Tubular localization of silent calcium channels in crustacean skeletal muscle fibers.

Ca2+-induced Ca2+ release (CICR) in the superficial abdominal flexor muscle of the crustacean Atya lanipes appears to be mediated by a local control mechanism similar to that of vertebrate cardiac muscle, but with an unusually high gain. Thus, Ca2+ influx increases sufficiently the local concentration of Ca2+ in the immediate vicinity of the sarcoplasmic reticulum Ca2+ release channels to trigger the highly amplified release of Ca2+ required for contraction, but is too low to generate a macroscopic inward current (i.e., the Ca2+ channels are silent). To determine the localization of the silent Ca2+ Channels, the mechanical, electrophysiological and ultrastructural properties of the muscle were examined before and after formamide treatment, a procedure that produces the disruption of transverse tubules of striated muscle. We found that tubular disruption decreased tension generation by about 90%; reduced inward current (measured as Vmax, the maximum rate of rise of Sr2+ action potentials) by about 80%; and decreased membrane capacitance by about 77%. The results suggest that ca. 80% of the silent Ca2+ channels are located in the tubular system. Thus, these studies provide further evidence to support the local control mechanism of CICR in crustacean skeletal muscle.

Silent calcium channels in skeletal muscle fibers of the crustacean Atya lanipes.

The superficial (tonic) abdominal flexor muscles of Atya lanipes do not generate Ca(2+) action potentials when depolarized and have no detectable inward Ca(2+) current. These fibers, however, are strictly dependent on Ca(2+) influx for contraction, suggesting that they depend on Ca(2+)-induced Ca(2+) release for contractile activation. The nature of the communication between Ca(2+) channels in the sarcolemmal/tubular membrane and Ca(2+) release channels in the sarcoplasmic reticulum in this crustacean muscle was investigated. The effects of dihydropyridines on tension generation and the passive electrical response were examined in current-clamped fibers: Bay K 8644 enhanced tension about 100% but did not alter the passive electrical response; nifedipine inhibited tension by about 70%. Sr(2+) and Ba(2+) action potentials could be elicited in Ca(2+)-free solutions. The spikes generated by these divalent cations were abolished by nifedipine. As the Sr(2+) or Ba(2+) concentrations were increased, the amplitudes of the action potentials and their maximum rate of rise, V(max), increased and tended towards saturation. Three-microelectrode voltage-clamp experiments showed that even at high (138 mm) extracellular Ca(2+) concentration the channels were silent, i.e., no inward Ca(2+) current was detected. In Ca(2+)-free solutions, inward currents carried by 138 mm Sr(2+) or Ba(2+) were observed. The currents activated at voltages above -40 mV and peaked at about 0 mV. This voltage-activation profile and the sensitivity of the channels to dihydropyridines indicate that they resemble L-type Ca(2+) channels. Peak inward current density values were low, ca. -33 microA/cm(2) for Sr(2+) and -14 microA/cm(2) for Ba(2+), suggesting that Ca(2+) channels are present at a very low density. It is concluded that Ca(2+)-induced Ca(2+) release in this crustacean muscle operates with an unusually high gain: Ca(2+) influx through the silent Ca(2+) channels is too low to generate a macroscopic inward current, but increases sufficiently the local concentration of Ca(2+) in the immediate vicinity of the sarcoplasmic reticulum Ca(2+) release channels to trigger the highly amplified release of Ca(2+) required for tension generation.

Heterogeneous distribution of acetylcholine receptors in chick myocytes induced by cholesterol enrichment.

The cholesterol concentration at the cell surface of cultured chick myocytes was increased in order to determine the effects of high levels of cholesterol on the ion channel properties of the nicotinic acetylcholine receptor. Single channel recordings and fluorescence polarization studies using 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed under equivalent conditions for normal and cholesterol enriched myocytes. In cell attached patches from myocytes with a cholesterol to phospholipid molar ratio (c/p) of 0.24 and a microviscosity of 1.35 poise a single conductance of 51 pS was detected. The cholesterol enriched myocytes with a c/p of 0.52 and a microviscosity of 2.05 poise showed two conductances, a 54 pS and a 39 pS channel: both were blocked by alpha-bungarotoxin. The 39 pS channel was detected with the simultaneous appearance of a slow component of tau m (modulation time) for DPH fluorescence measured by phase demodulation. The 80% reduction in the open time constant (tau 2) of the 39 pS channel suggest an inhibition of the normal conformational state. The combined results suggest that cholesterol enrichment may induced a more heterogeneous lipid environment and that the two types of channel properties could result from the distribution of the receptors in different domains.

