Voltage-clamp Steps Research Articles

Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, I: experimental studies.

Pacing-induced heart failure in the dog recapitulates many of the electrophysiological and hemodynamic abnormalities of the human disease; however, the mechanisms underlying altered Ca2+ handling have not been investigated in this model. We now show that left ventricular midmyocardial myocytes isolated from dogs subjected to 3 to 4 weeks of rapid pacing have prolonged action potentials and Ca2+ transients with reduced peaks, but durations approximately 3-fold longer than controls. To discriminate between action potential effects on Ca2+ kinetics and direct changes in Ca2+ regulatory processes, voltage-clamp steps were used to examine the time constant for cytosolic Ca2+ removal (tauCa). tauCa was prolonged by just 35% in myocytes from failing hearts after fixed voltage steps in physiological solutions (tauCa control, 216+/-25 ms, n=17; tauCa failing, 292+/-23 ms, n=22; P<0.05), but this difference was markedly accentuated when Na+/Ca2+ exchange was eliminated (tauCa control, 282+/-30 ms, n=13; tauCa failing, 576+/-83 ms, n=11; P<0. 005). Impaired sarcoplasmic reticular (SR) Ca2+ uptake and a greater dependence on Na+/Ca2+ exchange for cytosolic Ca2+ removal was confirmed by inhibiting SR Ca2+ ATPase with cyclopiazonic acid, which slowed Ca2+ removal more in control than in failing myocytes. beta-Adrenergic stimulation of SR Ca2+ uptake in cells from failing hearts sufficed only to accelerate tauCa to the range of unstimulated controls. Protein levels of SERCA2a, phospholamban, and Na+/Ca2+ exchanger revealed a pattern of changes qualitatively similar to the functional measurements; SERCA2a and phospholamban were both reduced in failing hearts by 28%, and Na+/Ca2+ exchange protein was increased 104% relative to controls. Thus, SR Ca2+ uptake is markedly downregulated in failing hearts, but this defect is partially compensated by enhanced Na+/Ca2+ exchange. The alterations are similar to those reported in human heart failure, which reinforces the utility of the pacing-induced dog model as a surrogate for the human disease.

Functional expression of the human cardiac Na+/Ca2+exchanger in Sf9 cells: rapid and specific Ni2+transport

Although inhibition of the Na+/Ca2+exchanger normally increases [Ca2+]iin neonatal cardiac myocytes, application of the inhibitor Ni2+appears to reduce [Ca2+]imeasured by fluo-3. To investigate how the apparent reduction in [Ca2+]ioccurs we examined Ca2+transport by the human Na+/Ca2+exchanger expressed in Sf9 cells. Transport of Ca2+by the Na+/Ca2+exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+exchange current (INaCa) using patch clamp methods. INaCawas elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+exchanger. In addition to measurements of INaCa, characterization includes Ca2+transport via the Na+/Ca2+exchanger and the voltage dependence of Ca2+transport. Application of Ni2+blocked INaCabut, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+was transported via the Na+/Ca2+exchanger at a rate comparable to the Ca2+transport. Once inside the cells, Ni2+reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.

Opposite effects of cooling on twitch contractions of skeletal muscle isolated from tropical toads (Leptodactylidae) and northern frogs (Ranidae).

Cooling increases the twitch force of frog skeletal muscle (Rana temporaria; Rana pipiens), but decreases the twitch force of tropical toad muscle (Leptodactylus insularis). Action potentials and intramembranous charge movement in frog and toad fibers were slowed identically by cooling. Cooling increased the integral of twitch Ca2+ detected by aequorin in frog fibers (1.4-fold), while also decreasing the peak and slowing the rate of decay. Conversely, cooling decreased the integral (0.6-fold) and the peak of twitch Ca2+ in toad fibers, without affecting the rate of decay. The difference in entire Ca2+ transients may account for cold-induced twitch potentiation in frogs and twitch paralysis in toads. In sustained contractions of toad fibers, cooling markedly decreased maximum force caused by: (i) tetanic stimulation, (ii) two-microelectrode voltage clamp steps, (iii) high [K+], or (iv) caffeine. Maximum force in sustained contractions was decreased moderately by cooling frog fibers. Rapid rewarming and simultaneous removal of high [K+] or caffeine during a sustained contraction, caused toad muscle force to rise towards the value corresponding to the warm temperature. This did not occur after removing high [K+] or caffeine from toad fibers kept in the cold. Transmission electron micrographs showed no relevant structural differences. Parvalbumins are thought to promote relaxation of frog muscle in the cold. The unique parvalbumin isoforms in toad muscle apparently lack this property.

