Separation of sodium and calcium channels in the surface membrane of molluscan nerve cells

By intracellular dialysis of isolated neurons of the mollusksHelix pomatia andLimnaea stagnalis and by a voltage clamp technique the characteristics of transmembrane ionic currents were studied during controlled changes in the ionic composition of the extracellular and intracellular medium. By replacing the intracellular potassium ions by Tris ions, functional blocking of the outward potassium currents was achieved and the inward current distinguished in a pure form. Replacement of Ringer's solution in the extracellular medium with sodium-free or calcium-free solution enabled the inward current to be separated into two additive components, one carried by sodium ions, the other by calcium ions. Sodium and calcium inward currents were found to have different kinetics and different potential-dependence: _mNa=1±0.5 msec, _mCa=3±1 msec, _hNa=8±2 msec, _hCa=115±10 msec (V_m=0), G_Na=0.5 (V_m=–21±2 mV), G_Ca=0.5 (V_m=–8±2 mV). Both currents remained unchanged by tetrodotoxin, but the calcium current was specifically blocked by cadmium ions (2·10^–3 M), verapamil, and D=600, and also by fluorine ions if injected intracellularly. All these results are regarded as evidence that the soma membrane of the neurons tested possesses separate systems of sodium and calcium ion-conducting channels. Quantitative differences are observed in the relative importance of the systems of sodium and calcium channels in different species of mollusks.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 183–191, March–April, 1976.     

Effects of calcium and potassium channel blockers on changes in transepithelial potential and spike responses in Lorenzi ampullary electroreceptors in Raja clavata

The effects of TEA, 4-AP, Co⁺⁺, Cd⁺⁺, Cs⁺, EDTA, and verapamil on the sensory epithelium of ampullae of Lorenzini were studied inRaja clavata (Black Sea skate). During voltage clamping, transepithelial application of TEA to the basal surface caused oscillations in transepithelial potentials in response to presentation of an excitatory stimulus, which had been suppressed by Co⁺⁺, Cd⁺⁺, and EDTA. Application of Cs⁺ was followed by complete or partial suppression of spike response adaptation. When applied to the apical epithelial surface, TEA produced an increase in spike response to stimulation and highly accelerated adaption. No substantial changes took place after apical application of Co⁺⁺, Cd⁺⁺, and verapamil. The mechanisms of these phenomena are discussed.Deceased.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 652–659, September–October, 1985.     

Effects of quinine on calcium current in mollusk neurons

The effects of quinine on the peak amplitude and the decay of calcium currents (I_Ca) were investigated in nonidentified neurons isolated fromHelix pomatia. A concentration of 1×10^–5–5×10^–4 M quinine was found to produce a reversible dose-dependent deceleration in the decline of I_Ca ("lead" effect) and a reversible, slowly evolving dose-dependent reduction in I_Ca amplitude ("lag" effect). A reduction in amplitude down to half control level is observed at a quinine concentration of 6 ×10^–5 M, while the current-voltage relationship of I_Ca shifts by 5–10 mV towards negative potentials. Results show that quinine successfully blocks calcium channels inHelix pomatia neurons.Institute of Brain Research, All-Union Mental Health Research Center, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 413–417, May–June, 1987.     

Expression of low voltage-activated calcium channels inXenopus oocytes

Calcium channels were expressed inXenopus oocytes by means of messenger RNA extracted from the rat thalamo-hypothalamic complex, mRNA(h). Inward barium currents,I _Ba, were recorded in Cl^–-free extracellular solution with 40 mM Ba²⁺ as a charge carrier, using two-microelectrode technique. Depolarizations from a very negative holding potential (V _h=–120 mV) began to activateI _Ba at about –80 mV; this current peaked at –30 to –20 mV and reversed at +50 mV, indicating that I _Ba may be transferred through the low voltage-activated (LVA) calcium channels. The time-dependent inactivation of the current during a prolonged depolarization to –20 mV was quite slow, followed a single exponential decay with a time constant of 1550 msec, and contained a residual component constituting 30% of the maximum amplitude. The current could not be completely inactivated at any holding potential. As expected for LVA current, a steady-state inactivation curve was shifted towards negative potentials. It could be described by the Boltzmann's equation with the half-inactivation potential of –78 mV, slope factor of 15 mV, and residual level of 0.3. ExpressedI _Ba could be blocked by flunarizine (K _d=0.42 µM), nifedipine (K _d=10 µM), and amiloride at a 500 µM concentration. Among the inorganic Ca²⁺ channel blockers, the most potent was La³⁺ (K _d=0.48 µM), while Cd²⁺ and Ni²⁺ were not very selective and almost thousand-fold less effective (K _d=0.52 mM andK _d=0.62 mM, respectively) than La³⁺. Our data show that mRNA(h) induces expression in the oocytes of almost exclusively LVA Ca²⁺ channels with voltage-dependent and pharmacological properties very similar to those observed for T-type Ca²⁺ current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 183–189, May–June, 1995.     

