Depolarizing Voltage Clamp Pulses Research Articles

Inhibitory Effect of a Late Sodium Current Blocker, NCC-3902, on the Automaticity of the Guinea Pig Pulmonary Vein Myocardium.

The effect of blocking the persistent component of the sodium channel current (late INa) on the automatic activity of the isolated guinea pig pulmonary vein myocardium was examined. NCC-3902 blocked late INa, but did not affect other major ion channel currents stably expressed in cell lines. In isolated pulmonary vein cardiomyocytes, NCC-3902 blocked the late INa induced by a ramp depolarizing voltage clamp pulse similar to that of the pacemaker depolarization observed in the pulmonary vein myocardium. In isolated pulmonary vein tissue, NCC-3902 decreased the frequency of automatic firing of the myocardium through a reduction of the pacemaker depolarization slope. In isolated pulmonary vein cardiomyocytes, NCC-3902 significantly reduced the firing frequency of Ca2+ transients, but had no effect on Ca2+ sparks. NCC-3902 affected neither the spontaneous beating rate of the right atrium nor the contractile force of the ventricular myocardium. Selective blockers of late INa like NCC-3902, which inhibit the automatic activity of the pulmonary vein myocardium, appear to be promising as drugs for the pharmacological treatment of atrial fibrillation.

The Cole-Moore Effect: Still Unexplained?

In the first issue, on the first page of the Biophysical Journal in 1960, Cole and Moore provided the first confirmation of the Hodgkin and Huxley formulation of the sodium and potassium conductances that underlie the action potential. In addition, working with the squid giant axon, Cole and Moore noted that strong hyperpolarization preceding a depolarizing voltage-clamp pulse delayed the rise of the potassium conductance: once started, the time course of the rise was always the same but after significant hyperpolarization there was a long lag before the rise began. This phenomenon has come to be known as the Cole-Moore effect. Their article examines and disproves the hypothesis that the lag reflects the time required to refill the membrane with potassium ions after the ions are swept out of the membrane into the axoplasm by hyperpolarization. The work by Cole and Moore indirectly supports the idea of a membrane channel for potassium conductance. However, the mechanism of the Cole-Moore effect remains a mystery even now, buried in the structure of the potassium channel, which was completely unknown at the time.

Mechanism of Postinhibitory Rebound in Molluscan Neurons1

Postinhibitory rebound (PIR) is an intrinsic property of many neurons but the underlying mechanism is not well understood. We studied PIR and its relationship to spike adaptation in B-cells isolated from the buccal ganglia of Aplysia. These neurons exhibit PIR following inhibitory synaptic input and following direct membrane hyperpolarization. Hyperpolarizing and depolarizing voltage clamp pulses from the resting potential evoke slow changes in membrane current that persist in the form of tail currents following the pulses. A subtraction method was used to isolate slow tail currents for study. Current-voltage measurements indicate that slow outward tail currents following depolarizing pulses result from increases in membrane conductance, while inward tail currents following hyperpolarizations to −50 and −60 mV result from conductance decreases. The reversal potential of both outward and inward tail current is between −60 and −70 mV. Tail currents activated by pulses more positive than −60 mV are sensitive to the external K concentration and blocked by injection of Cs and TEA. When Ca2 influx is prevented by bathing cells in Ca2 free saline or by adding Co2 or Ni2 , the tail currents are reduced but a significant fraction of the current is insensitive to these treatments. More negative conditioning pulses activate a second component of inward tail current that is weakly sensitive to K but more strongly effected by substitution of N-methyl glucamine or Li for external Na . We conclude that both PIR and adaptation result from slow changes in a voltage dependent, non-inactivating K conductance that is active at voltages near the resting potential and is not tightly coupled to Ca2 influx. In addition, a second inward current is activated by large hyperpolarizing pulses that results from an increase in Na and K conductance. This second process is likely to contribute to PIR under particular circumstances.

Requirements for LTP induction by pairing in hippocampal CA1 pyramidal cells.

