Improved high-performance liquid chromatographic method for the determination of domoic acid and analogues in shellfish: effect of pH.

Domoic acid (DA) is a naturally-occurring amino acid that causes a form of human intoxication called amnesic shellfish poisoning (ASP) following the consumption of shellfish. A rapid and sensitive HPLC-UV method has been developed for analysis of DA and analogues in shellfish without the need for SPE clean-up. Isocratic chromatographic separation of DA and its isomers from shellfish matrix interferences and from the prevalent amino acid, tryptophan, was achieved by careful control of the mobile phase pH. The optimised pH was found to be 2.5 when using a Luna(2) C18 column. Sample extraction was verified with control extracts from shellfish spiked at 5.0 and 10.0 microg/g of DA and with certified reference material. The average extraction efficiency was 98.5%. The calibration, based on mussel tissue spiked with DA standard, was linear in the range 0.05-5.0 microg/ml (r = 0.9999) and the detection limit (signal:noise 3:1) was better than 25 ng/ml. The DA assay achieved good precision; %RSD = 1.63 (intra-day, n = 6) and %RSD = 3.7 (inter-day, n = 8). This method was successfully applied to a variety of shellfish species, allowing the rapid screening of a large number of samples per day (20-30), without the need for SPE clean-up. Quantitative data were obtained for shellfish samples containing domoic acid in the concentration range 0.25-330 microg/g. Using the same chromatographic conditions, LC-MS3 was used to determine DA and its isomers, isodomoic acid D and epi-domoic acid, in scallop tissues.

A functional assay for paralytic shellfish toxins that uses recombinant sodium channels.

Saxitoxin (STX) and its derivatives are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. During algal blooms ("red tides"), shellfish accumulate saxitoxins leading to paralytic shellfish poisoning (PSP) in human consumers. PSP is a consequence of the high-affinity block of voltage-dependent Na channels in neuronal and muscle cells. PSP poses a significant public health threat and an enormous economic challenge to the shellfish industry worldwide. The standard screening method for marine toxins is the mouse mortality bioassay that is ethically problematic, costly and time-consuming. We report here an alternative, functional assay based on electrical recordings in cultured cells stably expressing a PSP target molecule, the STX-sensitive skeletal muscle Na channel. STX-equivalent concentration in the extracts was calibrated by comparison with purified STX, yielding a highly significant correlation (R=0.95; N=30) between electrophysiological determinations and the values obtained by conventional methods. This simple, economical, and reproducible assay obviates the need to sacrifice millions of animals in mandatory paralytic shellfish toxin screening programs.

Modal gating in neuronal and skeletal muscle ryanodine-sensitive Ca2+ release channels.

The bursting behavior of ryanodine-sensitive single Ca2+ release channels present in chicken cerebellum endoplasmic reticulum (ER), rat hippocampus ER, and frog and rabbit skeletal muscle sarcoplasmic reticulum was established. Unconditional dwell time distributions fitted by the maximum likelihood method reveal at least three open and closed exponential components. Trains of low open probability (P(o)) bursts were interspersed with trains of high P(o) bursts (> or = 0.8) in all the ryanodine receptor isotypes tested. The gating kinetics of the Ca2+ release channels were defined in long recordings by analyzing burst sequences and gamma distributions of average intraburst open (T(o)) and closed times (Tc). The gamma distributions of T(o) had two gamma components, suggesting the existence of two distinct burst types. In contrast, the gamma distributions of Tc had only one component. The correlation between consecutive burst pairs was defined in terms of T(o) and then statistically tested by 2 x 2 matrix contingency analysis. The probability that the ubiquitous sequential burst pattern was generated by random occurrence was < 0.01 (two-tailed Fisher's exact test). Temporal correlations were observed in all ryanodine receptor isotypes under a variety of experimental conditions. These data strongly suggest that single Ca2+ release channels switch slowly between modes of gating. We propose that the effects of agonists of Ca2+ release channels such as Ca2+ itself can be explained as concentration-dependent changes in the availability of each mode.

Functionally heterogenous ryanodine receptors in avian cerebellum.