Dose-inward current relationships for current-carrying calcium and strontium ions in isolated Limnea stagnalis neurons

The dose-inward current relationships for current-carrying Ca⁺⁺ and Sr⁺⁺ ions were studied by means of intracellular dialysis and voltage clamp methods in isolated neurons ofLimnea stagnalis. These relationships rose to a ceiling for these ions. The data are explained in terms of an activation theory using a double-barrier model of the calcium channels. Relative values of velocity constantsv _X for passage of ion X²⁺ across the second energy barrier and of the constant K_X, defining the binding effectiveness at the entrance to the channel were determined:vCa:vSr = 1:2; K_Ca = 1400 M^–1; K_Sr = 270 M^–1.Research Institute of Physiology and Pathology of the Cardiovascular System, Medical Institute, Ministry of Higher Middle and Specialized Education of the Lithuanian SSR, Kaunas; Institute of Biophysics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 11, No. 4, pp. 362–366, July–August, 1979.     

Theoretical description of the calcium channel in rat spinal ganglion neurons

The surface charge density (σ′₀) and the binding constant of Ca⁺⁺ with charged groups on the outer surface of the membrane (K_Ca) were calculated from experimentally determined values of the shift of the current-voltage characteristic curves of calcium currents in the membrane of rat spinal ganglion neurons: σ′₀ = 0.15 ± 0.05 e/nm² and K_Ca = 70 ± 10 liters/mole. Using a three-barrier model the energy profile of the calcium channel of the membrane of these neurons was calculated for Ca⁺⁺, Ba⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and verapamil. The calcium current was shown to be determined mainly by the depth of the potential hole corresponding to the outer binding site of the calcium channel. It is concluded from the results that the outer binding site of the calcium channel contains only one carboxyl group.     

Nifedipine-sensitive low voltage-activated calcium current in neurons of the rat laterodorsal thalamic nucleus

The kinetic and pharmacological properties of low voltage-activated (LVA) Ca²⁺ channels were studied in neurons of the laterodorsal (LD) thalamic nucleus in brain slices from 12-day-old rats. A homogeneous population of LVA Ca²⁺ channels was found in the tested neurons. LVA Ca²⁺ current evoked by a step depolarization from a holding potential more negative than −70 mV was found to be sensitive to nifedipine (K _d=2.6 (M). This current gained its maximum at −55 mV and demonstrated fast monoexponential decay with the time constant of 32.3±4.0 msec (n=15). Lanthanum (1 μM) effectively blocked LVA Ca²⁺ current, while nickel (25 μM) did not affect this current. It is concluded that the channels that, according to their pharmacological properties, provide the studied LVA Ca²⁺ current cannot be regarded as T-type Ca²⁺ channels and belong to some other type of LVA Ca²⁺ channels.     

Voltage-gated ion channels of inflowing current expressed in Xenopus oocytes after injection of rat brain mRNA

The expression of two types of voltage-gated ion channels of the inflowing current ("fast" sodium channels, sensitive to tetrodotoxin, and high-threshold calcium channels) was detected by electrophysiological methods in the membrane ofXenopus oocytes, after injection of poly(A)⁺-mRNA from the brains of 18- to 20-day-old rats. When Cd²⁺ (200 µmoles/liter) was added to the extracellular solution, the barium current through the expressed calcium channels was completely suppressed, but no sensitivity to D-600 (20 µmoles/liter) and nitrendipine (50 µmoles/liter) was exhibited. A peptide blocker of the high-threshold calcium channels of the neuron membrane, -conotoxin GVIA, in a concentration of 1 µmole/liter led to 20–40 min suppression of the barium current expressed in the oocyte. Steady-state inactivation of this current could be described by the Boltzman formula, using the values of the half-inactivation potential V_1/2=–50 mV and the steepness factor k=14 mV. It is concluded that in potential-dependent and pharmacological properties, the calcium channels expressed in the oocyte, despite the absence of any appreciable time-dependent inactivation, most resemble the high-threshold inactivatable (HTI- or N-type) calcium channels of the neuron membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 344–353, May–June, 1991.     