Inactivation of macroscopic late Na+ current and characteristics of unitary late Na+ currents in sensory neurons.

Na+ currents in adult rat large dorsal root ganglion neurons were recorded during long duration voltage-clamp steps by patch clamping whole cells and outside-out membrane patches. Na+ current present >60 ms after the onset of a depolarizing pulse (late Na+ current) underwent partial inactivation; it behaved as the sum of three kinetically distinct components, each of which was blocked by nanomolar concentrations of tetrodotoxin. Inactivation of one component (late-1) of the whole cell current reached equilibrium during the first 60 ms; repolarizing to -40 or -50 mV from potentials of -30 mV or more positive gave rise to a characteristic increase in current (tau >/= 5 ms), attributed to removal of inactivation. A second component (late-2) underwent slower inactivation (tau > 80 ms) at potentials more positive than -80 mV, and steady-state inactivation appeared complete at -30 mV. In small membrane patches, bursts of brief openings (gamma = 13-18 pS) were usually recorded. The distribution of burst durations indicated that two populations of channel were present with inactivation rates corresponding to late-1 and late-2 macroscopic currents. The persistent Na+ current in the whole cell that extended to potentials more positive than -30 mV appeared to correspond to sporadic, brief openings that were recorded in patches (mean open time approximately 0.1 ms) over a wide potential range. None of the three types of gating described corresponded to activation/inactivation gating overlap of fast transient currents.

Blockade by 18beta-glycyrrhetinic acid of intercellular electrical coupling in guinea-pig arterioles.

1. Intercellular electrical communication between smooth muscle and endothelial cells was examined in guinea-pig mesenteric arterioles using the whole-cell patch-clamp method. The time course of the current required to impose a 10 mV voltage clamp step was used to determine the extent of electrical coupling between them. Currents recorded from both smooth muscle and endothelial cells relaxed in a multi-exponential manner, indicating the existence of electrical coupling between cells. 2. 18beta-Glycyrrhetinic acid, a gap junction blocker, quickly blocked electrical communication at 40 microM, while neither heptanol nor octanol did so at concentrations of up to 1 mM. 3. In the current clamp mode, repetitive spikes, induced by 10 mM Ba2+ solutions, could be recorded from both kinds of cells. After blocking gap junctions, spikes could only be recorded from the smooth muscle cell layer, indicating that they had been conducted through myoendothelial junctions. 4. In endothelial cells, acetylcholine (ACh, 3 microM) induced hyperpolarizing responses, which had two phases (an initial fast and a second slower phase) in the current clamp condition. This ACh response persisted in the presence of 18beta-glycyrrhetinic acid, although this compound seemed to make the membrane slightly leaky. 5. After blocking gap junctions, the membrane potential of a single cell in a multicellular preparation could be well clamped. Thus, 18beta-glycyrrhetinic acid may be useful in studying the function of both arteriolar smooth muscle and endothelial cells while they remain located within a multicellular preparation.

Excitability of the squid giant axon revisited.

The electrical properties of the giant axon from the common squid Loligo pealei have been reexamined. The primary motivation for this work was the observation that the refractoriness of the axon was significantly greater than the predictions of the standard model of nerve excitability. In particular, the axon fired only once in response to a sustained, suprathreshold stimulus. Similarly, only a single action potential was observed in response to the first pulse of a train of 1-ms duration current pulses, when the pulses were separated in time by approximately 10 ms. The axon was refractory to all subsequent pulses in the train. The underlying mechanisms for these results concern both the sodium and potassium ion currents INa and IK. Specifically, Na+ channel activation has long been known to be coupled to inactivation during a depolarizing voltage-clamp step. This feature appears to be required to simulate the pulse train results in a revised model of nerve excitability. Moreover, the activation curve for IK has a significantly steeper voltage dependence, especially near its threshold (approximately -60 mV), than in the standard model, which contributes to reduced excitability, and the fully activated current-voltage relation for IK has a nonlinear, rather than a linear, dependence on driving force. An additional aspect of the revised model is accumulation/depeletion of K+ in the space between the axon and the glial cells surrounding the axon, which is significant even during a single action potential and which can account for the 15-20 mV difference between the potassium equilibrium potential EK and the maximum afterhyperpolarization of the action potential. The modifications in IK can also account for the shape of voltage changes near the foot of the action potential.