The functional heterogeneity of the ryanodine receptor (RyR) channels in avian cerebellum was defined. Heavy endoplasmic reticulum microsomes had significant levels of ryanodine and inositol 1,4,5-trisphosphate binding. Scatchard analysis and kinetic studies indicated the existence of at least two distinct ryanodine binding sites. Ryanodine binding was calcium-dependent but was not significantly enhanced by caffeine. Incorporation of microsomes into planar lipid bilayers revealed ion channels with pharmacological features (calcium, magnesium, ATP, and caffeine sensitivity) similar to the RyR channels found in mammalian striated muscle. Despite a wide range of unitary conductances (220-500 picosiemens, symmetrical cesium methanesulfonate), ryanodine locked both channels into a characteristic slow gating subconductance state, positively identifying them as RyR channels. Two populations of avian RyR channels were functionally distinguished by single channel calcium sensitivity. One population was defined by a bell-shaped calcium sensitivity analogous to the skeletal muscle RyR isoform (type I). The calcium sensitivity of the second RyR population was sigmoidal and analogous to the cardiac muscle RyR isoform (type II). These data show that there are at least two functionally distinct RyR channel populations in avian cerebellum. This leads to the possibility that these functionally distinct RyR channels are involved in different intracellular calcium signaling pathways.

Properties of the ryanodine-sensitive release channels that underlie caffeine-induced Ca2+ mobilization from intracellular stores in mammalian sympathetic neurons.

The most compelling evidence for a functional role of caffeine-sensitive intracellular Ca2+ reservoirs in nerve cells derives from experiments on peripheral neurons. However, the properties of their ryanodine receptor calcium release channels have not been studied. This work combines single-cell fura-2 microfluorometry, [3H]ryanodine binding and recording of Ca2+ release channels to examine calcium release from these intracellular stores in rat sympathetic neurons from the superior cervical ganglion. Intracellular Ca2+ measurements showed that these cells possess caffeine-sensitive intracellular Ca2+ stores capable of releasing the equivalent of 40% of the calcium that enters through voltage-gated calcium channels. The efficiency of caffeine in releasing Ca2+ showed a complex dependence on [Ca2+]i. Transient elevations of [Ca2+]i by 50-500 nM were facilitatory, but they became less facilitatory or depressing when [Ca2+]i reached higher levels. The caffeine-induced Ca2+ release and its dependence on [Ca2+]i was further examined by [3H]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high-affinity binding site for ryanodine with a dissociation constant (KD = 10 nM) similar to that previously reported for brain microsomes. However, the density of [3H]ryanodine binding sites (Bmax = 2.06 pmol/mg protein) was at least three-fold larger than the highest reported for brain tissue. [3H]Ryanodine binding showed a sigmoidal dependence on [Ca2+] in the range 0.1-10 microM that was further increased by caffeine. Caffeine-dependent enhancement of [3H]ryanodine binding increased and then decreased as [Ca2+] rose, with an optimum at [Ca2+] between 100 and 500 nM and a 50% decrease between 1 and 10 microM. At 100 microM [Ca2+], caffeine and ATP enhanced [3H]ryanodine binding by 35 and 170% respectively, while binding was reduced by > 90% with ruthenium red and MgCl2. High-conductance (240 pS) Ca2+ release channels present in ganglionic microsomal membranes were incorporated into planar phospholipid bilayers. These channels were activated by caffeine and by micromolar concentrations of Ca2+ from the cytosolic side, and were blocked by Mg2+ and ruthenium red. Ryanodine (2 microM) slowed channel gating and elicited a long-lasting subconductance state while 10 mM ryanodine closed the channel with infrequent opening to the subconductance level. These results show that the properties of the ryanodine receptor/Ca2+ release channels present in mammalian peripheral neurons can account for the properties of caffeine-induced Ca2+ release. Our data also suggest that the release of Ca2+ by caffeine has a bell-shaped dependence on Ca2+ in the physiological range of cytoplasmic [Ca2+].

Activation of inositol trisphosphate-sensitive Ca2+ channels of sarcoplasmic reticulum from frog skeletal muscle.

1. The modulation by Ca2+ of the activation by inositol 1,4,5-trisphosphate (IP3) of Ca2+ channels present in native sarcoplasmic reticulum membranes from frog skeletal muscle was studied after channel incorporation into planar phospholipid bilayers in the presence of Ca2+ or Ba2+ as current carrier species. 2. Channel activity expressed as fractional open time (Po) was low (less than or equal to 0.15) in the presence of varying free Ca2+ concentrations bathing the myoplasmic face of the channel (cis side), and did not increase significantly between 0.01 and 30 microM-Ca2+. 3. Channel activation mediated by IP3 could be elicited from free Ca2+ levels similar to those of resting skeletal muscle (about 0.1 microM) and was found to be strongly regulated by the free Ca2+ concentration present at the myoplasmic moiety of the channel. 4. Channel activation by 10 microM-IP3 depended on the Ca2+ concentration on the cis side. Po reached a maximum between pCa 7.0 and 6.0, but decreased at higher concentrations of free Ca2+. Thus, Ca2+ exerted a modulatory influence on IP3-mediated activation in a concentration range where the channel was insensitive to Ca2+. 5. The results indicate that Ca2+ ions act as modulators of IP3 efficacy to open the channel. This could arise from an interaction of Ca2+ with the channel gating mechanism or with the agonist binding site.