Membrane Currents Carried by Ca, Sr, and Ba in Barnacle Muscle Fiber During Voltage Clamp

Membrane currents associated with voltage clamp of the giant muscle fibers of a barnacle, Balanus nubilus, were analyzed in terms of currents of the Ca and K channels. Although the activation of the K channel occurs more slowly than that of the Ca channel, both currents show a significant temporal overlap. The currents carried by Ca⁺⁺, Sr⁺⁺, or Ba⁺⁺ through the Ca channel were compared under the conditions at which this overlap was the least. When only one divalent cation is present in the solution, Ba⁺⁺ carries more current than Ca⁺⁺ or Sr⁺⁺ and the sequence of the current is Ba > Sr ≈ Ca. When the external solution contains a relatively high concentration of Co⁺⁺, which is a blocking agent for the Ca channel, inversion of the sequence occurs, to Ca > Sr > Ba. This is due to the fact that the blocking effect differs depending on which ion carries current through the Ca channel. The Ba current is most sensitive and the Ca current is least affected. Ba suppresses the current of the K channel, independently of its current-carrying function through the Ca channel.     

Selectivity of edta-modified calcium channels to monovalent cations

The characteristics of slow inward sodium currents arising in response to membrane depolarization were studied in experiments on isolated dialyzed neurons of the snailHelix pomatia when the calcium-chelating agent EDTA was added to the calcium-free external solution. Values of the relative permeability of the corresponding ionic channels, determined from the shift of the equilibrium potential, were: P_Na+:P_Li+: +=1.00:0.80:0.55:0.21. The ratio between these values for "fast" sodium channels was 1.00:1.04:0.44:0.19. The induced sodium current was blocked by D-600 and nifedipine, which block calcium channels, more effectively than the calcium current of the same membrane (the corresponding dissociation constants were 10^–5 and 0.8·10^–5 mole/liter for the induced sodium current compared with 2.6·10^–5 and 2.3·10^–5 mole/liter for the calcium current). It is postulated on the basis of these data that the calcium channels have a principal selective filter similar to that of sodium channels, but also an additional binding site for bivalent cations, which prevents entry of monovalent cations into the channel. The addition of calcium-chelating agents to the calcium-free external solution liberates this site and thereby modifies the calcium channel into a sodium channel.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 491–498, September–October, 1982.     

Ca2+-activated K+ currents inNecturus choroid plexus

Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of –59±2 mV and a whole cell resistance of 56±6 M were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca²⁺, stepping the membrane potential from a holding value of –50 to –10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca²⁺-activated K^_ channels. Based on the voltage dependence of the activation of Ca²⁺-activated K⁺ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K⁺ as their reversal potential at different K⁺ concentration gradients followed the Nernst potential for K⁺. These currents were reduced by the addition of TEA⁺ to the bath solution and were eliminated when Cs⁺ or Na⁺ replaced intracellular K⁺. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t _1/2) to the steady value. Increasing the pipette Ca²⁺ also decreased the delay and decreasedt _1/2. For instance, when pipette Ca²⁺ was increased from 5 to 500nm, the delay andt _1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K⁺ currents through Ca²⁺-activated K⁺ channels.At the resting membrane potential of –60 mV, Ca²⁺-activated K⁺ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca²⁺. Thus the Ca²⁺-activated K⁺ channels may play a specific role in maintaining intracellular K⁺ concentrations during CSF secretion.     