The induction of long-term potentiation (LTP) in the hippocampal CA1 region requires both presynaptic activity and large postsynaptic depolarization. A standard protocol for inducing LTP using whole-cell recording is to pair low-frequency synaptic stimulation (100-200 pulses, 1-2 Hz) with a depolarizing voltage-clamp pulse (1-3 min duration). In this standard protocol, a Cs(+)-based internal solution is used to improve the fidelity of the depolarization produced by voltage-clamp. In an attempt to induce LTP more rapidly, we tried to induce LTP by pairing high-frequency stimulation (200 pulses, 20-100 Hz) with a short depolarization ( approximately 15 s). Surprisingly, we found that this protocol failed to induce LTP, even though large LTP ( approximately 300% of baseline) could be induced by a subsequent standard protocol in the same cell. Pairing brief high-frequency stimulation at the beginning of a long depolarization (3 min) also did not induce LTP. However, the same high-frequency stimulation at the end of the long depolarization did induce LTP. When similar experiments were done with a K(+)-based internal solution, pairing high-frequency stimulation with a short depolarization did induce LTP. This indicates that the requirement for long depolarization is related to the use of Cs(+). We speculate that, when recording is made with Cs(+), a tetanus given at the beginning of depolarization initiates a process that inhibits N-methyl-D-aspartate (NMDA)-dependent LTP. This inhibitory process itself decays away during prolonged depolarization.

Depolarization-evoked increases in cytosolic calcium concentration in isolated smooth muscle cells of rat portal vein.

1. Ca2+ current through voltage-dependent Ca2+ channels (ICa) and intracellular free Ca2+ concentration ([Ca2+]i) were measured simultaneously in rat portal vein smooth muscle cells using conventional whole-cell voltage clamp technique and high temporal resolution microfluorimetry. 2. The relationship between depolarization-evoked ICa and rise in [Ca2+]i was examined. The extracellular Ca2+ concentration dependence and the voltage dependence of the depolarization-evoked increases in ICa and [Ca2+]i were similar. Both ICa and increased [Ca2+]i were blocked to a similar extent by nimodipine and cadmium and augmented by Bay K 8644. Furthermore, the time course of the measured increase in [Ca2+]i, closely followed the increase in [Ca2+]i expected from the time-integrated ICa. These observations suggest that the depolarization-evoked rise in [Ca2+]i was tightly coupled to ICa. 3. The cytosolic Ca2+ buffering capacity, determined as the ratio of the expected increase in [Ca2+]i (from ICa) divided by the measured increase in [Ca2+]i, was over 100. Therefore, less than 1 out of 100 Ca2+ ions entering the cell appears as a free Ca2+. 4. Ryanodine (30 microM), a blocker of the Ca(2+)-induced Ca2+ release mechanism, had little effect on buffering capacity measured over the first 200 ms of the depolarizing voltage clamp pulse. Ryanodine also had little effect on the buffering capacity during 800-1000 ms of the depolarizing voltage clamp pulse. Therefore, it was concluded that there is little Ca(2+)-induced Ca2+ release from the stores in rat portal vein smooth muscle cells during depolarization-evoked Ca2+ entry. 5. During brief depolarizations, the largest [Ca2+]i increase and ICa occurred at 0 mV. However, during steady-state depolarization, the largest increase in [Ca2+]i occurred around -30 mV, and we estimate the peak steady-state ICa to be about 0.6 pA.

Cation regulation of anion current activated by cell swelling in two types of human epithelial cancer cells.

1. In epithelial cells, hyposmotic stress induces visible cell swelling and large Cl- currents, which deactivate on return to isotonic solutions and are abolished by 0.1-0.5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). During depolarizing voltage clamp pulses, the currents activate rapidly and show time-dependent relaxation with associated tail currents on return to negative potentials. 2. We used whole-cell and outside-out patch recording to study volume activation of Cl- currents in the epithelial cancer cell lines H69AR and HeLa S5. In a 210 or 160 mosmol l-1 hyposmotic bathing solution containing 90 mM NaCl, 1 mM Ca2+ and 1 mM Mg2+, current relaxation was rapid, occurred positive to the Cl- reversal potential and reduced current to < 30% of its peak level at +100 mV. 3. Replacement of most bath inorganic cations by N-methyl-D-glucamine (NMDG) at constant Cl- concentration and osmolarity eliminated most of the current relaxation and caused an increase in steady-state current levels. Steady-state current was 85 +/- 6% of peak current at +100 mV in NMDG-Cl bath solution. This ratio fell to 55 +/- 2% (n = 5) when 1 mM Mg2+ was re-added to the bath. 4. Re-addition of Mg2+ or other Group II metals (Ca2+, Sr2+, Ba2+) induced immediate changes in current relaxation in a dose- and species-dependent manner. Concentrations of Mg2+ as low as 0.1 mM were effective in causing Cl- current relaxation. The IC50 for steady-state current block by external Mg2+ was 1.75 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitation of calcium-dependent potassium current