NSC1: a novel high-current inward rectifier for cations in the plasma membrane of Saccharomyces cerevisiae

The plasma membrane of the yeast Saccharomyces cerevisiae possesses a non-specific cation `channel', tentatively dubbed NSC1, which is blocked by normal (mM) calcium and other divalent metal ions, is unblocked by reduction of extracellular free divalents below ∼10 μM, and is independent of the identified potassium channel and porters in yeast, Duk1p, Trk1p, and Trk2p. Ion currents through NSC1, observed by means of whole-cell patch recording, have the following characteristics: Large amplitude, often exceeding 1 nA of K +/cell at −200 mV, in tetraploid yeast, sufficient to double the normal intracellular K + concentration within 10 s; non-saturation at large negative voltages; complicated activation kinetics, in which ∼50% of the total current arises nearly instantaneously with a voltage-clamp step, while the remainder develops as two components, with time constants of ∼100 ms and ∼1.3 s; and voltage independence of both the activation time constants and the associated fractional current amplitudes.

Na-Ca Exchange and the Trigger for Sarcoplasmic Reticulum Ca Release: Studies in Adult Rabbit Ventricular Myocytes

The importance of Na-Ca exchange as a trigger for sarcoplasmic reticulum (SR) Ca release remains controversial. Therefore, we measured whole-cell Ca currents ( I Ca), Na-Ca exchange currents (I NaCa), cellular contractions, and intracellular Ca transients in adult rabbit cardiac myocytes. We found that changing pipette Na concentration markedly affected the relationship between cell shortening (or Ca transients) and voltage, but did not affect the Ca current-voltage relationship. We then inhibited Na-Ca exchange and varied SR content (by changing the number of conditioning pulses before each test pulse). Regardless of SR Ca content, the relationship between contraction and voltage was bell-shaped in the absence of Na-Ca exchange. Next, we rapidly and completely blocked I Ca by applying nifedipine to cells. Cellular shortening was variably reduced in the presence of nifedipine. The component of shortening blocked by nifedipine had a bell-shaped relationship with voltage, whereas the “nifedipine-insensitive” component of contraction increased with voltage. With the SR disabled (ryanodine and thapsigargin pretreatment), I Ca could initiate late-peaking contractions that were ∼70% of control amplitude. In contrast, nifedipine-insensitive contractions could not be elicited in the presence of ryanodine and thapsigargin. Finally, we recorded reverse Na-Ca exchange currents that were activated by membrane depolarization. The estimated sarcolemmal Ca flux occurring by Na-Ca exchange (during voltage clamp steps to +30 mV) was ∼10-fold less than that occurring by I Ca. Therefore, Na-Ca exchange alone is unlikely to raise cytosolic Ca concentration enough to directly activate the myofilaments. We conclude that reverse Na-Ca exchange can trigger SR Ca release. Because of the sigmoidal relationship between the open probability of the SR Ca release channel and pCa, the effects of I Ca and I NaCa may not sum in a linear fashion. Rather, the two triggers may act synergistically in the modulation of SR release.

The transient outward current in mice lacking the potassium channel gene Kv1.4.