Imaging of calcium transients in skeletal muscle fibers.

Epifluorescence images of Ca2+ transients elicited by electrical stimulation of single skeletal muscle fibers were studied with fast imaging techniques that take advantage of the large fluorescence signals emitted at relatively long wavelengths by the dyes fluo-3 and rhod-2 in response to binding of Ca2+ ions, and of the suitable features of a commercially available CCD video camera. The localized release of Ca2+ in response to microinjection of InsP3 was also monitored to demonstrate the adequate space and time resolutions of the imaging system. The time resolution of the imager system, although limited to the standard video frequency response, still proved to be adequate to investigate the fast Ca2+ release process in skeletal muscle fibers at low temperatures.

Activation of calcium channels in sarcoplasmic reticulum from frog muscle by nanomolar concentrations of ryanodine.

Sarcoplasmic reticulum vesicles isolated from fast-twitch frog skeletal muscle presented two classes of binding sites for ryanodine, one of high affinity (Kd1 = 1.7 nM, Bmax1 = 3.3 pmol per mg) and a second class with lower affinity (Kd2 = 90 nM, Bmax2 = 7.0 pmol per milligram). The calcium channels present in the sarcoplasmic reticulum membranes were studied in vesicles fused into lipid bilayers. Low concentrations of ryanodine (5 to 10 nM) activated a large conductance calcium channel after a short delay (5 to 10 min). The activation, which could be elicited from conditions of high or low fractional open time, was characterized by an increase in channel fractional open time without a change in conductance. The open and closed dwell time distributions were fitted with the sum of two exponentials in the range of 4 to 800 ms. The activating effect of ryanodine was due to an increase of both open time constants and a concomitant decrease in the closed time constants. Under conditions of low fractional open time (less than 0.1), the time spent in long closed periods (greater than 800 ms) between bursts was not affected by ryanodine. Higher concentrations of ryanodine (250 nM) locked the channel in a lower conductance level (approximately 40%) with a fractional open time near unity. These results suggest that the activating effects of nanomolar concentrations of ryanodine may arise from drug binding to high affinity sites. The expression of the lower conductance state obtained with higher concentrations of ryanodine may be associated with the low affinity binding sites observed in frog sarcoplasmic reticulum.

Electrical properties of cultured dorsal root ganglion neurons from normal and trisomy 21 human fetal tissue.

Dorsal root ganglion (DRG) neurons, in 22 degrees C tissue culture containing nerve growth factor, taken from normal and trisomy 21 human fetal tissues, were subjected to current and voltage clamp measurements using a tight-seal whole-cell recording technique. Measurements were made between 1 and 2 weeks in culture, when the electrical properties of both neuron groups were shown to be constant and when mean values for passive electrical parameters did not differ significantly between groups. The duration of the action potential was significantly less in trisomic than in control neurons, and both depolarization and repolarization were accelerated. Tetraethylammonium (5 mM), which partially blocked outward currents, prolonged the rate of repolarization of the action potential in both neuron groups, and abolished the difference in the rate between the groups. Furthermore, the activation rate constants of two model-defined outward potassium currents were significantly higher in trisomic than in control neurons, suggesting that acceleration of repolarization of the action potential in trisomic neurons was due to shorter activation time-constants of outward potassium currents.

Inositol (1,4,5)-trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes.