The activation of Ca-dependent K+ current, Ic, was studied in macropatches on the cell bodies of molluscan neurons. When a depolarizing voltage-clamp pulse was applied repeatedly, Ic facilitated in a manner that resembled the facilitation of synaptic transmitter release. Facilitation was characterized by an increase in Ic amplitude, a progressive increase in instantaneous outward current, and a decrease in utilization time. Experiments were done to investigate the mechanism responsible for Ic facilitation. Facilitation was reduced by microinjection of an exogenous Ca2+ buffer into the cytoplasm, indicating that facilitation is a Ca(2+)-dependent process. It was also reduced at elevated temperatures. Conversely, facilitation was greatly potentiated by blocking the Na/Ca exchange mechanism. It is concluded that the facilitation of Ca-dependent K+ current results from the accumulation of Ca2+ at the inner face of the membrane during the repeated activation of Ca2+ channels by depolarization. The Ca2+ indicator fluo-3 was used in fluorescence imaging experiments to measure changes in [Ca]i near the cell membrane during repeated depolarizing pulses and the interpretation of these results was aided by numerical simulations of Ca2+ accumulation, diffusion, and buffering in the peripheral cytoplasm. These experiments showed that the time course of Ic facilitation matches the time course of Ca2+ accumulation at the membrane. It was found that the strength of Ic facilitation varies among patches on the same neuron, suggesting that the accumulation of Ca2+ is not uniform along the inner surface of the membrane and that gradients in [Ca]i develop and are maintained during trains of depolarizing pulses. Potential mechanisms that may lead to local differences in Ca2+ accumulation and Ic facilitation are discussed.

Diacylglycerol-induced activation of protein kinase C attenuates Na+ currents by enhancing inactivation from the closed state.

The causes of attenuation of Na+ currents by diacylglycerol (DAG)-induced protein kinase C (PKC) activation in mouse neuroblastoma N1E-115 cells were investigated using the cell-attached patch, and the perforated-patch (nystatin based) whole-cell voltage-clamp techniques. Activation of PKC by DAG attenuated Na+ currents. Attenuation occurred in the absence of significant changes in the time-course of Na+ currents. However, the steady-state inactivation curve of these currents shifted to more negative voltages by approximately 20 mV. Here we demonstrate that the time-course of inactivation is accelerated by treatment with DAG-like substances in a voltage-dependent manner (time constant of inactivation decreased by 2- and 3.6-fold at -60, and -30 mV, respectively). In cell-attached patches, treatment with DAG compounds increased the percentage of current traces showing no single Na+ channel openings in response to depolarizing voltage-clamp pulses. Moreover, the average of current traces containing single Na+ channel openings was essentially the same in control conditions and after treatment with DAG compounds. Removal of Na+ channel inactivation by the alkaloid batrachotoxin prevented the attenuation of Na+ currents by PKC activation via DAGs. Taken together, these data strongly suggest that PKC-induced attenuation of Na+ currents is linked to an enhancement of Na+ channel inactivation. This attenuation is caused by an increase in the number of Na+ channels inactivating directly from the closed state(s). This inactivation pathway represents a simple and efficient physiological mechanism by which PKC activation might modulate the electrical activity of excitable cells.

Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes

Intracellular passive Ca2+, buffering was measured in voltage-clamped rat ventricular myocytes. Cells were loaded with indo-1 (K+ salt) to an estimated cytosolic concentration of 44 +/- 5 microM (Mean +/- SEM, n = 5), and accessible cell volume was estimated to be 24.5 +/- 3.6 pl. Ca2+ transport by the sarcoplasmic reticulum (SR) Ca-ATPase and sarcolemmal Na-Ca exchange was inhibited by treatment with thapsigargin and Na-free solutions, respectively. Extracellular [Ca2+] was maintained at 10 mM and, in some experiments, the mitochondrial uncoupler "1799" was used to assess the degree of mitochondrial Ca2+ uptake. To perform single cell titrations, intracellular Ca2+ ([Ca2+]i) was increased progressively by a train of depolarizing voltage clamp pulses from -40 to +10 mV. The total Ca2+ gain with each pulse was calculated by integration of the Ca current and then analyzed as a function of the rapid change in [Ca2+]i during the pulse. In the range of [Ca2+]i from 0.1 to 2 microM, overall cell buffering was well described as a single lumped Michaelis-Menten type species with an apparent dissociation constant, KD, of of 0.63 +/- 0.07 microM (n = 5) and a binding capacity, Bmax, of 162 +/- 15 mumol/l cell H2O. Correction for buffering attributable to cytosolic indo-1 gives intrinsic cytosolic Ca2+ buffering parameters of KD = 0.96 +/- 0.18 microM and Bmax = 123 +/- 18 mumol/l cell H2O. The fast Ca2+ buffering measured in this manner agrees reasonably with the characteristics of known rapid Ca buffers (e.g., troponin C, calmodulin, and SR Ca-ATPase), but is only about half of the total Ca2+ buffering measured at equilibrium. Inclusion of slow Ca buffers such as the Ca/Mg sites on troponin C and myosin can account for the differences between fast Ca2+ buffering in phase with the Ca current measured in the present experiments and equilibrium Ca2+ buffering. The present data indicate that a rapid rise of [Ca2+]i from 0.1 to 1 microM during a contraction requires approximately 50 microM Ca2+ to be added to the cytosol.

Na(+)-Ca2+ exchange current in latent pacemaker cells isolated from cat right atrium.

1. Single latent pacemaker cells were isolated from cat right atrium, and studied in a whole-cell configuration using a nystatin-perforated patch recording method. The nystatin method avoids alterations in intracellular Ca2+, cellular constituents and run-down of ionic currents. 2. Depolarizing voltage clamp pulses from -40 mV elicited L-type Ca2+ current (ICa) that exhibited an initial rapid phase of inactivation followed by a secondary slower inward current component that decayed over about 100 ms. The secondary inward component appeared as a slowly decaying inward tail current following short (10-40 ms) depolarizing clamp steps. 3. Slowly decaying inward currents were abolished by internally dialysing pacemaker cells with 2 mM EGTA using a ruptured patch recording method. Inward tail currents were also abolished by exposure to 1 microM ryanodine and significantly decreased by replacing 85% of external Na+ with lithium, without effect on peak ICa. These findings identify a Na(+)-Ca2+ exchange current (INa-Ca) that is mediated by sarcoplasmic reticulum (SR) Ca2+ release. 4. Properties of INa-Ca and ICa differed significantly: (i) ICa exhibited a bell-shaped voltage dependence that peaked at 0 mV and decreased at more positive voltages. INa-Ca was maximal at -10 mV and remained relatively constant at more positive voltages; (ii) a paired pulse protocol showed that the time course of INa-Ca recovery (5 s) was significantly longer than that of ICa (2 s); (iii) cadmium (50 microM) induced an inhibition of ICa that did not correlate in time with changes in INa-Ca. 5. The duration of depolarizing steps between 10 and 120 ms had no effect on the time course of INa-Ca tail currents. 6. Isoprenaline > or = 5 x 10(-8) M significantly increased peak ICa amplitude, peak INa-Ca amplitude, accelerated INa-Ca rate of decay and decreased the absolute time of INa-Ca decay. 7. Free-running pacemaker action potentials were clamped during diastole at either -40 or -70 mV (maximum diastolic potential) for variable periods of time. At times between 0.2 and 1 s, INa-Ca exhibited a voltage-dependent increase in amplitude over time, i.e. INa-Ca recovered more rapidly from -70 mV than from -40 mV. At times > 2 s, INa-Ca exhibited a voltage-dependent decline in amplitude over time, i.e. from -40 mV INa-Ca decreased by 10% of maximum whereas from -70 mV INa-Ca decreased by 60% of maximum.(ABSTRACT TRUNCATED AT 400 WORDS)

