Voltage clamp analysis of embryonic heart cell aggregates

The double-microelectrode voltage clamp technique was applied to small spheroidal aggregates of heart cells from 7-d chick embryos. A third intracellular electrode was sometimes used to monitor spatial homogeneity. On average, aggregates were found to deviate from isopotentiality by 12% during the first 3--5 ms of large depolarizing voltage steps, when inward current was maximal, and by less than 3% thereafter. Two components of inward current were recorded: (a) a fast, transient current associated with the rapid upstroke of the action potential, which was abolished by tetrodotoxin (TTX); and (b) a slower inward current related to the plateau, which was not affected by TTX but was blocked by D600. The magnitudes, kinetics, and voltage dependence of these two inward currents and a delayed outward current were similar to those reported for adult cardiac preparations. From a holding potential of -60 mV, the peak fast component at the point of maximal activation (-20 mV) was -185 microA/cm2. This value was about seven times greater than the maximal slow component which peaked at 0 mV. The ratio of rate constants for the decay of the two currents was between 10:1 and 30:1.     

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.     

Effect of nonachlazine on transmembrane ionic currents in the frog atrial trabeculae

The effect of the antianginal drug nonachlazine displaying antiarrhythmic properties on transmembrane ionic currents in the frog atrial fibers was studied in experiments on isolated trabeculae of the frog atria. The transmembrane ionic currents were measured by a voltage clamp technique based on a double sucrose gap arrangement. Nonachlazine (1.03 X 10(-5) mol/l) decreased the amplitude of the fast inward current whatever the magnitude of membrane potential. The drug inhibited the slow inward current and prevented the adrenaline-increased permeability of the slow sodium-calcium channel if external sodium ions were replaced by choline chloride. Nonachlazine (1.03 X 10(-5) mol/l) diminished the amplitude of the inward ionic current in a calcium-free medium as well. The stimulatory effect of prostacycline (2 X 10(-7) mol/l) on the fast inward ionic current was inhibited by nonachlazine. The data obtained suggest that the antiarrhythmic effect of nonachlazine might be linked with the inhibition of the fast sodium inward current and the slow calcium inward current.     

巨孔匙(虫戚)(Megathura)未受精卵细胞膜离子流的特征

用双微电极电压钳技术在巨孔匙(虫戚)(Megathura)未受精卵细胞膜上记录到多种离子流。主要有一种内向的两价离子流和几种钾离子流:包括钡离子激活的钾离子流,迅速激活又迅速失活的钾离子流(类似于I_A)和异常整流钾离子流。不同细胞的离子流大小不同。在一些卵可能会缺少其中某一种离子流。此外,还观察到浴槽溶液中氯和钠离子浓度改变对膜电位及膜电导的影响。     

Two different tetrodotoxin-separable inward sodium currents in the membrane of isolated cardiomyocytes

Isolated ventricular myocytes of 3 to 5 weeks old rats were studied under conditions of intracellular perfusion and voltage clamp. The existence of two inward sodium currents with different TTX-sensitivities and different properties was shown. The fast sodium current was more sensitive to TTX (Kd about 8 X 10(-8) mol/l). The block of the slow sodium current by TTX was less specific (Kd about 7 X 10(-6) mol/l). There was an about four fold difference in the inactivation time constants between these currents. The maximum on the I-V curve of the slow sodium current was shifted along the voltage axis by about 15 mV in the positive direction as compared with that of the fast sodium current. A slow current carried by calcium ions was observed in sodium-free solution. The kinetics and TTX-sensitivity of this current were similar to those of the slow sodium current. The amplitude of this current was 15 to 20 times lower as compared with the slow sodium current observed in Na-containing solution with 10(-6) mol/l TTX (a concentration which completely blocked the fast sodium current). It is suggested that the slow voltage-gated TTX-sensitive channels described are not highly selective and pass both sodium and calcium ions.     

