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1. The effects of chlorpromazine on the voltage-activated sodium and type I (transient) calcium channels were studied in cultured mouse neuroblastoma cells (N1E-115) using the whole-cell patch-clamp technique. 2. Chlorpromazine (2-10 x 10(-6) M) blocked both the sodium channel current and the calcium channel current as carried by Ba2+ in a reversible and dose-dependent manner. 3. The block was not associated with any change in the time course of the activation and inactivation of the sodium and calcium channel currents. 4. The dose-response relationships for the block of these channels measured with a holding potential of -120 mV indicated a 1:1 binding stoichiometry with apparent dissociation constants of 2.5 +/- 10(-6) M and 1.5 +/- 10(-5) M for the sodium and calcium channels, respectively. 5. The block was dependent on the holding potential for both channel currents. The apparent dissociation constant for the sodium channel was decreased to 0.65 +/- 10(-6) M when the membrane was held at -80 mV. The apparent dissociation constant for the calcium channel was decreased to 3.2 +/- 10(-6) M when the membrane was held at -60 mV. 6. The steady-state inactivation curve for the sodium channel was shifted by 12.4 +/- 1.8 mV to more negative potentials by exposure to 1 x 10(-6) M-chlorpromazine. The inactivation curve for the calcium channel was also shifted by 15.4 +/- 3.2 mV to more negative potentials by exposure to 1 x 10(-5) M-chlorpromazine. These results indicate a greater affinity of chlorpromazine for the inactivated state of the channels than for the resting state. 7. Chlorpromazine caused a marked use-dependent block of the sodium channel current, as demonstrated by a cumulative increase of the block during a train of depolarizing pulses. The use-dependent block was observed even with an interpulse interval as long as 2 s. 8. On the other hand, the block of the calcium channel current did not notably accumulate during a train of depolarizing pulses even when extremely prolonged (1 s) pulses were applied at a very short interpulse interval (200 ms). 9. The marked use dependence of the sodium channel block was due to a very slow repriming of the drug-bound sodium channels from inactivation, whereas the lack of use dependence of the calcium channel block was due to a rapid repriming of the drug-bound calcium channels.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The unfertilized egg of the tunicate, Halocynthia roretzi, was intracellularly perfused with various solutions. 2. The perfusion apparatus consisted of lower and upper compartments which were connected by a small glass funnel. A denuded egg cell without chorion was dropped into the funnel and brought into close contact with the glass wall of the funnel. The membrane of the egg faced to the lower compartment was ruptured by a slight difference of hydrostatic pressure and the inside of the egg was perfused with the internal solution flowing through the lower compartment. The current across the upper membrane was analysed by voltage-clamp technique. 3. The egg cell in contact with 400 mM-Na external solution and perfused intracellularly with 400 mM-Na for 30 min showed a relatively low Na reversal potential, +6 mV, in comparison with +60 mV in the intact egg in standard artificial sea water. The exchange efficiency was monitored by observing the shift of Na reversal potential during perfusion with high Na internal perfusate. 4. The internal perfusate containing F- ions stabilized the egg membrane and kept the excitability for 1--2 hr during the intracellular perfusion. With the internal F- perfusate the intracellular cationic content was changed to 400 mM-Na, K, Rb or Cs (external solution of 400 mM-Na) and permeability ratios of the egg Na channel were estimated as PNa:PK:PRb:PCs=1.0:0.14:0.05:0.04. The internal F- perfusate abolished Ca current which was consistently observed in the intact egg, while the internal Cl- perfusate kept both Na and Ca current as in the intact egg. However with the internal Cl- perfusate the egg cell could not be kept in good condition more than 20-30 min. 6. The effects of intracellular free Ca ions upon the egg Na and Ca channels were analysed by using Ca ion-buffered internal Cl- and high Na perfusate. The results showed that internal Ca ions above 10(-6) reduced the Ca current and enhanced the Na current at the same time. In the range between 10(-5) and 10(-4) M the Ca current became half of the control obtained with zero free Ca perfusate while the Na conductance at the zero current level doubled. The internal Ca ions above one mM seemed to abolish the Ca current and to reduce the Na current as well. The reciprocal effect of intracellular Ca ions upon the egg Na and Ca channels was demonstrated in the concentration range from 10(-6) to 10(-3) M.