Sarcoplasmic reticulum membrane vesicles isolated from frog skeletal muscle display high conductance calcium channels when fused into phospholipid bilayers. The channels are selective for calcium and barium over Tris. The fractional open time was voltage-independent (-40 to +25 mV), but was steeply dependent on the free cis [Ca2+] (P0 = 0.02 at 10 microM cis Ca2+ and 0.77 at 150 microM Ca2+; estimated Hill coefficient: 1.6). Addition of ATP (1 mM; cis) further increased P0 from 0.77 to 0.94. Calcium activation was reversed by addition of EGTA to the cis compartment. Magnesium (2 mM) increased the frequency of rapid closures and 8 mM magnesium decreased the current amplitude from 3.4 to 1.2 pA at 0 mV, suggesting a reversible fast blockade. Addition of increasing concentrations of inositol (1, 4, 5)-triphosphate (cis), increased P0 from 0.10 +/- 0.01 (mean +/- SEM) in the control to 0.85 +/- 0.02 at 50 microM in an approximately sigmoidal fashion, with an apparent half-maximal activation at 15 microM inositol (1, 4, 5)-trisphosphate in the presence of 40 microM cis Ca2+. Lower concentrations of this agonist were required to produce a significant increase in P0 when 10 microM or less cis Ca2+ were used. The channel was blocked by the addition to the cis compartment of either 0.5 mM lanthanum, 0.5 microM ruthenium red, or 200 nM ryanodine, all known inhibitors of Ca2+ release from sarcoplasmic reticulum vesicles. These results demonstrate the presence of calcium channels in the sarcoplasmic reticulum from frog skeletal muscle with a pharmacological profile consistent with a role in excitation contraction coupling and with the hypothesis that inositol ( 1,4,5)-trisphosphate is a physiological agonist in this process.

A soluble factor (less than 4000 Da) from chick spinal cord blocks slow hyperpolarizing afterpotentials in cultured rat muscle cells.

The slow hyperpolarizing afterpotential (slow HAP) following an evoked action potential in cultured rat muscle cells is inhibited by coculture with spinal cord neurons, spinal cord-conditioned medium (CM) and by a low-molecular-weight fraction (less than 4000 Da) prepared from this CM. The incidence, amplitude and duration of the slow HAPs decreased significantly after 24 h of incubation. In contrast, chick retinal neuron cocultures or CM did not alter the slow HAP.

The interaction of electrically stimulated twitches and spontaneous contractile waves in single cardiac myocytes.

Spontaneous myofilament motion that propagates within cells as a contractile wave is a manifestation of localized Ca2+ release from sarcoplasmic reticulum (SR). At 37 degrees C, when bathing [Ca2+] (Cao) is 1.0 mM, rat myocytes exhibit contractile waves at rest and the interwave interval averages 9.1 +/- 1.5 s (n = 6). We determined whether there was an interaction between this type of SR Ca2+ release and that induced by electrical stimulation to cause a twitch, and whether such an interaction had functional significance. Progressive decreases in SR Ca2+ loading effected by graded concentrations of caffeine produced proportional decreases in the mechanical amplitude of the twitch and of the spontaneous contractile wave. Regular electrical stimulation in physiologic Cao abolished the waves and, after stimulation, waves did not reappear for a period of time (delay interval). Over a range of stimulation frequencies (6-72 min-1), the delay interval ranged from 11.4 +/- 3.6 to 12.4 +/- 1.7 s and was usually greater than the interwave interval at rest. The delay interval for a wave to occur after a twitch was reduced in the presence of increased Cao, glycosides, or catecholamines. When the interstimulus interval exceeded the delay interval, waves could appear between twitches and had a marked effect of shortening the duration of the action potential and decreasing the amplitude of the subsequent twitch. The magnitude of this effect varied inversely with time (up to 2 s) between the onset of the spontaneous diastolic wave and the subsequent stimulated twitch. A reduction of the interstimulus interval to less than the delay interval prevented the occurrence of diastolic waves. These results demonstrate the presence of an interaction between spontaneous and action potential-mediated Ca2+ release, which can be interpreted on the basis of a common Ca2+ pool and perhaps common release mechanisms. This interaction can explain many of the known effects of electrical stimulation on phenomena that are thought to result from spontaneous Ca2+ oscillations in intact tissue.

Single calcium channels in native sarcoplasmic reticulum membranes from skeletal muscle.

Electrical properties of native sarcoplasmic reticulum membranes from rabbit skeletal muscle were investigated using the patch-clamp technique. Bilayers were assembled at the tip of patch pipettes from monolayers formed at the air-water interface of sarcoplasmic reticulum membrane suspensions. The membranes were found to contain a spontaneously active cation channel of small conductance (5 pS in 200 mM CaCl2, symmetrical solutions) that was selective for Ca2+ and Ba2+. Between 50 and 200 mM CaCl2 (symmetrical) the increase in conductance as a function of [Ca2+] fit a hyperbola (K0.5, 83 mM, and gamma max, 7.9 pS) that extrapolated to a single-channel conductance of 0.5 pS at physiological Ca2+ levels. The channel opened in bursts followed by long silent periods of up to a minute. During a burst the channel fluctuated very rapidly with time constants in the millisecond range. The mean burst duration was voltage dependent, increasing from 1.8 s at a pipette voltage of +60 mV to 4.1 s at +80 mV. Over this range, burst frequency decreased with increasing voltage such that the fraction of time spent in the open state (fb) remained constant. Application of 1.6 mM caffeine resulted in activation of the channel that appeared as an increase in mean burst duration. In contrast, 50 microM dantrolene significantly decreased burst frequency, whereas 10 microM nitrendipine had no effect. The functional and pharmacological properties of this Ca2+ channel suggest that it may be important in mediating Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling.