Dihydropyridine-sensitive skeletal muscle Ca channels in polarized planar bilayers. 3. Effects of phosphorylation by protein kinase C

The effects of protein kinase C (PKC) were studied on dihydropyridine (DHP)-sensitive Ca channels from rabbit skeletal muscle T tubule membranes. To determine which channel subunits become phosphorylated under the conditions used for electrophysiological studies, we first performed biochemical studies of phosphorylation. T tubular membranes were fused with vesicles of the lipid mixture used in the planar bilayers, and phosphorylation was assessed using the same concentrations of PKC, adenosine 5'-triphosphate, and buffers as were used in the electrophysiological experiments. The alpha 1 subunit of the DHP receptors was phosphorylated by PKC to an extent of 1 mol phosphate/mol protein. The beta subunit was also phosphorylated but to a significantly lesser extent. The DHP-sensitive Ca channel activity was studied after fusing T tubule membranes with planar bilayers (Ma, J., C. Mundiña-Weilenmann, M. M. Hosey, and E. Ríos. 1991. Biophys. J. 60:890-901). The bilayers were held at -80 mV and activated by depolarizing voltage clamp pulses. The observed Ca channels exhibited two open states (tau o1 = 5 ms and tau o2 = 25 ms). On addition of purified PKC to the intracellular side, the proportion of the longer open state increased threefold. The average open probability during a 2-s, maximally activating pulse (Pmax) increased from 10 to 15%. The voltage dependence of activation was not changed by PKC; the Boltzmann parameters were V1 = -20.5 mV and K = 10.5 mV, which were not significantly different from the reference channels. The deactivation (closing) time constant was increased from 7 to 12 ms after PKC. The inactivation time constant during the pulse was slightly increased(from 1.2 to 1.6 s), and the channel availability at the holding potential was decreased from 76 to 71%. Taken together, the results revealed that PKC increased Pmax largely through a shift in the voltage independent open-close equilibrium of the fully activated channels.This is in contrast with the effect of phosphorylation by PKA (Mundir'a-Weilenmann, C., J. Ma, E. Rios, and M. M. Hosey. 1991. Biophys.J. 60:902-909), which also increases Pmax but mostly by increasing the availability of channels and slowing inactivation during the pulse.

Regional variations in action potentials and transient outward current in myocytes isolated from rabbit left ventricle.

1. Regional variations in the shape of early repolarization of the action potential have been correlated to differences in transient outward K+ current, I(t), in myocytes isolated from the epicardial surface, the endocardial trabeculae and the papillary muscles of rabbit left ventricles. Temperature was 35 degrees C during whole-cell, and 22-23 degrees C during cell-attached experiments. 2. Membrane resting potentials were very similar regionally. At 0.1 Hz stimulation the action potential plateau amplitude in papillary muscle cells was significantly higher (104.7 mV) than in epicardial cells (96.47 mV). Exposure to 4-aminopyridine or increases in the rate of stimulation from 0.1 Hz to 3.3 Hz increased plateau height and diminished the initial notch on repolarization. These effects were correlated to the magnitude of I(t) in these cells. At low rates of stimulation I(t) caused a 'spike and dome' morphology of the action potential. 3. Voltage clamp experiments confirmed a higher current density of I(t) in epicardial cells (7.66 pA/pF at +20 mV) than in endocardial (6.45 pA/pF) or papillary muscle cells (3.69 pA/pF). I(t) at 35 degrees C was faster and larger than previously reported and individual currents inactivated almost completely during 100 ms pulses to plateau potentials. No differences in the kinetics or voltage dependence of whole-cell currents were found. Thus, the half-inactivation potential was -37.8 mV in cells from all three regions. 4. Cell-attached recordings from endocardial and epicardial cells showed very similar single-channel amplitudes, burst open probabilities and ensemble averages. The peak channel open probability soon after the start of depolarizing voltage clamp pulses did not change between cell types (P approximately 0.8). The slope conductance of I(t) channels was 13.0 pS with an intercept near the resting potential of the cell. 5. We conclude that regional variations in the shape of initial repolarization in cells from rabbit left ventricle are caused by variations in the magnitude of the transient outward K+ current, I(t). Epicardial cells have the largest, and papillary muscle cells the smallest I(t). The differences are not explained by alterations in the whole-cell kinetics or single-channel kinetics and conductance. The most likely explanation for variations in whole-cell current density is therefore a decrease in channel density in endocardium and papillary muscle compared with epicardial tissue. We estimate the density of I(t) channels per cell to be 1495 (one per 3-4 micron2) in epicardium, 1175 (one per 4-5 micron2) in endocardium, and 875 (one per 6 micron2) in papillary muscle cells.

