Voltage-sensitive calcium flux promoted by vesicles in an isolated cardiac sarcolemma preparation

Summary The effect of membrane potential on the vesicular uptake of calcium in an isolated cardiac sarcolemma preparation from canine ventricle was evaluated. Membrane potentials were developed by the establishment of potassium gradients across the vesicular membranes. In the presence of valinomycin, the fluorescence changes of the voltage sensitive dye, diS-C₃-(5) were consistent with the development of potassium equilibrium potentials. Using EGTA to remove endogenous calcium from the preparation and to maintain a low intravesicular calcium concentration over time, the uptake of calcium was linear from 5 to 100 sec, in the absence of sodium, at both –98 and –1 mV. The rate of calcium uptake (calcium influx) was approximately twofold greater at –1 mV than at –98 mV, and prepolarization of the membrane potential to –98 mV did not enhance calcium influx upon subsequent depolarization to –1 mV. Hence, calcium influx was voltage-sensitive but not depolarization-induced and did not inactivate with time. Furthermore, the calcium influx was not inhibited by the organic calcium antagonists, which suggests that this flux did not occur via the transient calcium channel. Evaluation of calcium influx over a wide range of membrane potentials produced a profile consistent with the hypothesis that calcium entered the vesicles through the pathway responsible for the persistent inward current observed in voltage-clamped isolated myocytes. A model was proposed to account for these results.     

Two calcium currents in the somatic membrane of mollusc neurons

Two new types of calcium channels were discovered during research in ionic currents in the somatic membrane ofHelix pomatia neurons, using an intracellular perfusion technique. Apart from the principal calcium current described in the literature with a holding potential of about –110 mV, an additional calcium current was observed activated at depolarizations of –40 to –80 mV and was not reduced when the cell was perfused with solutions containing fluoride anions. The kinetics of this current were well described in the context of the Hodgkin and Huxley model with a time constant of activation of 6–8 msec and of inactivation of 300–600 msec. It increased in amplitude as the Ca⁺⁺ rose in the cellular environment but was reduced by extracellular addition of the Ca⁺⁺ antagonists Co⁺⁺, Ni⁺⁺, and Cd⁺⁺, and the organic blockers nifedipine and verapamil. The association constants of these substances with corresponding channels determined from the maximum of the current-voltage relationship were 2 (Ca⁺⁺), 3 (Co⁺⁺), 0.06 (nifedipine), and 0.2 mM (verapamil). The properties detected in this component of calcium conductance are compared with those of calcium channels in other excitatory formations and its possible functional role is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 627–633, September–October, 1985.     

Voltage dependence of calcium current deactivation in the somatic membrane of mouse sensory neurons

Voltage dependence of the deactivation kinetics of calcium inward currents was investigated in the somatic membrane of murine spinal ganglia neurons. It was found that deactivation of high threshold calcium current has a slower component (=0.80–0.85 msec at a repolarizing potential of –80 mV) as well as the principal transient exponential component (130 sec at the same potential repolarizing level). A dissimilar relationship exists between amplitudes of the transient and slower exponential components, describing deactivation of high threshold calcium current and degree of activation of the depolarizing shift in membrane potential; the former dependence is expressed by a sigmoid and the latter by a V-shaped curve. The slower component of deactivation of high threshold current was inhibited substantially by perfusing the cell with a Tris-PO₄-containing solution. Low-threshold calcium tail current undergoes slower deactivation (=1.1–1.2 msec) at a repolarizing potential of –160 mV. A relationship between the time constant of low threshold current deactivation and the type of penetrating cation used was observed. A kinetic model of calcium current deactivation is suggested, taking account of the three different types of calcium channels, (one low and two high threshold) present in the somatic membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 185–193, March–April, 1988.     