1. The cleavage of the embryos of an ascidian, Halocynthia roretzi, was arrested with cytochalasin B (1 microgram/ml.) at the 16- or 64-cell stage. These cleavage-arrested embryos were still able to develop membrane excitability. 2. In the cleavage-arrested 64-cell embryos at the time when control embryos became hatched larvae, Ca spikes were evoked in the presumptive muscle blastomeres, and Na- and Ca-dependent action potentials were induced in some ectodermal blastomeres. 3. Membrane currents of the cleavage-arrested 16-cell embryos were recorded with the voltage-clamp technique and analysed as a function of developmental time at 15 degrees C. For this purpose, intact eggs, 4- and 8-cell embryos were also used. The cleavage-arrested embryos behaved electrically like single cells, due to tight electrical coupling between blastomeres. After the 25-hr stage decoupling occurred. 4. Both Na and Ca currents decreased during the initial 10 hr. Na current became less than one third and Ca current almost disappeared. At 17 hr both Na and Ca currents increased again. 5. The potential-dependence of the Na and Ca currents after 17 hr was similar to that in the egg, although substantial parallel shifts in the current-voltage relations were observed: a 5 mV positive shift for the Na current and a 15-20 mV negative shift for the Ca current. 6. Delayed (outward) K current developed gradually until 20 hr and then increased abruptly. The activation level for the delayed rectification was markedly negative (around -10 mV) in comparison with that of the egg (around + 100 mV). Anomalous (inward) K current, on the other hand, increased gradually without changes in the potential-dependence throughout development. 7. The results suggest that the differentiation of excitable membranes in the ascidian embryo does not involve changes in the properties of the individual channels, but rather changes in the numbers of various kinds of ion channels.

The Ca current of the egg cell membrane of a certain tunicate, Halocynthia roretzi Drasche, was studied by the voltage-clamp technique. 2. The Ca current in the standard artificial sea water (ASW) was produced at the critical membrane potential of -10 mV after inactivating the Na current by conditioning depolarization, -30 to -15 mV. The Ca current was abolished by replacing Ca in ASW with Mg2+ or Mn2+. The Ca current was not significantly influenced by replacing Na in ASW with choline or Cs. 3. The relation of Ca current to the external Ca concentration was a monotonously increasing function, but was not linear. The current tended to saturate above 50 mM-Ca. In 100 mM-Ca ASW, the maximum peak inward current of Ca ranged from 1 to 7 X 10(-9) A. 4. The kinetics of Ca current was accurately analysed because of the small contribution of K outward current and was found to be relatively slow in comparison with the Na current. The peak time and the half-decay time of the maximum Ca current at about 25 mV were about 25 and 100 msec respectively in 100 mM-Ca ASW at 15 degrees C. 5. Addition of 20 mM-Co2+ to 100 mM-Ca ASW reduced Ca current to one fourth and 1 mM-La3+ to 100 mM-Ca ASW abolished the current. 6. Sr and Ba could substitute for Ca in Ca channels. The selectivity ratios for the 'Ca channels's were Ca (1-00):Sr(1-17):Ba(0-71) at a potential level of +40 mV. The Ca current in the egg cell membrane appeared to be essentially the same as the Ca current in the common excitable membranes, such as the crustacean muscle fibre. 7. The polyvalent cations including Ca ion and monovalent H+ ion showed the stabilizing effect upon both Na and Ca currents, by shifting V-I relations along the voltage axis. From the prediction of a theory of the diffuse double layer, the shift in the V-I relation induced by those cations should be directly related to their binding powers to the membrane. Thus, the sequence of the binding powers was inferred as H+ greater than La3+ greater than Co2+ greater than Mn2+ greater than Ca2+ greater than Sr2+ larger than or equal to Ba2+ greater than Mg2+. 9. In Na-free ASW, such as isotonic Ca ASW, Ca current was composed of two components. The one component was the Ca current described in 1 to 6. The other was also dependent upon the external Ca concentration, but showed the more negative critical membrane potential and the faster kinetics. It was concluded that this component should be the Ca current through Na channels. 10. The selectivity among Ca, Sr and Ba for 'Ca' current through 'Na channels' was significantly different from that of 'Ca' current through 'Ca channels', being Ca greater than Sr larger than or equal to Ba = 0.