High activity extracellular glucose (xylose) isomerase from a Chainia species

Summary A sclerotia-forming actinomycete of the genus Chainia secreted high levels of glucose (xylose) isomerase when grown in submerged culture on a wheat bran - yeast extract medium. Maximum activity (4 units/ml) was obtained after 3–4 days when the cell bound activity was 0.19 units/ml. The two enzymes differed significantly in pH optima (extracellular, 9.5; cell-bound, 7.0) and in their adsorption behaviour on CM and DEAE celluloses. Both Mg⁺⁺ and Co⁺⁺ are required by the cell-bound enzyme for its optimum activity while either Mg⁺⁺ or Co⁺⁺ is necessary for the extracellular enzyme.NCL Communication 3320     

Long-lasting inward current in snail neurons in barium solutions in voltage-clamp conditions

Summary The inward membrane current was recorded under voltage clamp from nonbursting neurons of the snailHelix pomatia in Na-free solutions containing Ba ions but no other divalent cations. The inward current was separated into two components: (i) an early fast inactivating component and (ii) a smaller long-lasting component. Both components were dependent on the external Ba concentration. It is concluded that both components of the inward current are carried by Ba ions. The activation of the early fast inactivating component of the inward current occurred at more positive membrane potential than that of the long-lasting component. The shape of the inactivation curve for the peak value of the inward current was similar to that for the long-lasting component. The potentials of half-inactivation for the peak value of the inward current and for its long-lasting component were –28 and –22 mV, respectively. The blocking effect of Co⁺⁺ on the early fast inactivating component was substantially greater. In some neurons after treatment with 15mm Co⁺⁺ only the long-lasting component was recorded. The activation kinetics of the long-lasting component of the inward current were analyzed using the Hodgkin-Huxley equations. The results could be explained by assuming that two components of the inward current in Na–Ca-free solution with Ba ions flowed through the two different channels. The significance of the long-lasting inward current for the normal spike generation is discussed.     

Regulation of excitation-secretion coupling by thyrotropin-releasing hormone (TRH): Evidence for TRH receptor-ion channel coupling in cultured pituitary cells

Summary The electrophysiological and secretory properties of a well-studied clonal line of rat anterior pituitary cells (GH₃) have been compared with a new line of morphologically distinct cells derived from it (XG-10). The properties of the latter cells differ from the parent cells in that they do not have receptors for thyrotropin-releasing hormone and their basal rate of secretion is substantially higher (ca. three- to fivefold). While both cell types generate Ca⁺⁺ spikes, the duration of the spike in XG-10 cells (ca. 500 msec) is about 2 orders of magnitude longer than that in GH₃ cells (5–10 msec). The current-voltage characteristics of the two cell types are markedly different; the conductance of GH₃ cells is at least 20-fold higher than XG-10 cells when cells are depolarized to more positive potentials than the threshold for Ca⁺⁺ spikes (–35 mV). While treatment of GH₃ cells with the secretagogues tetraethylammonium chloride or thyrotropin-releasing hormone decreases the conductance in this voltage region to approximately the same as that for XG-10 cells, the electrophysiological and secretory properties of XG-10 cells are unaffected by treatment with either of these agents. Results of this comparative study suggest that XG-10 cells lack tetraethylammonium-sensitive K⁺ channels. The parallel loss of thyrotropin-releasing hormone receptor binding activity and of a K⁺ channel in XG-10 cells implies that the thyrotropin-releasing hormone receptor may be coupled with, or be an integral part of, this channel. Apparently thyrotropin-releasing hormone, like tetraethylammonium chloride, acts by inhibiting K⁺ channels resulting in a prolongation of the action potential, promoting Ca⁺⁺ influx and subsequently enhancing hormone secretion.     

Identification of a Phosphoprotein Expressed During Somatic Embryogenesis in Wheat Leaf Base Cultures