Analysis of adenosine actions on Ca2+ currents and synaptic transmission in cultured rat hippocampal pyramidal neurones.

1. The role of adenosine receptors in reducing calcium currents (ICa) and in triggering presynaptic inhibition was studied using whole-cell patch-clamp techniques to record ICa and synaptic currents from the cell bodies of cultured rat hippocampal pyramidal neurones. Recordings of intracellular Ca2+ using the indicator dye Fura-2 were used to obtain further insights into the actions of adenosine agonists. 2. The adenosine analogue 2-chloroadenosine (2-CA) reduced ICa in these neurones. This action was also evident when Ba2+ was used as the charge carrier through Ca2+ channels. Adenosine also reduced the influx of Ca2+ into the cell body during a depolarizing voltage-clamp pulse as measured with Fura-2. The potency of various adenosine receptor agonists was as follows: cyclopentyladenosine greater than cyclohexyl-adenosine greater than or equal to R-phenylisopropyladenosine greater than 2-CA greater than S-phenylisopropyladenosine, consistent with the pharmacological profile of an A1 adenosine receptor. 3. The specific A1 receptor antagonist cyclopentyltheophylline (CPT) blocked the actions of 2-CA on ICa in a competitive fashion. 4. The actions of 2-CA on ICa were abolished by pre-incubation of cultured cells with pertussis toxin (PTX; 250 ng/ml). Intracellular dialysis with the GTP analogue GTP-gamma-S (guanosine-5'-O-(3-thiotriphosphate] enhanced the actions of 2-CA and rendered the response irreversible. 5. Excitatory postsynaptic currents (EPSCs) were recorded from pyramidal neurones under whole-cell voltage clamp by stimulating nearby neurones with an extracellular electrode. 2-CA potently and reversibly reduced the amplitude of EPSCs. This action was shown to be due to presynaptic inhibition of neurotransmitter release. 6. The order of potency of different adenosine agonists in reducing EPSCs was as follows: cyclopentyladenosine greater than cyclohexyladenosine greater than or equal to R-phenylisopropyladenosine greater than 2-CA greater than S-phenylisopropyladenosine. CPT inhibited the action of 2-CA in a competitive fashion. 7. The effects of 2-CA on synaptic transmission were abolished by pre-treatment with 250 ng/ml PTX, indicating that a PTX-sensitive G-protein is involved in this action. 8. These results indicate that activation of adenosine receptors does induce a reduction in ICa in hippocampal pyramidal neurones. Furthermore, this effect and the reduction of excitatory synaptic transmission by adenosine analogues are both mediated by PTX-sensitive G-proteins and have identical pharmacological properties.

Analysis of calcium channel properties in cultured leech retzius cells by internal perfusion, voltage-clamp and single-channel recording

ABSTRACT The properties of voltage-dependent calcium channels have been measured in Retzius cells isolated from the central nervous system of the leech and maintained in tissue culture. Macroscopic divalent cation currents were isolated after blocking Na+ and K+ currents by bathing the cells with Na+-free solutions containing TEA+ and 4-AP, and internally perfusing them with Cs+ and TEA+. Depolarizing voltageclamp pulses activated inward currents that were larger for Sr2+ than for Ba2+ or Ca2+. The peak currents were observed at +15 mV for Ca2+ and Ba2+ and at +7 mV for Sr2+. Divalent cation currents were blocked by Cd2+ and Mn2+ but not by dihydropyridine blockers. The activation kinetics of Ba2+ currents was sigmoid. The inactivation was approximately 10% at the end of a 50ms depolarizing pulse. Decay of Sr2+ and Ca2+ currents was larger and showed two kinetic phases. Activation and inactivation of the calcium channels were not significantly influenced by the holding potential. Deactivation kinetics observed during tail currents consisted of two exponential components. At a closing voltage of −60 mV, the time constant was ≈200 ps for the fast component and 1.9 ms for the slow component. Both time constants of deactivation were voltage-dependent over the range from −80 to −20 mV, and increased at more depolarized closing voltages. Single-channel activity was recorded with cell-attached patches in solution containing 75 mmol l−1 Ba2+. Taken together, the results define the characteristics of a distinctive type of calcium channel in isolated Retzius cells.