1. The transient outward current (Ito) plays a prominent role in the repolarization phase of the cardiac action potential. Several K+ channel genes, including Kv1.4, are expressed in the heart, produce rapidly inactivating currents when heterologously expressed, and may be the molecular basis of Ito. 2. We engineered mice homozygous for a targeted disruption of the K+ channel gene Kv1.4 and compared Ito in wild-type (Kv1.4+/+), heterozygous (Kv1.4+/-) and homozygous 'knockout' (Kv1.4-/-) mice. Kv1.4 RNA was truncated in Kv1.4-/- mice and protein expression was absent. 3. Adult myocytes isolated from Kv1.4+/+, Kv1.4+/- and Kv1.4-/- mice had large rapidly inactivating outward currents. The peak current densities at 60 mV (normalized by cellular capacitance, in pA pF-1; means +/- s.e.m.) were 53.8 +/- 5. 3, 45.3 +/- 2.2 and 44.4 +/- 2.8 in cells from Kv1.4+/+, Kv1.4+/- and Kv1.4-/- mice, respectively (P < 0.02 for Kv1.4+/+ vs. Kv1.4-/-). The steady-state values (800 ms after the voltage clamp step) were 30.9 +/- 2.9, 26.9 +/- 3.8 and 23.5 +/- 2.2, respectively (P < 0.02 for Kv1.4+/+ vs. Kv1.4-/-). The inactivating portion of the current was unchanged in the targeted mice. 4. The voltage dependence and time course of inactivation were not changed by targeted disruption of Kv1.4. The mean best-fitting V (membrane potential at 50 % inactivation) values for myocytes from Kv1.4 +/+, Kv1.4+/- and Kv1. 4-/- mice were -53.5 +/- 3.7, -51.1 +/- 2.6 and -54.2 +/- 2.4 mV, respectively. The slope factors (k) were -10.1 +/- 1.4, -8.8 +/- 1.4 and -9.5 +/- 1.2 mV, respectively. The fast time constants for development of inactivation at -30 mV were 27.8 +/- 2.2, 26.2 +/- 5. 1 and 19.6 +/- 2.1 ms in Kv1.4+/+, Kv1.4+/- and Kv1.4-/- myocytes, respectively. At +30 mV, they were 35.5 +/- 2.6, 30.0 +/- 2.1 and 28. 7 +/- 1.6 ms, respectively. The time constants for the rapid phase of recovery from inactivation at -80 mV were 32.5 +/- 8.2, 23.3 +/- 1.8 and 39.0 +/- 3.7 ms, respectively. 5. Nearly the entire inactivating component as well as more than 60 % of the steady-state outward current was eliminated by 1 mM 4-aminopyridine in Kv1.4+/+, Kv1.4+/- and Kv1.4-/- myocytes. 6. Western blot analysis of heart membrane extracts showed no significant upregulation of the Kv4 subfamily of channels in the targeted mice. 7. Thus, Kv1.4 is not the molecular basis of Ito in adult murine ventricular myocytes.

Voltage-gated and Ca2+-activated conductances mediating and controlling graded electrical activity in crayfish muscle.

Crayfish opener muscle fibers provide a unique preparation to quantitatively evaluate the relationships between the voltage-gated Ca2+ (ICa) and Ca2+-activated K+ (IK(Ca)) currents underlying the graded action potentials (GAPs) that typify these fibers. ICa, IK(Ca), and the voltage-gated K+ current (IK) were studied using two-electrode voltage-clamp applying voltage commands that simulated the GAPs evoked in current-clamp conditions by 60-ms current pulses. This methodology, unlike traditional voltage-clamp step commands, provides a description of the dynamic aspects of the interaction between different conductances participating in the generation of the natural GAP. The initial depolarizing phase of the GAP was due to activation of the ICa on depolarization above approximately -40 mV. The resulting Ca2+ inflow induced the activation of the fast IK(Ca) (<3 ms), which rapidly repolarized the fiber (<6 ms). Because of its relatively slow activation, the contribution of IK to the GAP repolarization was delayed. During the final steady GAP depolarization ICa and IK(Ca) were simultaneously activated with similar magnitudes, whereas IK aided in the control of the delayed sustained response. The larger GAPs evoked by higher intensity stimulations were due to the increase in ICa. The resulting larger Ca2+ inflow increased IK(Ca), which acted as a negative feedback that precisely controlled the fiber's depolarization. Hence IK(Ca) regulated the Ca2+-inflow needed for the contraction and controlled the depolarization that this Ca2+ inflow would otherwise elicit.