Inward and outward currents in isolated dendrites of crustacea coxal receptors

Segments from the nonspiking peripheral dendrites of a crustacean coxal receptor (T fiber) were studied using the voltage clamp technique. The peripheral endings of the T fiber are sensitive to stretch applied to a specialized receptor muscle by rotation of the coxa. The intraganglionary portion of the T fiber is presynaptic to the motor neurons innervating the coxal muscle. Depolarizing commands activated three separate fast channels: (i) a transient inward sodium current, INa, which is blocked by tetrodotoxin (TTX); (ii) a transient outward current, Io1 , having the same voltage-dependent characteristics as INa; and (iii) a second, longer-lasting, outward current, Io2 . Both INa and Io1 were inactivated when segments were clamped at voltages more positive than -50 mV, whereas Io2 could be activated at voltages more positive than -50 mV. Io1 and Io2 were blocked by 4-aminopyridine (4-AP) and by tetraethylammonium (TEA), although Io2 shows a greater sensitivity to TEA than Io1 . It is suggested that Io1 may be a factor in determining the nonspiking behavior of the dendrites and that Io2 may limit the stretch-induced depolarization in the dendrite to a value more negative than that at which the maximum rate of transmitter release occurs. In addition to the three fast currents, the presence of a slow inward and slow outward current could also be demonstrated. The effects of the slow currents were longer in segments cut from the proximal part of the dendrites.     

Ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis

The ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis were examined by using conventional two-electrode voltage clamp and whole-cell patch clamping methods. Several separable currents were identified. These include: (1) a transient and (2) a steady-state voltage-activated inward current; both are tetrodotoxin (TTX) and saxitoxin (STX) insensitive, partly reduced by decreasing external Ca2+ or Na+ or by addition of 5 mM Co2+, D-600 or verapamil and are totally blocked with 5 mM Cd2+; (3) an early, transient, cation-dependent, outward K+ current (IKCa/Na); (4) a transient, voltage-activated, outward K+ current provisionally identified as IA; (5) a delayed, steady-state, voltage-activated outward K+ current (IK) and (6) a late, transient, outward K+ current which is blocked by Cd2+ and evident only during long voltage pulses. Despite their phylogenic origin, most of these currents are similar to currents identified in many vertebrate smooth and cardiac muscle preparations, and other excitable cells in higher animals.     

Three types of slow inward currents as distinguished by melittin in 3-day-old embryonic heart

Membrane slow inward currents of 3-day-old embryonic chick single heart cells were investigated using the whole-cell patch clamp technique. In a solution containing only Na+ ions and in the presence of tetrodotoxin and Mn2+, the inward current-voltage relationship presented two maxima, confirming the existence of two different voltage-dependent slow inward currents. The first type, a fast transient slow inward current (Isi (ft], was activated from a holding potential of -80 mV and showed fast activation and inactivation. This current was highly sensitive to melittin (10(-8) M) and insensitive to low concentrations of desmethoxyverapamil [-)D888, 10(-9)-10(-6) M). Depolarizing voltage steps from a holding a potential of -50 mV activated two components of the slow inward current, i.e., a slow and a sustained current (Isi(sts] that showed a slow inactivation followed by a slow inactivation and a sustained component. Melittin at a high concentration (10(-4)M) completely blocked the slow transient component (Isi(st] and left unblocked the sustained component (Isi(s]. Both components (Isi(st) and Isi(s] were blocked by verapamil (10(-5)M) and low concentrations of (-)D888 (10(-8)-10(-6)M).     