Axonal contribution to subthreshold currents inaplysia bursting pacemaker neurons

The contribution of axonal activity to the ionic currents which generate bursting pacemaker activity was studied by using the two-electrode voltage-clamp technique in Aplysia bursting neuron somata in conjunction with intraaxonal voltage recordings. Depolarizing voltage-clamp pulses applied to bursting cell somata triggered axonal action potentials. The voltage-clamp current recording exhibited transient inward current "notches" corresponding to each of the axonal spikes. The addition of 50 microM tetrodotoxin (TTX) to the bathing medium blocked the fast axonal spikes and current notches, revealing a slower axonal spike which was blocked by the replacement of external Ca2+ with Co2+. The inward current evoked by applying a depolarizing voltage-clamp pulse in the soma is distorted by the occurrence of the axonal Ca2+ spike. Elimination of the axonal spike, by injecting hyperpolarizing current into the axon, changes both the time course and the magnitude of the inward current. The axonal Ca2+ spikes are followed by a series of Ca2+-dependent afterpotentials: a rapid postspike hyperpolarization, a depolarizing afterpotential (DAP) and, finally, a long-lasting postburst hyperpolarization. The long-lasting hyperpolarization is not blocked by 50 mM external tetraethyl ammonium, an effective blocker of Ca2+-activated K+ current [IK(Ca)], and does not appear to reverse at EK. Hence, the axonal long-lasting hyperpolarization may not be due to IK(Ca). Somatic voltage-clamp pulses in bursting neurons are followed by a slow inward tail current, which is sometimes coincident with a DAP in the axon. In some cells, the amplitude of the slow inward tail current is greatly reduced if axonal spikes and DAPs are prevented by hyperpolarization of the axon, while, in other cells, elimination of axonal activity has little effect. Therefore, the slow inward tail current is not necessarily an artifact of poor voltage-clamp control over the axonal membrane potential but probably results from the activation of an ionic conductance mechanism located partly in the axon and partly in the soma.

Kinetics of veratridine action on Na channels of skeletal muscle.

Veratridine bath-applied to frog muscle makes inactivation of INa incomplete during a depolarizing voltage-clamp pulse and leads to a persistent veratridine-induced Na tail current. During repetitive depolarizations, the size of successive tail currents grows to a plateau and then gradually decreases. When pulsing is stopped, the tail current declines to zero with a time constant of approximately 3 s. Higher rates of stimulation result in a faster build-up of the tail current and a larger maximum value. I propose that veratridine binds only to open channels and, when bound, prevents normal fast inactivation and rapid shutting of the channel on return to rest. Veratridine-modified channels are also subject to a "slow" inactivation during long depolarizations or extended pulse trains. At rest, veratridine unbinds with a time constant of approximately 3 s. Three tests confirm these hypotheses: (a) the time course of the development of veratridine-induced tail currents parallels a running time integral of gNa during the pulse; (b) inactivating prepulses reduce the ability to evoke tails, and the voltage dependence of this reduction parallels the voltage dependence of h infinity; (c) chloramine-T, N-bromoacetamide, and scorpion toxin, agents that decrease inactivation in Na channels, each greatly enhance the tail currents and alter the time course of the appearance of the tails as predicted by the hypothesis. Veratridine-modified channels shut during hyperpolarizations from -90 mV and reopen on repolarization to -90 mV, a process that resembles normal activation gating. Veratridine appears to bind more rapidly during larger depolarizations.

Sodium currents in the giant axon of the crab Carcinus maenas.