2,4-D mediated induction of somatic embryogenesis in wheat is enhanced in the presence of Ca⁺⁺ and its removal by EGTA reduces the response significantly. Changes that occur at the polypeptide level following 2,4-D treatment were analysed. Intense cell division activity was discernable in the leaf base explants within an hour of treatment. Changes in protein profiles were prominent in the membrane fraction as compared to the soluble fraction. The protein profile of the leaf base culture with somatic embryos was distinct from the calli induced from mature embryos on a 2,4-D containing medium. The role of Ca²⁺ in the induction of somatic embryogenesis was demonstrated by the use of EGTA (a calcium chelator), verapamil, nifedipine (calcium channel blockers), W7 (calmodulin antagonist) and Li (PI inhibitor). ^{In vitro} protein phosphorylation studies showed that 2,4-D, calcium and related treatments inhibit phosphorylation of proteins. In the membrane fraction proteins, accumulation of polypeptides at the low molecular weight range was seen in samples treated with verapamil and W7, and a 30 kO polypeptide in the samples treated with calmodulin antagonist, W7. Autoradiography of membrane fraction proteins displayed the presence of a 16 kO protein phosphorylated in samples treated with verapamil, nifedipine and W7. It thus appears that 2,4-D and Ca⁺⁺ prevent the phosphorylation of this phosphoprotein. These results thus indicate the action of 2,4-D via the Ca²⁺-CaM signaling pathway in triggering the induction of somatic embryogenesis.     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) significantly increased potential difference (PD) and short-circuit current (SCC) across isolated frog skin when added to the outside Ringer's solution at 10^–4, 10^–3 and 5×10^–3 m concentration. Resistance was reduced by 10^–4 m Cd⁺⁺ but not significantly changed by the higher concentrations. When SCC was first stimulated by vasopressin, 10^–4 and 10^–3 m Cd⁺⁺ produced additive stimulation which was reversible by washing with Cd⁺⁺-free Ringer's. If SCC was first stimulated by Cd⁺⁺, further stimulation by vasopressin was additive with 10^–4 m Cd⁺⁺ but completely inhibited by 10^–3 m Cd⁺⁺. Elevating the calcium ion (Ca⁺⁺) concentration of the outer Ringer's from 10^–3 m to 5×10^–3 m or 10^–2 m prior to Cd⁺⁺ treatment did not reduce the magnitude of SCC stimulation by Cd⁺⁺. Removal of Ca⁺⁺ from the outside Ringer's with 2×10^–3 m EDTA increased SCC as predicted. Subsequent addition of 5×10^–3 m Cd⁺⁺ drastically reduced SCC below control levels while equimolar concentrations of Cd⁺⁺ and EDTA reduced SCC only to control levels. These results suggest that Cd⁺⁺ interacts with the components of the apical plasma membranes of epithelial cells which are associated with the stimulation of SCC by vasopressin and Ca⁺⁺ removal and may be a useful probe for elucidating these components.     

Changes produced in intracellular calcium concentration and transmembrane currents by iontophoretic injection of cAMP in Helix pomatia neurons

Transmembrane currents and changed [Ca²⁺]_in produced by iontophoretic injection of cAMP were investigated in voltage clampedHelix pomatia neurons. The Fura-2 fluorescence probe technique was used to measure [Ca²⁺]_in. Injection of cAMP was found to produce a protracted rise in the latter at a membrane potential range of –40 to –100 mV in conjunction with transmembrane inward current. Duration of the changes in [Ca²⁺]_in largely dependent on neuronal size and varied between 50 and 500 sec (parameters for neurons with somata of around 100 and 40 µm respectively). In a medium with Ca²⁺ replaced by Mg²⁺ (as well as after addition of EDTA, a calcium chelator) both transmembrane current and the pattern of increase in [Ca²⁺]_in remained unchanged. Inward current usually declined substantially but degree of change in [Ca²⁺]_in remained the same when Na⁺ was eliminated from the solution by replacing its Tris⁺. Addition of 2 mM Cd²⁺ to the external medium hardly affected current level and increase in [Ca²⁺]_in. Neither procaine, a local anesthetic, nor ryanodine (which inhibits release of calcium from the intracellular store) changed the cAMP effects observed. A concentration of 1 mM La³⁺ depressed both inward current and the [Ca²⁺]_in increase. Findings would imply the occurrence of cAMP-dependent release of calcium from the intracellular store in the neurons tested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 396–402, May–June, 1989.     

Role of external calcium in sodium and chloride transport in the gills of seawater-adaptedMugil capito