1. Two types of voltage-sensitive calcium channels were identified and studied in the neuroblastoma cell line N1E-115. Calcium channel currents as carried by Ba2+ (50 mM) were recorded using the whole-cell variation of the patch-electrode voltage-clamp technique. 2. A transient (type I) inward Ba2+ current was evoked by a step depolarization from a holding potential of -80 mV to potentials more positive than -50 mV. The current amplitude became maximum around -20 mV. 3. A depolarization to potentials more positive than -20 mV evoked a long-lasting (type II) component of the inward Ba2+ current. This component reached its maximum around +10 mV and did not inactivate during a prolonged depolarizing pulse lasting 400 ms. 4. When preceded by a 5 s conditioning pulse to -30 mV, step depolarization failed to evoke a transient current due to inactivation. However, it induced a long-lasting current. 5. A transient current isolated as the component sensitive to conditioning depolarization became faster in its time course and smaller in its amplitude with membrane depolarization. The current direction was still inward at +60 mV. 6. From the differential voltage sensitivity and the independent channel activity described above, calcium channels responsible for the transient current (type I channel) and those responsible for the long-lasting current (type II channel) were considered to be two different entities. 7. Cd2+ preferentially blocked type II channels, whereas La3+ was a highly potent blocker for both types of calcium channels. 8. The relative potency for block by polyvalent cations was as follows (apparent dissociation constant in microM): La3+, 1.5 much greater than Ni2+, 47 greater than Cd2+, 160 = Co2+, 160 for type I channels, and La3+, 0.9 greater than Cd2+, 7.0 much greater than Ni2+, 280 greater than Co2+, 560 for type II channels. 9. The two types of calcium channels were equally sensitive to the temperature. The current amplitude was reduced by cooling below 30 degrees C. The temperature coefficient (Q10) value was estimated to be 3.0 between 20 and 30 degrees C, and 15.0 below 20 degrees C. Above 30 degrees C, warming reduced the amplitude slightly. 10. External application of dibutyryl adenosine 3',5'-phosphate (dibutyryl cyclic AMP) (1 mM) caused an increase in the amplitude of the type II current by 30-50%, while failing to enhance the type I component.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Ionic currents of the egg membrane of a certain tunicate. Halocynthia roretzi Drashe, were studied by the voltage-clamp technique. 2. The membrane depolarization beyond -55mV in standard artificial sea water induced mainly transient inward current and slight outward currents, when the holding potential was kept at -99 mV. 3. The transient inward current was composed of two components; the major one showed a faster time course, a more negative critical level of about -55 mV, and a reversal potential around +60 mV and the minor one showed a slower time course, a less negative critical level o -10 mV, and no definite reversal potential. 4. The major component became maximum at about -25 mV with the peak time of 6-9 msec at 15 degrees C, and the maximum currents ranged from 0-5 to 1-5 X 10(-5) A/cm2. 5. The major component of the inward current was abolished by the replacement of Na with choline or Tris or Cs ions, while it was almost unaltered by the replacement with Li. The minor component was independent of Na concentration in the external solution. 6. The major component showed the activation and inactivation identical with those of Na current of other excitable membranes. A conditioning depolarization over -90 mV inactivated the Na current and the half inactivation of the major inward current was obtained by a conditioning pulse to -56 mV, when the pulse duration was 400 msec and the temperature was at 15 degrees C. 7. The time course of the Na current was formulated with m and h parameters in the following equations: (see article). 8. The kinetic parameters taum and tauh of egg Na current were calculated and compared with those of the squid axon. The potential dependence of taum and tauh was almost identical with that of the axon, but the absolute values of both taum and tauh were ten- to twentyfold larger than those of the axon in any range of the membrane potential. 9. The temperature depdence of the kinetic parameters taum, tauh and of the chord conductance gNa was studied. The Q10's for taum and tauh were both 4-0, while the Q10 for gNa was 2-0 in the temperature range from 5 to 20 degrees C. 10. The outward and inward rectifying conductances of egg membrane were remarkably activated at the potential level above +100 mV and below -70 mV respectively in standard artificial sea water. Both increased currents were subsequently subject to inactivation. 11. It was suggested that Na, Ca, K inward rectifying and K outward rectifying conductances all exist separately in the egg cell membrane and the Na current was essentially identical with that through the Na channel in other excitable membranes.