Measurements were made of the kinetics and steady-state properties of the sodium conductance changes in the giant axon of the crab Carcinus maenas. The conductance measurements were made in the presence of small concentrations of tetrodotoxin and as much electrical compensation as possible in order to minimize errors caused by the series resistance. After an initial delay of 10-150 microsec, the conductance increase during depolarizing voltage clamp pulses followed the Hodgkin-Huxley kinetics. Values of the time constant for the activation of the sodium conductance lay on a bell-shaped curve with a maximum under 180 microsec at -40 mV (at 18 degrees C). Values of the time constant for the inactivation of the sodium conductance were also fitted using a bell-shaped curve with a maximum under 7 msec at -70 mV. The effects of membrane potential on the fraction of Na channels available for activation studied using double pulse protocols suggest that hyperpolarizing potentials more negative than -100 mV lock a fraction of the Na channels in a closed conformation.

Functional significance of voltage-dependent conductances in Limulus ventral photoreceptors.

The influence of voltage-dependent conductances on the receptor potential of Limulus ventral photoreceptors was investigated. During prolonged, bright illumination, the receptor potential consists of an initial transient phase followed by a smaller plateau phase. Generally, a spike appears on the rising edge of the transient phase, and often a dip occurs between the transient and plateau. Block of the rapidly inactivating outward current, iA, by 4-aminopyridine eliminates the dip under some conditions. Block of maintained outward current by internal tetraethylammonium increases the height of the plateau phase, but does not eliminate the dip. Block of the voltage-dependent Na+ and Ca2+ current by external Ni2+ eliminates the spike. The voltage-dependent Ca2+ conductance also influences the sensitivity of the photoreceptor to light as indicated by the following evidence: depolarizing voltage-clamp pulses reduce sensitivity to light. This reduction is blocked by removal of external Ca2+ or by block of inward Ca2+ current with Ni2+. The reduction of sensitivity depends on the amplitude of the pulse, reaching a maximum at or approximately +15 mV. The voltage dependence is consistent with the hypothesis that the desensitization results from passive Ca2+ entry through a voltage-dependent conductance.

Simultaneous changes in the equilibrium potential and potassium conductance in voltage clamped Ranvier node in the frog.

1. In voltage clamped myelinated nerve fibres, the K+ conductance has been calculated from current recordings obtained in low and high K+ media, taking into account the changes in EK resulting from accumulation of depletion of K+ ions near to nodal membrane. 2. At the end of a depolarization, the instantaneous K+ current reverses at a potential (instantaneous reversal potential) differing from the Nernst potential calculated using the external and internal bulk concentrations (theoretical Nernst potential). During a depolarization, EK, as estimated from the instantaneous reversal potential, changes continuously. This change depends on the size, the duration and the direction of the time dependent K+ current. The variation of EK is attributed to continuous changes in K+ concentration near the membrane during voltage pulses which turn on the K+ conductance. 3. The chord conductance [GK = IK/(E-EK), as calculated using the instantaneous reversal potential values for EK, has been analysed as a function of time and membrane potential. As previously reported it increases with the initial K+ concentration in the external medium. 4. The time course of the K+ current depends on both the kinetics of the conductance increase and the rate of change in the driving force for K. The kinetics of the conductance increase can satisfactorily be described by a single exponential function following a delay after the onset of the depolarizing voltage clamp pulse. 5. This delay increases when the holding potential is made more negative. It decreases with membrane depolarization and it is independent of the external K+ concentration. At a given membrane potential, the turning on of the K+ conductance is found to be faster at high than at low external K+ concentrations. 6. At repolarization the turning off of the conductance cannot be described by a single exponential function. It is faster at low than at high external K+ concentrations. 7. The results suggest that the change in K+ conductance proceeds in a multi-step transition or (and) that the K+ conductance is determined by several types of K+ channels.

Membrane currents following activity in canine cardiac Purkinje fibers

Recent experiments in canine Pukinje fibers (Gadsby and Cranefield, 1979) have shown that following a period of sodium loading in K+-free solution a slowly decaying outward current is observed. This current has been attributed to the activity of the electrogenic Na+-K+ exchange pump. In the present paper we show that similar slowly decaying outward currents are observed following prolonged periods of overdrive with action potentials or with brief depolarizing voltage clamp pulses. The dependent of the prolonged outward current on the duration and frequency of the preceding period of overdrive and on the potential following overdrive is reported. We also present results which indicate that a large portion of this current can be induced by phasic Na+ loading through the fast-inward channel.