Action Potential Prolongation and Potassium Currents in Left-Ventricular Myocytes Isolated from Hypertrophied Rabbit Hearts

We have studied the action potential characteristics and potassium currents in single left ventricular myocytes isolated from control and hypertrophied rabbit hearts. Left-ventricular hypertrophy (LVH) was induced following perinephritis-induced hypertension. Control animals underwent a sham operation. Animals were killed at 10 weeks post-operation. Left-ventricular myocytes were isolated by an enzyme dissociation technique. Action potential duration (APD) at 50 and 90% repolarisation was prolonged in myocytes obtained from hypertrophied compared to control hearts over the range of stimulation frequencies (0.1–1.5 Hz). This prolongation in APD was more pronounced in epicardial compared to endocardial myocytes. Steady-state ionic current, measured at the end of voltage clamp steps of 3-s duration, stepping at intervals of 10 mV, from a holding potential of −40 mV, was similar in control and hypertrophied myocytes. However, when normalised for capacitative cell surface area, steady-state current was significantly smaller in hypertrophied myocytes over the voltage range −40 to −60 mV and at potentials greater than +10 mV. Inward rectifier potassium current (IKl), identified as the barium chloride (0.1 mm)-sensitive current, contributed to the steady state current at negative potentials. Normalised IKlwas significantly smaller in hypertrophied compared to control myocytes at potentials negative to −60 mV. Peak transient outward potassium current (Ito) density was reduced in hypertrophied compared to control myocytes at 0 and +10 mV, from a holding potential −80 mV (12.9±2.3v24.9±3.9μA cm2, at +10 mV,P<0.05). Steady-state inactivation of Itowas similar in control and hypertrophied myocytes. In conclusion, LVH induced by perinephritis hypertension in the rabbit is associated with a prolongation in APD. Reductions in IKl, sustained outward current and Itomay contribute to the prolongation in APD.

The restriction of diffusion of cations at the external surface of cardiac myocytes varies between species

In cardiac muscle sarcolemmal structures such as T-tubules, caveolae and negatively charged protein-polysaccharides may affect the rate of cation exchange on the external surface of the cells. To test this hypothesis, we examined the rate of external cation exchange in adult rabbit and rat ventricular myocytes using a rapid solution switcher to change the bulk external solution within 4 ms. To assess the rate of diffusion of monovalent cations, we increased [K+]o from 4.4 to 6.6 or 8.8 mM and measured the time required to achieve a stable membrane depolarization. In rat myocytes, the mean time to 90% depolarization (t90) was significantly longer than that in rabbit myocytes (137 and 64 ms, respectively) and the difference in t90 was not associated with the cell size. To assess the time course of exchange of external Ca2+, we rapidly exposed the myocytes to 0 Ca2+-2 mM EGTA solution at specific time points before action potentials or voltage clamp steps, and measured the rate of alteration of the normalized peak [Ca2+]i transient (Flux-3) or Ca2+ current. Exposure to 0 Ca2+-2 mM EGTA solution caused a decline in the intracellular calcium transient. In rat myocytes, the rate of decline in the [Ca2+]i transient was much slower (t90 > 1500 ms, the time required for 90% decline) than for the rabbit (t90 = 295 ms). Also, the rate of decline in the Ca2+ current was prolonged in rat myocytes (t90 = 910 ms) compared with rabbit myocytes (t90 = 241 ms). These data indicate that there is a restricted space on the external surface of sarcolemma which limits diffusion of divalent cations more markedly than monovalent cations. The extent of this limitation of cation diffusion varies between species, and may have functional significance.

Estimating the Time Course of the Excitatory Synaptic Conductance in Neocortical Pyramidal Cells Using a Novel Voltage Jump Method

We introduce a method that permits faithful extraction of the decay time course of the synaptic conductance independent of dendritic geometry and the electrotonic location of the synapse. The method is based on the experimental procedure of Pearce (1993), consisting of a series of identical somatic voltage jumps repeated at various times relative to the onset of the synaptic conductance. The progression of synaptic charge recovered by successive jumps has a characteristic shape, which can be described by an analytical function consisting of sums of exponentials. The voltage jump method was tested with simulations using simple equivalent cylinder cable models as well as detailed compartmental models of pyramidal cells. The decay time course of the synaptic conductance could be estimated with high accuracy, even with high series resistances, low membrane resistances, and electrotonically remote, distributed synapses. The method also provides the time course of the voltage change at the synapse in response to a somatic voltage-clamp step and thus may be useful for constraining compartmental models and estimating the relative electrotonic distance of synapses. In conjunction with an estimate of the attenuation of synaptic charge, the method also permits recovery of the amplitude of the synaptic conductance. We use the method experimentally to determine the decay time course of excitatory synaptic conductances in neocortical pyramidal cells. The relatively rapid decay time constant we have estimated (tau approximately 1.7 msec at 35 degrees C) has important consequences for dendritic integration of synaptic input by these neurons.