Electrophysiological and Theoretical Analysis of Depolarization-Dependent Outward Currents in the Dendritic Membrane of an Identified Nonspiking Interneuron in Crayfish

Depolarization-dependent outward currents were analyzed using the single-electrode voltage clamp technique in the dendritic membrane of an identified nonspiking interneuron (LDS interneuron) in situ in the terminal abdominal ganglion of crayfish. When the membrane was depolarized by more than 20 mV from the resting potential (65.0 ± 5.7 mV), a transient outward current was observed to be followed by a sustained outward current. Pharmacological experiments revealed that these outward currents were composed of 3 distinct components. A sustained component (I _s) was activated slowly (half rise time > 5 msec) and blocked by 20 mM TEA. A transient component (I _t1) that was activated and inactivated very rapidly (peak time < 2.5 msec, half decay time < 1.2 msec) was also blocked by 20 mM TEA. Another transient component (I _t2) was blocked by 100 M 4AP, activated rapidly (peak time < 10.0 msec) and inactivated slowly (half decay time > 131.8 msec). Two-step pulse experiments have revealed that both sustained and transient components are not inactivated at the resting potential: the half-maximal inactivation was attained at –21.0 mV in I _t1, and –38.0 mV in I _t2. I _s showed no noticeable inactivation. When the membrane was initially held at the resting potential level and clamped to varying potential levels, the half-maximal activation was attained at –36.0 mV in I _s, –31.0 mV in I _t1 and –40.0 mV in I _t2. The activation and inactivation time constants were both voltage dependent. A mathematical model of the LDS interneuron was constructed based on the present electrophysiological records to simulate the dynamic interaction of outward currents during membrane depolarization. The results suggest that those membrane conductances found in this study underlie the outward rectification of the interneuron membrane as well as depolarization-dependent shaping of the excitatory synaptic potential observed in current-clamp experiments.     

Contribution of a H+ pump in determining the resting potential of neuroblastoma cells

The aim of this work was to examine the effects of changes in external K⁺ concentration (K _o ) around its physiological value, of various K⁺ channels blockers, including internal Cs⁺, of vacuolar H⁺-ATPase inhibitors and of the protonophore CCCP on the resting potential and the voltage-dependent K⁺ current of differentiated neuroblastoma x glioma hybrid NG108-15 cells using the whole-cell patch-clamp technique. The results are as follows: (i) under standard conditions (K _o =5 mm) the membrane potential was –60±1 mV. It was unchanged when K _o was decreased to 1 mm and was depolarized by 4±1 mV when K_o was increased to 10 mm. (ii) Internal Cs⁺ depolarized the membrane by 21±3 mV. (iii) The internal application of the vacuolar H⁺-ATPase inhibitors N-ethylmaleimide (NEM), NO ₃ ^– and bafilomycin A1 (BFA) depolarized the membrane by 15±2, 18±2 and 16±2 mV, respectively, (iv) When NEM or BFA were added to the internal medium containing Cs⁺, the membrane was depolarized by 45±1 and 42±2 mV, respectively. (v) The external application of CCCP induced a transient depolarization followed by a prolonged hyperpolarization. This hyperpolarization was absent in BFA-treated cells. The voltage-dependent K⁺ current was increased at negative voltages and decreased at positive voltages by NEM, BFA and CCCP. Taken together, these results suggest that under physiological conditions, the resting potential of NG108-15 neuroblastoma cells is maintained at negative values by both voltage-dependent K⁺ channels and an electrogenic vacuolar type H⁺-ATPase.This work was supported by a grant from INSERM (CRE 91 0906).     

Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells

Summary Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidlyinactivating component had a time constant of 75±5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inanctivating component had a time constant of 2750±280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between –100 and –40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 m) when a holding potential of –100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1–10 m) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.     

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.     

Oxidation-reduction midpoint potentials of mitochondrial flavoproteins and their intramitochondrial localization

Spectrophotometric and fluorimetric substrate couple titrations and potentiometric spectrophotometric titrations were used to determine the oxidation-reduction potentials of components showing absorbance or fluorescence at the wavelengths attributable to the flavoproteins of mitochondria fractionated using digitonin together with sonication. A pure mitoplast fraction devoid of cytochrome b₅ contamination could be obtained using 230 µg digitonin/mg of mitochondrial protein. The digitonin-soluble fraction contained a species havingE _m 7 .4=–123 mV and probably represents the outer membrane flavoproteins. The inner membrane-matrix fraction, treated with ultrasound, provided evidence of a flavoprotein species with low redox potential (E _m 7 .4=–302 mV) in the matrix fraction. The –302 mV component is probably lipoamide dehydrogenase. A high redox potential species withE _m 7 .4=+19 mV in titrations with the succinate fumarate couple was located in the inner membrane vesicles and is probably identical with succinate dehydrogenase. The electron-transferring flavoprotein (ETF) was isolated from bovine heart mitochondria and itsE _m 7 .4=–74 mV determined. The component in the matrix fraction with an apparentE _m 7 .4=–56 mV probably represents ETF, and that in the inner membrane fraction with an apparentE _m 7 .4=–43 mV the NADH dehydrogenase flavoprotein. A component in an apparently low concentration withE _m 7 .4=+30 mV was detected in the inner membrane fraction. This probably represents the ETF-dehydrogenase flavoprotein. The origin of the flavoprotein fluorescence of mitochondria and intact tissues is discussed.     