Forskolin and antidiuretic hormone stimulate a Ca2+-activated K+ channel in cultured kidney cells.

Single channels in the apical cell membrane of primary cultured chick kidney cells were studied using the patch clamp technique. Cell-attached recordings revealed the presence of a 107 +/- 6 pS channel that increased fractional open time upon depolarization. Experiments with inside-out excised patches indicated that the channel is K+ selective, Ca2+ activated, and inhibited by Ba2+. The addition of forskolin or antidiuretic hormone (ADH) to the bath during cell-attached recordings caused an increase in the fractional open time of the channel. The activation of a K+ channel by increases in cAMP may be one way in which K+ secretion in the kidney is stimulated by ADH in vivo.

Blockers of calcium permeability inhibit neurite extension and formation of neuromuscular synapses in cell culture.

Growth of neurites from trypsin-dissociated retinal neurons from chick embryos is very sensitive to the extracellular calcium concentration. Blockers of calcium permeability such as Co2+, Mn2+, La3+ and nitrendipine, when added to dissociated neurons, decrease the fraction of cells that extend neurites and the rate of neurite growth without influencing cell-substratum adhesion or survival capacity. Inhibition is concentration dependent, is related to the age of the donor chick embryo and can be prevented by increasing the extracellular calcium concentration. 50% inhibition in 8-day neurons is produced by 120 microM Co2+, 250 microM Mn2+, 50 microM La3+ and 10 microM nitrendipine in medium containing 1.8 mM Ca2+. Inhibition of neurite extension is accompanied by a concentration dependent inhibition of synapse formation between retinal neurons and muscle cells in culture, as determined by intracellular recording. 50% inhibition in the fraction of innervated myotubes is produced by 0.4 mM Co2+ and 4 mM Mn2+. These results suggest that: (1) a voltage-dependent calcium flux is a signal not only for growth cone expansion but also for neurite extension in primary dissociated neurons; and (2) that neurite extension is a prerequisite for synaptogenesis between neurons and muscle cells in culture.

Channel properties of the purified acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayer membranes.

The electrophysiological properties of the cation channel of the purified nicotinic acetylcholine receptor (AChR) reconstituted in planar lipid bilayers were characterized. Single-channel currents were activated by acetylcholine, carbamylcholine and suberyldicholine. The single channel conductance (28 pS in 0.3 M NaCl) was ohmic and independent of the agonist. Single channel currents increased with Na+ concentration to a maximum conductance of 95 pS and showed a half-saturation point of 395 mM. The apparent ion selectivity sequence, derived from single-channel current recordings, is: NH+4 greater than Cs+ greater than Rb+ greater than or equal to Na+ Cl-, F-, SO2-(4). The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of at least two distinct open states. The time constants depend on the choice of agonist, being consistently longer for suberyldicholine than for carbamylcholine. Similar channel properties were recorded in bilayers formed from monolayers at the tip of patch pipets . Single-channel currents occur in paroxysms of channel activity followed by quiescent periods. This pattern is more pronounced as the agonist concentration increases, and is reflected in histograms of channel-opening frequencies. Computer simulations with a three-state model, consisting of two closed (unliganded and liganded) and one open state, do not resemble the recorded pattern of channel activity, especially at high agonist concentration. Inclusion of a desensitized liganded state reproduces the qualitative features of channel recordings. The occurrence of paroxysms of channel activity thus seems to result from the transit of AChR through its active conformation, from which it can open several times before desensitizing.

Single-channel recordings from purified acetylcholine receptors reconstituted in bilayers formed at the tip of patch pipets.

