electrode. At 1.4 mM Cao in the perfused electrode tip, spontaneous EPSCs were reduced at least by a factor of 4, and elicited EPSCs by a factor of 16. Application of Cd2+ had similarly strong effects on the EPSCs. Also the decay of EPSCs was shortened substantially in 1.4 mM Cao or 5 mM Cd2+. The inhibitory Cl(-)-channel of crayfish muscle, activated by glutamate or GABA, also was studied in outside-out patches. The openings of this channel persisted in 0 Cao solutions; the block of channel openings in low Cao thus is a specific property of the excitatory channel. The action of Cao on the excitatory channel may be described as that of a cofactor to glutamate. A possible reaction scheme is proposed.

Single glutamate activated ionic channels were recorded with the patch clamp technique from untreated crayfish muscle fibres with M omega seals, and after treatment with collagenase, with G omega seals. In regions with single channel activity spontaneous synaptic currents could also be recorded, and the channels were therefore identified as synaptic. The single channel current amplitude was -7 to -8 pA at the resting potential of -70 mV, representing a conductance of 100 pS. The amplitudes decreased by a factor of two when the temperature was lowered by 10 degrees C. Openings occurred in bursts, and the mean burst length varied between 0.3 ms (50 microM glutamate in the pipette) and 0.8 ms (1 mM glutamate in the pipette). After treatment with collagenase, G omega seals could be formed. The conductance of the channel and the mean burst length was not affected by the enzyme, but after treatment active spots could be found easier and they were distributed more uniformly along the fibre. After treatment the concentrations of glutamate necessary to elicit channel openings were higher (100 microM compared to 20-50 microM) and simultaneous openings of two or more channels were observed very rarely. Synaptic currents could not be recorded from preparations cleaned by collagenase (2 mg/ml) for longer than 60 min.

Excitatory single channel currents triggered by L-glutamate were measured in outside-out excised patches of crayfish muscle membrane. If an 'intracellular' solution was present in the pipette and normal extracellular solution with added glutamate (10(-3) M) passed the outside of the patch, the single channel currents, i1, had amplitudes of -8 pA at a patch potential of -70 mV. If in the extracellular solution Na+ was replaced by Li+ or Ca2+, the amplitudes of single channel currents were reduced by about 30%. Only about 20% of the channel current amplitude remained on replacement of Na+ by choline. Replacement of Na+ reduced the variance of channel amplitude distributions to the level of the baseline. Presence of Na+ thus induces an additional variance of open channel current. When the proportions of Na+/choline were varied, the resulting channel currents could be separated in Na+, Ca2+ and choline components. The amplitude of the Na+ component, i1,Na, could be described by a constant channel permeability pi Na = 110 10(-15) cm3 s-1 according to the constant field equation. Ba2+ could replace Ca2+ without change in single channel current, while replacement of Ca2+ by Mg2+ reduced the channel currents by 20%. The following permeabilities of the single channel were estimated (in 10(-15) cm3 s-1): pi Na = 110, pi K = 86, pi Ca = 30, pi Mg = 24, pi Ba = 30, pi Li = 84 and pi choline = 11. These permeabilities were obtained inserting ionic concentrations. The respective permeabilities are listed also as calculated on the basis of ionic activities.(ABSTRACT TRUNCATED AT 250 WORDS)