An Assessment of the Double Sucrose-Gap Voltage Clamp Technique as Applied to Frog Atrial Muscle

The homogeneity of voltage clamp control in small bundles of frog atrial tissue under double sucrose-gap voltage clamp conditions was assessed by intracellular microelectrode potential measurements from cells in the test node region. The microelectrode potential measurements demonstrated that (1) good voltage control of the impaled cell existed in the absence of the excitatory inward currents (e.g., during small depolarizing clamp pulses of 10-15 mV), (2) voltage control of the impaled cell was lost during either the fast or slow excitatory inward currents, and (3) voltage control of the impaled cell was regained following the inward excitatory currents. Under nonvoltage clamp conditions the transgap recorded action potential had a magnitude and waveform similar to the intracellular microelectrode recorded action potentials from cells in the test node. Transgap impedance measured with a sine-wave voltage of 1,000 Hz was about 63% of that measured either by a sine-wave voltage of 10 Hz or by an action potential method used to determine the longitudinal resistance through the sucrose-gap region. The action potential data in conjunction with the impedance data indicate that the extracellular resistance (R_s) through the sucrose gap is very large with respect to the longitudinal intracellular resistance (R_i); the frequency dependence of the transgap impedance suggests that at least part of the intracellular resistance is paralleled by a capacitance. The severe loss of spatial voltage control during the excitatory inward current raises serious doubts concerning the use of the double sucrose-gap technique to voltage clamp frog atrial muscle.     

Possible existence of transient TTX-sensitive chloride current in the membrane of isolated cardiomyocytes

Cardiomyocytes enzymatically isolated from rat and guinea pig ventricular tissue were investigated under conditions of intracellular perfusion and voltage clamp at 18-20 degrees C. Perfusion with 135 mmol/l Tris(HF), pH 7.2 was used to eliminate outward potassium currents. The dependence of inward current (elicited by depolarizing pulses from a holding potential level of--120 mV) on low external TTX concentrations (from 10(-13) to 10(-10) mol/l) was studied. Similar TTX concentrations increased the amplitude of the inward current and changed its kinetics in a large number of cells tested. The effect was fully reversible. The effect could be evaluated in a net form by digital subtraction of the current obtained after the application of a low external TTX concentration from the initial current in a TTX-free solution. The TTX concentration dependence of the difference current could be fitted by one-to-one binding curve with Kd = (1.0 +/= 0.4) x 10(-12) mol/l. TTX-induced current changes were absent in low sodium or chloride-free external solutions. The outward current (a block of which by TTX produced the inward current changes observed) showed a reversal potential consistent with the chloride nature of such a current. The existence of a transient TTX-sensitive Na-dependent potential gated chloride current in the membrane of isolated cardiomyocytes is postulated.     

Membrane currents in taste cells of the rat fungiform papilla. Evidence for two types of Ca currents and inhibition of K currents by saccharin

Taste buds were isolated from the fungiform papilla of the rat tongue and the receptor cells (TRCs) were patch clamped. Seals were obtained on the basolateral membrane of 281 TRCs, protruding from the intact taste buds or isolated by micro-dissection. In whole-cell configuration 72% of the cells had a TTX blockable transient Na inward current (mean peak amplitude 0.74 nA). All cells had outward K currents. Their activation was slower than for the Na current and a slow inactivation was also noticeable. The K currents were blocked by tetraethylammonium, Ba, and 4-aminopyridine, and were absent when the pipette contained Cs instead of K. With 100 mM Ba or 100 mM Ca in the bath, two types of inward current were observed. An L-type Ca current (ICaL) activated at -20 mV had a mean peak amplitude of 440 pA and inactivated very slowly. At 3 mM Ca the activation threshold of ICaL was near -40 mV. A transient T-type current (ICaT) activated at -50 mV had an average peak amplitude of 53 pA and inactivated with a time constant of 36 ms at -30 mV. ICaL was blocked more efficiently by Cd and D600 than ICaT. ICaT was blocked by 0.2 mM Ni and half blocked by 200 microM amiloride. In whole-cell voltage clamp, Na-saccharin caused (in 34% of 55 cells tested) a decrease in outward K currents by 21%, which may be expected to depolarize the TRCs. Also, Na-saccharin caused some taste cells to fire action potentials (on-cell, 7 out of 24 cells; whole-cell, 2 out of 38 cells responding to saccharin) of amplitudes sufficient to activate ICaL. Thus the action potentials will cause Ca inflow, which may trigger release of transmitter.     