1. The effects of alterations of the intracellular ionic composition on the properties of anomalous (or inward) rectification of the egg membrane of the starfish, Mediaster aequalis, were studied by using an intracellular perfusion technique. The following results were obtained, analysing the membrane current with the voltage-clamp technique. 2. The inward rectification of the K conductance depends only on the membrane potential, V, when the K equilibrium potential, VK, is altered by changing the internal K+ concentration at a fixed external K+ concentration, while it depends on V-VK when VK is altered by changing the external K+ at a fixed internal K+ concentration. 3. From the above the conclusion is reached that the gating of the K channel of the inward rectification depends on V and external but not internal K+ concentration. 4. The conductance of the K channel at a given voltage is roughly proportional to the square root of [K+]i when the latter is altered at a fixed external K+ concentration. 5. Since the conductance is proportional to the square root of [K+]o when this is altered at a fixed internal K+ concentration, the final conclusion is that this conductance is proportional to the geometric mean of the external and internal K+ concentrations. 6. Intracellular Na+ ions are necessary for the activation of inward rectification; the K conductance increases sharply with internal Na+ concentration, reaching saturation at 200 mM. 7. A similar potentiating effect is found for Li+, although it is weaker. Rb+, Cs+ and organic cations such as arginine+ do not have this effect.

1. In the gastrula of the tunicate (Halocynthia aurantium) the resting potential of the embryonic membrane was about -70 mV in both std ASW and Na-free ASW.2. The potential responses to depolarizing current in the early gastrula embryo showed distinct potential jumps from about -50 to about +40 mV during the application of current and from about 0 to resting level after its cessation thus forming a plateau at about 0 mV. Those jumps were not altered significantly by the removal of both Na and Ca ions from ASW except for the duration of the plateau after the cessation of current.3. Blastomeres in an embryo were tightly coupled with each other electrotonically at the blastula and gastrula stages. The fact that the coupling ratio was nearly 1.0 made it reasonable to treat an embryo as one cell as far as electrical properties are concerned.4. The I-V relation of the embryonic membrane consisted of an inward rectifying region, outward rectifying region and transitional zone between the two, resulting in a S-shaped curve as in the egg cell membrane. But in the gastrula embryo, the transitional zone actually included the discontinuous part of the I-V curve (from about -50 to about +40 mV) due to the potential jump.5. The ionic current (I(i)) during the potential jump in Na-free ASW was estimated from the slope of the potential response based on the assumption of constant capacitance during the membrane potential changes. The calculated I(i)-V curve complemented the interrupted I-V curve smoothly and showed the existence of a negative resistance region from about -50 to about 0 mV in the steady-state I-V relation.6. The steady-state I-V relation was altered significantly by changing the K concentration in ASW, and the existence of the negative resistance can be reasonably explained by the marked inward-going rectification or anomalous rectification of K conductance.7. The replacement of K with Rb in ASW produced the marked suppression of the inward-going rectification of the embryonic membrane.

1. The effect of temperature upon the anomalous or inward rectification of the K conductance in the immature egg membrane of the starfish, Mediaster aequalis, was studied by using voltage clamp technique. 2. The K conductance decrease with a relatively small Q10 (1.62) down to about 10 degrees C; below 10 degrees C, the Q10 is much greater (5.8 at [K+]o = 25 mM). The smaller Q10 is independent of [K+]o, whereas the larger one depends on [K+]o. 3. The activation of the rectification depends on V-VK, rather than V alone, at all temperatures at constant internal K concentration. 4. The K conductance at a given V-VK is approximately proportional to the square root of [K+]o at a fixed [K]i above 10 degrees C while the conductance depends substantially less on [K+]o below this temperature. 5. The logarithm of the activation time constant of the inward rectification depends linearly on the membrane potential at all temperatures. 6. The slope of the relation is strongly temperature dependent above about 10 degrees C whilst the dependence is much less below 10 degrees C; i.e. the Q10 of the activation time constant is membrane potential-dependent above 10 degrees C. 7. The results suggest that the mechanism of ion permeation during anomalous rectification changes at about 10 degrees C.

1. The properties of the fast-inactivating or transient K current and the slowly inactivating or delayed K current of the membrane of immature eggs of the clonial marine coelenterate, Renilla Koellikeri, were studied by using voltage clamp and intracellular dialysis techniques. 2. The transient current is activated when the membrane potential becomes more positive than -25 approximately -20 mV (resting potential, -72 +/- 5 mV) whereas the activation potential of the delayed current is -10 approximately OmV. These potentials are independent of either [k+]o or [K+]i. 3. The inactivation of the transient current is rapid and is almost complete for membrane potentials more negative than the activation potential while it is slow for the delayed current and incomplete within a few seconds. 4. Both currents shows similar reversal potentials which are predominantly determined by the K concentration gradient across the membrane. 5. The sensitivities of the conductance upon the internal K concentration differ between the two currents, suggesting that the interaction between the site and ions in the membrane channels differ between them. 6. Neither current is a Ca-activated K current. 7. 4-AP suppresses the transient current at concentrations substantially smaller than those that suppress the delayed current while TEA shows no effect on either current. 8. Intracellular application of pronase or tannic acid at relatively high concentrations does not alter the inactivation of either current. 9. The membrane includes a voltage-dependent Ca permeability which results in action potentials under current-clamp conditions.