Dual actions of tetracaine on intramembrane charge in amphibian striated muscle

1. The effects of graded concentrations of tetracaine on the steady-state and kinetic properties of intramembrane charge were examined in intact voltage-clamped amphibian muscle fibres. 2. The micromolar tetracaine concentrations that were hitherto reported to abolish Ca2+ transients in skeletal muscle failed to affect significantly the steady-state charge. Maximal reductions of such intramembrane charge required relatively high, 1-2 mM, concentrations of tetracaine. 3. The plots of maximum charge against tetracaine concentration suggested a saturable 1:1 drug binding that spared a fixed amount of tetracaine-resistant (q beta) charge but inhibited a discrete fraction of susceptible (q gamma) charge with a KD between 0.1 and 0.2 mM. 4. The q beta charge thus isolated by 2 mM tetracaine was conserved through a wide range of applied test voltages and pulse durations and regardless of whether the imposed transition from the holding potential (-90 mV) to the test potential took place in one or more steps. 5. Similarly, 'on' and 'off' q beta currents that were elicited by voltage steps from fixed conditioning to varying test levels mapped onto non-linear phase-plane trajectories that nevertheless depended uniquely upon voltage. In contrast, the currents that followed voltage steps made from varying prepulse levels to fixed -90 or -20 mV test potentials mapped onto identical q beta phase-plane trajectories that were independent of the prepulse history. 6. The charge movements that followed strong depolarizing voltage clamp steps to test potentials in the range -50 to 0 mV approximated simple monotonic decays that could empirically be described by a single time constant. Nevertheless, a complete inhibition of a tetracaine-sensitive (q gamma) charge movement by 2 mM tetracaine that left only q beta charge, sharply altered both the magnitude and the voltage dependence of these time constants. This establishes a distinct contribution of the q gamma species to overall charge kinetics even at such test voltages. 7. Under such a criterion for the voltage dependence of charging kinetics, even the micromolar (0.05-0.2 mM) tetracaine concentrations that failed to markedly alter the steady-state charge consistently increased the charging time constants yet did not influence their voltage sensitivity. 8. These findings demonstrate the existence of separate kinetic and steady-state effects of tetracaine on intramembrane charge movements, at micromolar and millimolar anaesthetic concentrations, respectively. These parallel earlier effects of tetracaine that have been reported upon the transient and sustained components of sarcoplasmic reticular Ca2+ release. They also establish that maximally effective concentrations of tetracaine isolate a single distinct species of conserved (q beta) intramembrane charge.

Low-threshold, persistent sodium current in rat large dorsal root ganglion neurons in culture.

Dorsal root ganglion neurons from adult rats (> or = 200 g) were maintained in culture for between 1 and 3 days. Membrane currents generated by large neurons (50-75 microns apparent diameter) were recorded with the whole cell patch-clamp technique. Large neurons generated transient Na+ currents and at least two types of inward current that persisted throughout 200-ms voltage-clamp steps to +20 mV. One persistent current activated close to -35 mV (high threshold), whereas in about half of the cells another persistent current began to activate negative to -70 mV (low threshold). The high-threshold persistent current was identified as a Ca2+ current, as previously described in these neurons. The low-threshold current was reversibly suppressed either by replacing external Na+ with tetramethylammonium ions or by reducing external Na+ concentration ([Na+]) and simultaneously raising external [Ca2+]. It was blocked by tetrodotoxin (TTX) with an apparent equilibrium dissociation constant in the single nanomolar range. We conclude that the low-threshold current is a TTX-sensitive, persistent Na+ current. The persistent TTX-sensitive current contributed to steady-state membrane current from at least -70 mV to 0 mV, a wider potential range than predicted by activation-inactivation gating overlap for transient Na+ current. Because of its low threshold and fast activation kinetics, the persistent Na+ current is expected to play an important role in determining membrane excitability.