Ionic mechanisms of the transmembrane current evoked by injection of cyclic amp into identified Helix pomatia neurons

Ionic mechanisms of the transmembrane current evoked by injection of cyclic AMP into identified neurons ofHelix pomatia were investigated by the voltage clamp method. Injection of cyclic AMP into neurons RPa3, LPa2, LPa3, and LPl1 was shown to cause the development of a two-component transmembrane (cyclic AMP) current. The current-voltage characteristic curve of the early component is linear in the region from –40 to –90 mV; the reversal potential of the early component, determined by extrapolation, lies between –5 and +20 mV; the current-voltage characteristic curve of the late component also is linear and has a reversal potential between –55 and –60 mV. A decrease in the sodium concentration in the external medium from 100 to 25 mM led to a decrease in amplitude of the cyclic AMP current and to a shift of the reversal potential for the early component by 30–32 mV toward hyperpolarization. It is suggested that the early component of the cyclic AMP current in neurons RPa3, LPa2, LPa3, and LPl1 is associated with an increase in permeability of the neuron membrane chiefly for sodium ions, whereas the late component is correspondingly connected with permeability for potassium ions. Injection of cyclic AMP also caused the appearance of a transmembrane inward current in neuron LPa8, but it was independent of the holding potential and was unaccompanied by any change in membrane permeability. It is suggested that this current may be due to a change in the activity of the electrogenic ion pump.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 12, No. 5, pp. 526–532, September–October, 1980.     

Effects of LTC4 and BW 755C,a blocker of its synthesis,on contraction in smooth muscle and operation of voltage-dependent calcium channels in nerve cells

The effects were investigated of LTC₄, a synthetic leukotriene, and BW 755C, a blocker of LTC₄ biosynthesis, on the operation of Ca channels at the cell membrane and on contraction of muscle fibers using intracellular dialysis and voltage clamping at the membrane of isolated nerve cells and by recording spontaneous contraction of the uterus in white rats at advanced stages of pregnancy. It was found that 1·10^–7 M LTC₄ stimulates the contraction of the uterus without altering its response to oxytocin application. The same concentration of LTC₄ was found to increase calcium conductance by 60±27%. At the same time, a 25±6 mV shift in peak current-voltage relationship along the voltage axis toward negative values was recorded for calcium current. BW 755C, a blocker of the key enzyme in the lipoxygenase metabolic pathway of arachidonic acid, exerts an action similar to leukotriene on calcium conductance, although brief contraction of the uterus is rapidly replaced by complete inhibition of this activity.Institute of Bioorganic Chemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 24–31, January–February, 1989.     

Action of neurotoxin from the sea anemoneHomostichanthus duerdemi on inward sodium current in mammalian neurons

The action of purified toxin from the sea anemoneHomostichanthus duerdemi (HTX-1) on the inward sodium current was studied in experiments on isolated neurons from rat spinal ganglia and neuroblastoma cells of clone N-18F1, by an intracellular perfusion and voltage clamp method. HTX-1 was found to delay inactivation of the tetrodotoxin-(TTX-)sensitive inward sodium current and to make it incomplete, but virtually without affecting its activation. The relationship between the fraction of sodium channels modified by the toxin and the HTX-1 concentration is described by a Langmuir isotherm with association constant of (1.1 ± 0.1)·10^–7 M (holding potential –100 mV). Under the influence of the toxin the peak inward sodium current was increased by about 80%. Binding of HTX-1 with TTX-sensitive sodium channels is distinguished by strong potential-dependence: at a holding membrane potential of 0 mV the binding constant was an order of magnitude less than at a potential of –100 mV. In the case of brief action of HTX-1 on the nerve cell membrane (under 5 min) the effect of the toxin was completely reversible, but if the time of action of HTX-1 exceeded 30 min, subsequent washing with normal solution for 90 min did not abolish the effect completely.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Pacific Institute of Bioorganic Chemistry, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 14, No. 4, pp. 402–409, July–August, 1982.     