Summary When the mulletMugil capito is transferred to medium lacking Ca⁺⁺ (either Ca⁺⁺-free seawater or distilled water) the passive permeability of the gill to Na⁺ and Cl^– is increased and the activating effect of external K⁺ on the Na⁺ and Cl^– effluxes in hyposaline media is inhibited. The permeability of the gill increases progressively in proportion to the time of Ca⁺⁺ deprivation; it declines when Ca⁺⁺ is added again to the external medium. The active mechanisms for ion excretion are not reversible. At external Ca⁺⁺ concentrations from 0.1 to 10 mM the Na⁺ permeability is constant but the activation of Na⁺ efflux by K⁺ shows a maximum at a Ca⁺⁺ concentration of about 1 mM. For activation of Cl^– efflux external bicarbonate must be present, in addition to Ca⁺⁺, suggesting the existence of a Cl^–/HCO ₃ ^– exchange. The mechanism by which Ca⁺⁺ controls the passive branchial permeability is thus probably different from that involved in K⁺ activation of ion excretion. The Ca⁺⁺ effect on the K⁺ sensitive ionic excretory mechanisms seems to be related to intracellular Ca⁺⁺ movements. Thus, on the one hand, substances such as Ruthenium Red and La⁺⁺⁺ which both inhibit Ca⁺⁺ exchange, in media containing Ca⁺⁺ and HCO ₃ ^– also inhibit K⁺ activation of Na⁺ and Cl^– effluxes; on the other hand, the ionophore A 23187, a stimulator of Ca⁺⁺ exchange, when added to these media, activates the Na⁺ and Cl^– effluxes; its maximal effect on the Na⁺ flux occurs at 2 mM Ca⁺⁺.Abbreviations ASW-Ca artificial seawater minus calcium - DW deionised water - DWCa deionised water with 1 mM Ca⁺⁺ added - DWCaHCO ₃ DW with calcium plus bicarbonate - DWHCO ₃ DW with 1 mM sodium bicarbonate added - FW freshwater (tap water) - FWK freshwater with K⁺ added - P. D. potential difference - SW seawater The experiments reported in this paper were done with Jean Maetz who tragically died in August 1977. It is the last report about several years of friendly collaboration     

Modification of single cardiac Na+ channels by DPI 201-106

Summary In inside-out patches from cultured neonatal rat heart cells, single Na⁺ channel currents were analyzed under the influence of the cardiotonic compound DPI 201-106 (DPI), a putative novel channel modifier. In absence of DPI, normal cardiac single Na⁺ channels studied at –30 mV have one open state which is rapidly left with a rate constant of 826.5 sec^–1 at 20°C during sustained depolarization., Reconstructed macroscopic currents relax completely with 7 to 10 msec. The current decay fits a single exponential. A considerable percentage of openings may occur during relaxation of the macroscopic current. In patches treated with 3×10^–6 m DPI in the pipette solution, stepping to –30 mV results in drastically prolonged and usually repetitive openings. This channel activity mostly persists over the whole depolarization (usually 160 msec in duration) but is abruptly terminated on clamping back the patch to the holding potential. Besides these modified events, apparently normal openings occur. The open time distribution of DPI-treated Na⁺ channels is the sum of two exponentials characterized by time constants of 0.85 msec (which is close to the time constant found in the control patches, 1.21 msec) and 12 msec. Moreover, DPI-modified Na⁺ channels exhibit a sustained high, time-independent open probability. Similar to normal Na⁺ channels, the mean number of open DPI-modified Na⁺ channels is voltage-dependent and increases on shifting the holding potential in the hyperpolarizing direction. These kinetic changes suggest an elimination of Na⁺ channel inactivation as it may follow from an interaction of DPI with Na⁺ channels.     

Voltage-dependent calcium and potassium conductances in striated muscle fibers from the scorpion,Centruroides sculpturatus

Ionic currents responsible for the action potential in scorpion muscle fibers were characterized using a three-intracellular microelectrode voltage clamp applied at the fiber ends (8–12°C). Large calcium currents (I _Ca) trigger contractile activation in physiological saline (5 mm Ca) but can be studied in the absence of contractile activation in a low Ca saline (2.5 mm). Barium (Ba) ions (1.5–3 mm) support inward current but not contractile activation.Ca conductance kinetics are fast (time constant of 3 msec at 0 mV) and very voltage dependent, with steady-state conductance increasing e-fold in approximately 4 mV. Half-activation occurs at –25 mV. Neither I _Ca nor I _Ba show rapid inactivation, but a slow, voltage-dependent inactivation eliminates I _Ca at voltages positive to –40 mV. Kinetically, scorpion channels are more similar to L-type Ca channels in vertebrate cardiac muscle than to those in skeletal muscle.Outward K currents turn on more slowly and with a longer delay than do Ca currents, and K conductance rises less steeply with voltage (e-fold change in 10 mV; half-maximal level at 0 mV). K channels are blocked by externally applied tetraethylammonium and 3,4 diaminopyridine.This work was supported by a grant from the NIH (NS-17510) to W.F.G. and a NRSA award to T.S. (GM-09921).