Analysis of current fluctuations during after‐hyperpolarization current in dentate granule neurones of the rat hippocampus.

1. We have studied macroscopic current fluctuations associated with the after-hyperpolarization current (IAHP) that follows a 200 ms voltage-clamp step to 0 mV in dentate granule (DG) neurones of the rat hippocampus. This maximally effective stimulus produced a peak IAHP of 205 +/- 20 pA. Background noise was minimized by using the whole-cell single-electrode voltage-clamp configuration. 2. Conventional current-variance analysis was performed on IAHP to obtain estimates of the unitary AHP channel current (i) and the maximal attainable AHP current (Imax). A second approach, utilizing changes in the power spectrum of IAHP 'noise' during the decay of IAHP, was employed to yield an independent estimate of Imax as well as an estimate of the mean open-state duration of AHP channels. 3. Changes in the power spectrum during IAHP decay revealed that the mean channel open time is fixed at 6.9 +/- 0.5 ms and that the decay is due to changes in channel closed-state duration. The same analysis gave a value for Imax of 320 +/- 20 pA (n = 7). 4. Current-variance analysis suggests that channels responsible for generation of IAHP have a unitary current of 0.29 +/- 0.08 pA at -45 mV in 5 mM extracellular potassium and an Imax of 400 +/- 180 (n = 7). Thus, both methods indicate that about 1200 channels are available to generate IAHP in DG neurones and that about 60% are open at the peak of a maximal IAHP. 5. Computer simulations of IAHP currents in a model neurone show that dendritic current sources will result in an underestimation of i while Imax is underestimated to a lesser extent. Estimates of Imax obtained from power-spectrum analysis are more accurate and less affected by neuronal electrotonic structure than estimates of Imax based on current-variance analysis.

Carbachol induces inward current in neostriatal neurons through M1-like muscarinic receptors

The effects of carbachol on rat neostriatal neurons were examined in the slice and the freshly dissociated neuron preparations using intracellular and whole-cell voltage-clamp recording methods. Superfusion of carbachol (30 microM) produced a depolarization concomitant with an increase in the rate of spontaneous action potentials. This depolarization was associated with an increase in the input resistance. The carbachol-induced membrane depolarization was blocked by pirenzepine (1 microM), a selective M1 muscarinic receptor antagonist. In other experiments, we observed that carbachol induced a transient inward current on the freshly dissociated neostriatal neuron at a holding potential of -60 mV in a concentration-dependent manner underlying the whole-cell voltage-clamp mode. The inward current caused by carbachol was not reduced by tetrodotoxin (1 microM), calcium-free recording solution or Cd2+ (100 microM). However, it was blocked by Ba2+ (100 microM). In addition, the carbachol-induced inward current reversed polarity at about the potassium equilibrium potential. The whole-cell membrane inward current in response to voltage-clamp step from -90 to -140 mV was reduced by 30 microM carbachol. With stronger hyperpolarization beyond the potassium equilibrium potential, carbachol produced a progressively greater reduction in membrane current. This inhibitory effect was also abolished by Ba2+ (100 microM). A concentration of 30 microM carbachol-induced inward current could be reversibly antagonized by the M1 muscarinic receptor antagonist pirenzepine (0.1-1 microM), with an estimated IC50 of 0.3 microM. However, other muscarinic receptor subtype (M2 or M3) antagonists could also block the carbachol-induced inward current. The rank order of antagonist potency was: pirenzepine (M1 antagonist) > 4-diphenylacetoxy-N,N-methyl-piperidine methiodide (M3/M1 antagonist) > gallamine (M2 antagonist). Based on these pharmacological data, we concluded that carbachol can act at M1-like muscarinic receptors to reduce the membrane K+ conductances and excite the neostriatal neurons.

Mode of Inhibition of Transient Outward Current by 1-Benzyl-1,2,3,4-Tetrahydroisoquinoline in Rat Ventricular Cells.