Sulfhydryl alkylating agents induce calcium current in skeletal muscle fibers of a crustacean (Atya lanipes).

Voltage-clamp experiments using the three-microelectrode voltage clamp technique were performed on ventroabdominal flexor muscles of the crustacean Atya lanipes. Potassium and chloride currents were found to underlie the normal, passive response of the muscle. Blocking potassium currents with tetraethylammonium and replacing chloride ions with methanesulfonate did not unmask an inward current. By treating the muscle with the sulfhydryl-alkylating agent 4-cyclopentene-1,3-dione an inward current was detected. The current induced by the agent is carried by Ca2+, since it is abolished in Ca(2+)-free solutions. The induced Ca2+ current is detected at about -40 mV and reaches a mean maximum value of -78 microA/cm2 at ca. -10 mV. At this potential the time to peak is close to 15 msec. The induced Ca2+ current inactivated with 1-sec prepulses which did not elicit detectable Ca2+ current; the fitted hx curve had a midpoint of -38 mV and a steepness of 5.0 mV. Measurements of isometric tension were performed in small bundles of fibers, and the effects of the sulfhydryl-alkylating agents 4-cyclopentene-1,3-dione and N-ethylmaleimide were investigated. Tetanic tension was enhanced in a strictly Ca(2+)-dependent manner by 4-cyclopentene-1,3-dione. The amplitude of K+ contractures increased after treatment with N-ethylmaleimide. It is concluded that Ca2+ channels are made functional by the sulfhydryl-specific reagents and that the increase in tension is probably mediated by an increase in Ca2+ influx through the chemically induced Ca2+ channels.

Ultrastructural and mechanical properties of electrically inexcitable skeletal muscle fibers of the crustacean Atya lanipes.

Examination of the ultrastructure and mechanical activation of the ventro-abdominal flexor muscle of the freshwater crustacean Atya lanipes shows that the fibers are of the long sarcomere, tonic type. The fibers possess an ample and well-organized internal membrane system, with extensive regions of T/SR dyad contacts near the ends of the A bands. An orbit of 10-12 thin filaments surrounds each thick filament. The lanthanum tracer method reveals a highly regular organization of the T-system, Z-tubules, and multiple internal clefts. Tension generation responds to extracellular potassium in a concentration dependent manner and is very slow. Mechanical activation is strictly dependent on extracellular Ca2+, even though these muscle fibers do not generate Ca2+ currents when depolarized. Tension development responds to caffeine and is also dependent on extracellular Na+, suggesting that Ca2+ release from the SR and Ca2+ influx via the Na/Ca exchanger intervene in mechanical activation.

Acetylcholine receptor channel gating and conductance involve extracellular disulfide bond(s).

Disulfide bonds are critical determinants of the function of the acetylcholine receptor at the vertebrate neuromuscular junction. In the present study, the role of these bonds in acetylcholine receptor channel gating and conductance was investigated at the single channel level. Disulfide bond reducing agents decreased the single channel conductance of both ligand-gated and spontaneously opening acetylcholine receptor channels, indicating that the observed decrease in conductance is not due to blockade of the channel lumen by agonist molecules. In addition, the reducing agents increased the opening frequency of both liganded and unliganded acetylcholine receptor channels. Use of inside-out patches and both membrane permeant and impermeant reducing agents demonstrated that the disulfide bonds involved are all extracellular. These findings indicate that both channel gating and conductance involve conformational changes in extracellular regions of the acetylcholine receptor.

Spontaneous openings of large and small conductance acetylcholine receptor channels in Xenopus myocytes.

Spontaneous openings of acetylcholine receptor channels were investigated using Xenopus myotomal muscle cells in culture and patch clamp recording methods. Two classes of channels with conductances close to 40 and 60 pS and reversal potentials near 0 mV were found to open in the absence of agonist. The open time of the low-conductance channel was about 3 times longer than that of the high-conductance channel. Open times were shorter by a factor of 3-5 than those of acetylcholine-activated channels in Xenopus myocytes and showed no significant voltage dependence. These studies indicate that the two predominant classes of acetylcholine receptor channels in this preparation can open spontaneously.

The nicotinic acetylcholine receptor channel in embryonic muscle of Xenopus laevis. A program to separate channel types.

The single channel activity of nicotinic acetylcholine receptor (AChR) channels from Xenopus laevis embryonic myotomes in culture, was studied with the patch clamp technique. Two types of single channel activity were observed at low agonist concentration (100 nM ACh-Cl), which were distinguished by their conductance (60 and 40 pS, approximately). Both channel types were recorded simultaneously and therefore to carry out the kinetic analysis, we separate them by using a program exploiting the difference in amplitude of the channels. By using the program CLASS the stationary kinetic characteristics of both types of channels can be obtained separately and evaluated.