Glass microelectrode measurements of sieve tube membrane potentials in internode discs and petiole strips of tomato (Solanum lycopersicum L.)

Summary In tissue slices of tomato (Solanum lycopersicum L.) sieve tube membrane potentials (E_m) were measured by use of glass microelectrodes. In internode discs, the potential differences (pd) of phloem cells near the cut surface fell into two distinct categories with average values of –66 and –109 mV. More distant from the cut surface the values decreased to averages of –71 and –140 mV. These pds were associated with phloem parenchyma cells and sieve tube/companion cell complexes, respectively. In petiole strips, pds were recorded from cells which were identified by iontophoretic injection of fluorescent dye. Averages in two different bathing media, were –140/–146mV, –149/–152mV, and –70/–68mV for sieve tubes, companion cells, and phloem parenchyma cells, respectively. The membrane potentials recorded from sieve tubes were transiently reduced upon sucrose addition. Reduction by CCCP and KCN was more permanent. Sieve tube E_ms recovered more slowly from potassium than from sucrose-induced depolarizations. Light/ dark (L/D) responses were minute (±3 mV). The limitations of the present experimentation are evaluated with special reference to the question as whether the recorded E_ms represent sieve tube membrane potentials occurring in the intact plant.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - D dark(ness) - E_m membrane potential - L light - LYCH Lucifer yellow CH - pd potential difference - SE standard error     

Inactivation of calcium currents in the molluscan neuron somatic membrane

The time course of weakening of inward calcium currents (inactivation) during prolonged (of the order of 1 sec) depolarizing shifts of membrane potential was studied in isolated dialyzed neurons of snailHelix pomatia. This decay of the current recorded in this way can be approximated by two exponential functions with time constants of 20–70 and 250–350 msec, respectively. With an increase in pH of the intracellular solution to 8.5 the fast component of the decay disappeared completely; the kinetics of the slow component in this case was very slightly retarded. It is concluded that the fast component of decay of the recorded current does not reflect a change in the calcium current but is due to parallel activation of the nonspecific outward current; the slow component, however, is true in activation of the calcium current. The rate of inactivation of this current was shown to be determined by its maximal value and not by the level of the depolarizing potential shift and it depends on the conditions of accumulation of calcium ions near the inner surface of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 525–531, September–October, 1982.     

Blocking of Ca-dependent outward current in molluscan neuron somatic membrane by raised intracellular pH

The action of a raised intracellular pH (pH_i) on transmembrane ionic currents was investigated on isolated unidentified neurons ofHelix pomatia under intracellular dialysis and membrane voltage clamping conditions. With a rise in pH_i from 7.3 to 9.0 and in the simultaneous presence of an inward calcium current, the outward potassium current was considerably reduced and the current-voltage characteristic curve was shifted toward more positive membrane potential values. The inward calcium current was practically unchanged in this case. If, however, the calcium current was inhibited by the action of cadmium ions, no decrease in the outward current was observed, only a shift of the I_K(V) curve toward more positive values of membrane potential. It is suggested that an increase in pH_i selectively blocks the Ca-dependent component of the outward potassium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 4, pp. 426–430, July–August, 1982.     

Cyclic GMP regulation of a voltage-activated K channel in dissociated Enterocytes