In this study, we examined the effects of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (S49) on a transient outward current (I(to)) in rat ventricular myocytes using the whole-cell patch-clamp technique. Depolarization of ventricular myocytes not only activated I(to), but sustained outward currents as well. S49 dose-dependently inhibited the amplitude or integral of I(to), with a 50% inhibitory concentration (IC(50)) of 4.3 and 2.7 &mgr;M, respectively. The inhibition of I(to) by S49 was associated with an acceleration of I(to) decay. However, S49 had no effect on half inactivation voltage (V(0.5)) and slope factor of the voltage-dependent steady-state inactivation curve of I(to). Time-course analysis revealed that S49 developed a block during the depolarizing voltage-clamp step in a monoexponential manner. The rate and magnitude of block were concentration dependent. The equilibrium dissociation constant (K(d)) used to inhibit I(to) induced by S49, as calculated from the time constant of developing block, was 3.4 &mgr;M. The time constant of recovery of I(to) from the inactivation state was prolonged by S49. Following treatment with quinidine, the process of I(to) recovery was divided into rapid and extremely slow recovery components. Also, the relief from quinidine- or S49-induced block was assessed by comparing the recovery processes of I(to) with or without drugs. That comparison revealed the relief from the block of I(to) channels by S49 to be more rapid. In summary, the inhibition of I(to) by S49 was dose dependent, time dependent but voltage independent. The mechanisms of action might be an open-state block. Copyright 1996 S. Karger AG, Basel

‘Intact’ dopaminergic midbrain neurons of the rat display unclamped dendritic Ca 2+ currents

Calcium channels play an important role in generating the complex electrophysiology of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNc). To directly study these calcium currents in dendritically intact neurons, two issues needed to be addressed: the identity of the neuron (DAergic versus non-DAergic) and control of putative dendritic calcium conductances. A measure of the anomalous rectifier ( I h) was used to identify DAergic neurons. Both groups of neurons produced large (∼1 nA), sustained inward currents that persisted as ‘plateau currents’ for hundreds of milliseconds after the termination of the depolarizing voltage clamp step. We conclude that intact DAergic neurons can be identified under conditions optimal for recording calcium currents (i.e. Cs + internal solution), but powerful dendritic currents limit rigorous biophysical analysis.

P-glycoprotein regulates a volume-activated chloride current in bovine non-pigmented ciliary epithelial cells.

1. The whole-cell patch clamp technique was used to investigate the swelling-activated currents in bovine non-pigmented ciliary epithelial (NPCE) cells. 2. Exposure to hypotonic solution activated a current that was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The I-V relationship was shifted in the direction expected for a Cl- current when the external Cl- was replaced by gluconate (permeability ratio P(gluconate)/PCl = 0.17). The inhibition of the current evoked by voltage clamp steps of +80 mV yielded an IC50 for NPPB of 13.4 microM. 3. The current was found to be dependent on ATP. With ATP in the patch pipette the current could be repeatedly activated by exposure to hypotonic solution but when ATP was omitted the current ran down with time. 4. The development of this current was associated with visible cell swelling and inhibitors of regulatory volume decrease in these cells, e.g. tamoxifen, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), also inhibited this current. 5. The volume-activated current was additionally blocked by NPPB, verapamil, quinidine and dideoxyforskolin. 6. The current was independent of external calcium and exhibited slight outward rectification and time-dependent inactivation at strong depolarizing potentials. 7. Disrupting the cytoskeleton and microtubules with cytochalasin B and colchicine had no effect on the activation of the Cl- current. 8. An antibody (C219) to the MDR1 gene product, P-glycoprotein, caused a functional block of the swelling-activated Cl- current when added to the patch pipette. 9. Immunofluorescence studies using the monoclonal antibodies C219 and JSB-1 demonstrated the presence of P-glycoprotein in the ciliary epithelial cells. The immunofluorescence was stronger on the non-pigmented than on the pigmented cells. 10. It is concluded that swelling in NPCE cells activates a Ca(2+)-independent, ATP-dependent Cl- current and that the activity of this current is associated with P-glycoprotein. 11. It is suggested that this Cl- current contributes to regulatory volume decrease and may participate in the secretory activity of these cells.