The channel of the purified acetylcholine receptor from Torpedo californica electric organ reconstituted in lipid vesicles was assayed by direct electrical recording using patch-clamp pipets. High-resistance seals were obtained by gentle suction of vesicles into the pipet or after the formation of lipid bilayers from monolayers at the tip of the pipet. Single-channel currents were activated by three cholinergic ligands: acetylcholine, carbamylcholine, and suberyldicholine. The single-channel conductance, gamma, was 40 +/- 5 pS in 0.5 M NaCl, irrespective of the agonist used. The distributions of channel open times were fitted by a sum of two exponentials. The lifetimes of the two exponential components were a factor of 2 longer for suberyldicholine than for acetylcholine or carbamylcholine. At desensitizing concentrations of agonists the single events appeared in paroxysms of channel activity followed by quiescent periods. These results suggest that the full cycle of solubilization, purification, and reconstitution of this membrane receptor can be achieved without impairment of channel function.

Activation and inactivation characteristics of the sodium permeability in muscle fibres from Rana temporaria.

1. The steady-state and kinetic characteristics of the processes of activation and inactivation of the Na(+) permeability, P(Na), were measured in cut skeletal muscle fibres from Rana temporaria under voltage-clamp conditions.2. The specific resistance, r(ss), in series with the surface sarcolemma, was estimated as 6 Omega cm(2) by measuring the initial value of the membrane potential transient in response to current pulses under current-clamp conditions. To reduce the error in the potential across the sarcolemma introduced by r(ss), Na(+) currents were recorded using positive feed-back compensation, in the presence of tetrodotoxin (2.4-5 nm).3. P(Na)(t) was fitted with m(3)h kinetics assuming a voltage-dependent delay, deltat, to the start of the activation process.4. The P(Na)-V(p) curve exhibited saturation at potentials more positive than 30 mV. m(infinity), calculated as (P(Na), (infinity)/ P(Na))((1/3)) as a function of V(p), was a sigmoid curve with a mid point at -35 mV. The slope, dm(infinity)/dV(p), at this point was 0.032 mV(-1).5. Using a double-pulse protocol a non-exponential time course for the development of fast inactivation at small depolarizations was observed.6. The time constant for activation, tau(m), as a function of V(p), and tau(h) as a function of V(p), could be fitted with an approximately bell-shaped function, maximum of 430 mus at -43 mV and 925 mus at -78 mV respectively, at 15 degrees C.7. The mid-point potential of the h(infinity)-V(l) curve occurred at -58 mV, and h(infinity) approached 1 for V(1) values more negative than -103 mV.8. Using a double-pulse procedure the development of a slow inactivation of the Na(+) current was demonstrated. Its time course could be described in terms of a single exponential function, time constant equal to 0.58 s. The recovery from slow inactivation could be described by a similar exponential for recovery times smaller than 1 s.

Fast charge movements in skeletal muscle fibres from Rana temporaria.

1. Fast charge movements were measured in cut skeletal muscle fibres from Rana temporaria.2. The initial time course of the current in response to a sudden displacement of the membrane potential from -110 to -60 mV was analysed in terms of an electrical equivalent circuit modified from Falk & Fatt (1964).3. The specific resistance in series with the sarcolemma was estimated as 7.4 Omega cm(2). The total capacity (surface sarcolemma plus tubular membrane) was estimated as 3.43 muF/cm(2).4. The asymmetry currents settling within 1 ms during depolarizing pulses of increasing size (on-response), from a holding potential around -120 mV, could be described in terms of a single exponential. The asymmetry currents after the pulses (off-response) exhibited at least two components.5. The integral of the on-response, Q(on), as a function of V(p), could be fitted using a function of the Boltzmann type. At the mid-point of the distribution curve, equal to -38 mV, the slope was 0.012 mV(-1). A saturating value of 28 pC was reached at 40 mV.6. The off-response to pulses not exceeding 3 ms exhibited two components. The first one had an exponential time course. The charge Q(off) associated with this fast component was always equal to Q(on).7. tau(on) (the relaxation time constant), as a function of membrane potential was asymmetrical, exhibiting a maximum value of 233 mus at about -38 mV.8. For V(p) values smaller than -20 mV the Q(on)-V(p) and tau(on)-V(p) curves could be analysed using the two-state transition model. From this analysis the average transition potential V' was estimated as -38 mV and the effective valence of the mobile charges as 1.36.9. Double-pulse protocols (duration of pre-pulses referred to as T in the range 0-3 s) showed that Q(on) and tau(on) decreased as T increased. Single transient analysis shows that the changes are confined to the transient for depolarizing pulses.10. This immobilization of the charges is reversible and follows a similar time course to the slow inactivation of the Na(+) conductance described in the preceding paper.11. A differential effect of the depolarizing pre-pulse on the ionic and asymmetry currents is seen in the decrease of tau(on) with increasing T while tau(m) remains constant.