Summary Enterrocytes from the intestinal epithelium of the winter flounder were isolated by collagenase digestion and incubated in flounder Ringer solution. Conventional whole-cell and amphotericin-perforated whole-cell recording techniques were used to characterize the properties of a voltage-activated K current present in dissociated cells. Resting membrane potentials and series resistances were significantly lower (from –23 to – 39 mV and 29 to 13 M, respectively) when amphotericin was used to achieve the whole-cell configuration. When cells were placed in flounder Ringer solution, held at –80 mV and subsequently stepped to a series of depolarizing voltages (from–70 to 0 mV), an outward current was observed that exhibited inactivation at voltages above –20 mV. This current was sensitive to holding potential and was not activated when the cells were held at –40 mV or above. When cells were bathed in symmetric K Ringer solution and the same voltage protocol was applied to the cell, inward currents were observed in response to the negative intracellular potentials. Reversal potentials at two different extracellular K concentrations were consistent with K as the currentcarrying ion. BaCl₂ (2 mM) and CsCl (0.5 mM) both produced voltage-dependent blockade of the current when added to the bathing solution. Charybdotoxin (300 nM extracellular concentration) completely blocked the current. The IC₅₀ for charybdotoxin was 50 nM. Cyclic. GMP inhibited the voltage-activated current in flounder Ringer and in symmetric K Ringer solution. The cyclic GMP analog, 8-Br cGMP, lowered the threshold for voltage activation and potentiated inactivation of the current at voltages above–40 mV. Previous studies with intact flounder epithelium showed that K recycling and net K secretion were inhibited by Ba²⁺ and by cGMP. We suggest that the channel responsible for the whole-cell current described in this study may be important in K recycling and secretion.     

Model experiments on squid axons and NG108-15 mouse neuroblastoma × rat glioma hybrid cells

Three types of ionic current essentially determine the firing pattern of nerve cells: the persistent Na⁺ current, the M current and the low-voltage-activated Ca²⁺ current. The present article summarizes recent experiments concerned with the basic properties of these currents. Keynes and Meves (Proc R Soc Lond B (1993) 253, 61–68) studied the persistent or steady-state Na⁺ current on dialysed squid axons and measured the probability of channel opening both for the peak and the steady-state Na⁺ current (PF_peak and PF_ss) as a function of voltage. Whereas PF_peak starts to rise at −50 mV and reaches a maximum at +40 to +50 mV, PF_ss only begins to rise appreciably at around 0 mV and is still increasing at +100 mV. This differs from observations on vertebrate excitable tissues where the persistent Na⁺ current turns on in the threshold region and saturates at around 0 mV. Schmitt and Meves (Pflügers Arch (1993) 425, 134–139) recorded M current, a non-inactivating K⁺ current, from NG108-15 neuroblastoma × glioma hybrid cells, voltage-clamped in the whole-cell mode, and studied the effects of phorbol 12,13-dibutyrate (PDB), an activator of protein kinase C (PKC), and arachidonic acid (AA). PDB and AA both decreased I_M, the effective concentrations being 0.1–1 μM and 5–25 μM, respectively; while the PDB effect was regularly observed, the M current depression by AA was highly variable from cell to cell. The PKC 19–31 peptide, an effective inhibitor of PKC, in a concentration of 1 μM almost totally prevented the effects of PDB and AA on M current, suggesting that both are mediated by PKC. Schmitt and Meves (Pflügers Arch (1994a) 426, Suppl R 59) measured low-voltage-activated (l-v-a) and high-voltage-activated (h-v-a) Ca²⁺ currents on NG108-15 cells and investigated the effect of AA and PDB on both types of current. At pulse potentials > −20 mV, AA (25–100 μM) decreased l-v-a and h-v-a I_Ca. The decrease was accompanied by a small negative shift and a slight flattening of the activation and inactivation curves of the l-v-a I_Ca. The AA effect was not prevented by 50 μM eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of AA metabolism, or PKC 19–31 peptide and not mimicked by 0.1–1 μM PDB. Probably, AA acts directly on the channel protein or its lipid environment. The physiological relevance of these three sets of observations is briefly discussed.     

Blocking action of calmidazolium and chlorpromazine on outward potassium current dependent on extra-cellular calcium ions

The effects of the calmodulin antagonists, calmidazolium (R 24571) and chlorpromazine on delayed outward potassium current at the somatic membrane were investigated in non-identified intracellularly perfused neurons isolated fromHelix pomatia. Voltage was clamped at the membrane. Extracellular application of these substances produced effective depression of the outward current. This effect even occurred at test substance concentrations of 10^–9–10^–8 M. Block-ade of delayed outward current was produced mainly as a result of suppressing the potassium current component dependent on intracellular potassium ions (I_k(Ca/in)). The possibility that the receptor for intracellular calcium responsible for modulating this current may be of a calmodulin-like nature is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 356–361, May–June, 1987.     