Organic calcium channel blockers generate the coexistence of two different types of action potentials in the same muscle membrane following chemical induction of excitability.

1. Following exposure to the sulfhydryl reagents known as alpha,beta-unsaturated carbonyl compounds, the ventroabdominal flexor muscles of the crustacean Atya lanipes, which are normally completely inexcitable, generate trains of overshooting calcium action potentials; the effects of organic calcium channel antagonists and potassium channel blockers on the chemically-induced trains of action potentials have been studied. 2. Verapamil and D600, at micromolar concentrations, elicit the appearance of slow, cardiac-like action potentials which coexist with the much faster chemically-induced calcium spikes, transforming the regular repetitive firing into a cyclic bursting pattern. 3. Bepridil (1 microM) decreases the frequency of firing of the action potentials, probably by increasing the threshold for the activation of a population of the chemically-induced calcium channels. 4. The potassium channel blockers, TEA (30-40 mM) and quinidine (100-200 microM), delayed the rate of repolarization of the chemically-induced action potentials; none of the potassium channel blockers, however, induced the appearance of repetitive spike activity.

Single acetylcholine channel currents recorded from Xenopus myocytes are influenced by the glass of the patch pipette.

The type of glass used to fabricate patch pipettes influences the kinetic properties of single-channel currents activated by acetylcholine in Xenopus myocytes. In patches formed with hard glass pipettes, the opening rate decreased in a time-dependent manner. Such decrease was most pronounced for the low conductance channel. In addition, longer duration events of this form of channel dropped out faster than shorter duration events and, as a consequence, the apparent channel open time became shorter during the recording period. These effects were not observed in patches formed with soft glass pipettes.

Single acetylcholine channel currents recorded from xenopus myocytes are influenced by the lass of the patch pipette

El tipo de vidrio que se utiliza para construir las pipetas de "patch clamp" altera las propiedades cinéticas de las corrientes unitarias activadas por acetilcolina en miocitos de Xenopus. En resgistros hechos con pipetas de vidrio duro, la velocidad de apertura disminuye con el transcurso del tiempo. Esta disminución es mas pronunciada para el canal de baja conductancia. Además, los eventos de mayor duración de esta forma del canal desaparecen mas rápidamente que los eventos mas cortos y, como consecuencia, el tiempo abierto del canal se acorta durante el período de registro. Estos efectos no se observan cuando los canales son registrados utilizando pipetas fabricadas con vidrio blando

Influence of the patch pipette glass on single acetylcholine channels recorded from Xenopus myocytes.

The type of glass used to fabricate patch pipettes influences the kinetic properties of single-channel currents activated by acetylcholine in Xenopus myocytes. In patches formed with hard glass pipettes, the opening rate decreased in a time-dependent manner. Such decrease was most pronounced for the low-conductance channel. In addition, longer-duration events of this form of channel dropped out faster than shorter-duration events and, as a consequence, the apparent channel open time became shorter during the recording period. These effects were not observed in patches formed with soft glass pipettes.

Further kinetic analysis of the chemically modified acetylcholine receptor.

The acethylcholine receptor was chemically modified using bisulfite to add a sulfonate group to a disulfide bond on the alpha subunit, and diamide, an oxidizing agent, to form an interchain disulfide bond between beta subunits of adjacent receptors. In previous work, both reagents increased mepc decay times but produced no change in mean channel open time or conductance as measured by spectral analysis of endplate current fluctuations (Steinacker and Zuazaga 1981). In the current work, we show that, while both chemical modifications increase the decay time of the miniature endplate current, only sulfonation increases the time to peak. Sulfonation also produced an effect on voltage jump current relaxation time, which parallels the increase in miniature endplate current decay time, and an increase in the ratio of the current relaxation amplitudes. Diamide had no effect on voltage jump current relaxation amplitudes or time constants. These data are analyzed in an attempt to correlate changes in specific rate constants to changes in the macroscopic current measurements.

Generation of calcium action potentials in crustacean muscle fibers following exposure to sulfhydryl reagents.

The ventroabdominal flexor muscles of the crustacean Atya lanipes, which are normally completely inexcitable, generate trains of overshooting calcium action potentials after exposure to the sulfhydryl reagents known as alpha, beta-unsaturated carbonyl compounds. The chemically induced action potentials are abolished by protein reagents specific for guanidino and amino groups. Attempts to induce excitability by the use of agents that block potassium conductance were without success. It is proposed that calcium channels are made functional by the covalent modification of a calcium protochannel, via the interaction between the introduced carbonyl group and existing arginine residues.