1. Threshold changes of Na and Ca currents due to various polyvalent cations (stabilizing cations) or H(+) ions were studied in the egg cell membrane of a tunicate, Halocynthia roretzi, by using the voltage-clamp technique.2. With an increase in [Ca](o) or a decrease in pH in the external solution, the current-voltage (I-V) relations for the peak of the Na and Ca currents shifted along the voltage axis in the positive direction. These voltage shifts in the I-V relations, measured at a potential of V((1/2)) where inward current attains its half-maximum, were shown to be identical to shifts in voltage-dependence of the time courses of Na and Ca currents, and also identical to shifts in the inactivation curves of Na current along the voltage axis.3. The shifts in V((1/2)) produced by various polyvalent cations or H(+) ions were analysed by the Gouy-Chapman equation for the diffuse double layer, by assuming that a change in V((1/2)) directly corresponds to a change in the surface double layer potential.4. The V((1/2))-divalent cation concentration relations of Na current were exactly described by the predictions of the theory with a constant value of the surface charge density of 1e(-)/(9 A)(2). The weak stabilizing effects of Mg(2+), Sr(2+) and Ba(2+) were quite similar to each other and were explained in terms of a ;screening' effect. Other divalent cations, such as Ca(2+), Mn(2+) and Ni(2+), showed various different stabilizing effects which were explained in terms of a ;binding' effect. The binding constants (K(1)'s) for Ca(2+), Mn(2+) and Ni(2+) were 0.21, 0.45 and 0.94 M(-1), respectively.5. H(+) ions showed a powerful stabilizing effect upon the Na current with a K(H) of 6 x 10(4)M(-1). This value indicates that the acidic sites around Na channels have a pK(a) of 4.78. La(3+) ions also acted as a strong stabilizer upon the Na current with a K(La) of 15 M(-1). For both H(+) and La(3+), the V((1/2))-concentration relations were also exactly described by the Gouy-Chapman equation with the same charge density of 1e(-)/(9 A)(2) as estimated by varying divalent cations.6. The stabilizing effect of permeant cations such as Ca(2+), Sr(2+) and Ba(2+) on Ca channel currents was analysed. The effect of lowering pH was also studied. It was found that the surface charge density of 1e(-)/(9 A)(2) estimated by Na current is also applicable to the explanation for the V((1/2))-divalent cation concentration or - pH relationships. The estimated binding constants for H(+), Ca(2+) and Sr(2+) were 1.2x10(5), 0.58 and 0.035 M(-1), respectively. Ba(2+) does not bind to charged sites near to the Ca channels.7. It was noticed that a considerable reduction in the conductances of Na and Ca currents occurred in parallel with a stabilizing effect. This reduction was ascribed to a decrease in the concentration of permeant cations at the external surface of the cell membrane, as predicted by the theory of the diffuse double layer. The Goldman, Hodgkin-Katz equation for ionic currents was applied to explain the conductance suppression.8. The conductance suppressions of Na and Ca channel currents due to Ca(2+), Sr(2+) and Ba(2+) were found to be apparent ones, only reflecting decreases in the surface concentration of permeant cations without any changes in the permeability. After correction for the apparent suppression, the real permeability ratio among Ca(2+), Sr(2+) and Ba(2+) for Ca channels was determined as 1.00, 0.56 and 0.21 respectively.9. The conductance suppression of Na current by lowering pH was explained in terms of a real suppression or blocking which is superimposed on the apparent suppression. Considering the surface [Na](o), the plot of P(Na) against the surface pH yielded a blocking curve of Na channel by H(+) ions, which implies that two H(+) ions are necessary to block each Na channel. For Ca channels no real blockage was observed in acidic pH.10. It was concluded from the present experiment that there exists a surface potential capable of affecting both gating and permeation mechanisms of ionic channels in this tunicate egg cell membrane.