Single calcium channels in neurons of rat spinal ganglia

Using a refined patch clamp technique, a study was made of single calcium channels of spinal ganglia neurons on a cell-attached membrane site in newborn rats; these convey the basic (high threshold) component of calcium current. Findings show that currents carried by calcium ions at a concentration of 60 mM in the recording pipet changes from 0.58±0.05 to 0.43±0.05 pA with a change in potential of 20 mV. This corresponds to a channel conductance of 7±0.5 pS. The distribution of open time was monoexponential with a time constant of about 0.75 msec, independent of membrane potential. Distribution of closed time approached a biexponential time course. The fast component (0.8 msec) was voltage-dependent, while the slow component decreased from 22 to 4 msec when depolarization increased by 20 mV. Using experimentally obtained time parameters which describe single calcium channel function, and assuming a three-tier model of the channel, the numerical values of the constants of transition rates between individual states were determined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 673–682, September–October, 1985.     

Action of ω-conotoxin on calcium currents of rat pituitary GH3 cells

The effect of -conotoxin (-CgTX) on calcium currents of rat pituitary GH3 cells was studied by the voltage clamp method on a whole cell, under tight junction conditions. Two different components of the inward calcium current were observed in a solution containing 15 mM Ca²⁺. The first was activated with a holding potential of –80 mV and by testing pulses more positive than –55 mV. A shift of holding potential to –40 mV led to steady-state inactivation of this low-threshold component of the current. -CgTX at the initial moment after its application had an activating action on both components of the calcium current: low-threshold and high-threshold, but the increase in the first was much greater. In the present experiments the currents increased as early as 30 sec after replacement of the external solution; later the drop of current took place with temporal parameters characteristic of the spontaneous current drop in the control solution during cell dialysis. Incubation of the cells in growth medium containing 5 µM -CgTX for 2 h led to an increase in the density of both types of calcium currents in the GH3 cells, which was reduced after incubation for 2 h in the same medium. Thus -CgTX was found to have an activating action on calcium currents of GH3 cells at the initial moment after application of the toxin. The absence of a marked blocking action of -CgTX on the calcium currents of the test cells confirms the high tissue specificity of action of the toxin as a blocker of high-threshold calcium channels in the nerve cell membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 2, pp. 199–205, March–April, 1991.     

Whole-cell and single-channel currents across the plasmalemma of corn shoot suspension cells

Summary Whole-cell sealed-on pipettes have been used to measure electrical properties of the plasmalemma surrounding protoplasts isolated from Black Mexican sweet corn shoot cells from suspension culture. In these protoplasts the membrane resting potential (V _m ) was found to be –59±23 mV (n=23) in 1mm K _o ^– . The meanV _m became more negative as [K^–] _o decreased, but was more positive than the K⁺ equilibrium potential. There was no evidence of electrogenic pump activity. We describe four features of the current-voltage characteristic of the plasmalemma of these protoplasts which show voltagegated channel activity. Depolarization of the whole-cell membrane from the resting potential activates time- and voltage-dependent outward current through K⁺-selective channels. A local minimum in the outward current-voltage curve nearV _m =150 mV suggests that these currents are mediated by two populations of K⁺-selective channels. The absence of this minimum in the presence of verapamil suggests that the activation of one channel population depends on the influx of Ca²⁺ into the cytoplasm. We identify unitary currents from two K⁺-selective channel populations (40 and 125 pS) which open when the membrane is depolarized; it is possible that these mediate the outward whole-cell current. Hyperpolarization of the membrane from the resting potential produces time- and voltage-dependent inward whole-cell current. Current activation is fast and follows an exponential time course. The current saturates and in some cases decreases at membrane potentials more negative than –175 mV. This current is conducted by poorly selective K⁺ channels, whereP _Cl/P _K=0.43±0.15. We describe a low conductance (20 pS) channel population of unknown selectivity which opens when the membrane is hyperpolarized. It is possible that these channels mediate inward whole-cell current. When the membrane is hyperpolarized to potentials more negative than –250 mV large, irregular inward current is activated. A third type of inward whole-cell current is briefly described. This activates slowly and with a U-shaped current-voltage curve over the range of membrane potentials –90<V _m <0 mV.