Membrane properties of isolated mudpuppy taste cells

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.     

Voltage Clamp of Cardiac Muscle in a Double Sucrose Gap: A Feasibility Study

A method of stabilizing the membrane potential of a small area of cardiac muscle membrane and the limitations of this method are described. Tiny bundles or strands, approximately 80 μm in diameter, of electrically interconnected fibers from the ventricles of rabbit hearts were used in a double sucrose gap. Current records associated with step changes in voltage were complicated by two capacitive surges of current of nodal and nonnodal origin and large “leakage” currents of nonnodal origin resulting mainly from the multifibered nature of the preparation and emphasized by the method. The transient, inward membrane currents in response to moderate depolarizing steps in command potential had the same duration as the upstroke of the action potential. In good runs, currents were smooth and free from notches. These initial currents behaved qualitatively like the initial sodium currents in squid axon and in other excitable membranes. A fraction of the initial sodium current persisted at least as long as 300 ms. The relationship between peak initial current and voltage was graded and linear in the positive direction. In the negative region the relationship was often very steep, indicating insufficient voltage control of all the membranes despite the squareness of the voltage record. Other indications of inadequacy of control could occur and thus even with this optimum preparation of cardiac muscle it was not feasible to analyze quantitatively either the initial or the prolonged sodium currents.     

Two Fast Transient Current Components during Voltage Clamp on Snail Neurons

Voltage clamp currents from medium sized ganglion cells of Helix pomatia have a fast transient outward current component in addition to the usually observed inward and outward currents. This component is inactivated at normal resting potential. The current, which is carried by K+ ions, may surpass leakage currents by a factor of 100 after inactivation has been removed by hyperpolarizing conditioning pulses. Its kinetics are similar to those of the inward current, except that it has a longer time constant of inactivation. It has a threshold close to resting potential. This additional component is also present in giant cells, where however, it is less prominent. Pacemaker activity is controlled by this current. It was found that inward currents have a slow inactivating process in addition to a fast, Hodgkin-Huxley type inactivation. The time constants of the slow process are similar to those of slow outward current inactivation.     

Currents related to the sodium-calcium exchange in squid giant axon

We report the measurement of a Cai-activated membrane current in dialyzed squid axon under membrane potential control with a low-noise voltage clamp. Two additional voltage clamp systems were used to clamp the external guard plates to a value that prevented the establishment of potential differences between the central and lateral compartments of the experimental chamber. This reduced to a minimum the contribution of membrane currents generated at the axon ends to the current measured in the central pool. This latter current was reduced by using internal and external solutions designed to diminish at a maximum membrane currents, while maintaining the conditions for optimal operation of the Na+-Ca2+ exchange. Thus TTX was used to block Na+ channels and prolonged exposure to K+-free media was used to eliminate K+ conductance. The maximum concentration of external sodium was 200 mM. The addition of fixed amounts of free ionic calcium to the internal solution, activated a current whose direction and magnitude depended on the thermodynamic driving forces for calcium and sodium. When the experimental conditions determined an inwardly directed current, this depended on the presence of external sodium, and lithium could not substitute for it. The Cai-activated current, was blocked by external lanthanum and showed a high temperature dependence. In experiments in which the reversal potential was measured for the Cai-activated current, it was found to be strikingly similar to the value calculated according to Er = 3ENa - 2ECa, suggesting that the current is the electrical manifestation of the Na+-Ca2+ exchange operating with an stoichiometry of 3Na+:1Ca2+.     

Extracellular recording of localized electrical activity in denervated frog slow muscle fibres

Pyriformis muscles of Rana temporaria were denervated by cutting the sciatic nerve in the pelvis. Slow muscle fibres were depolarized with intracellular current pulses, and the electrical activity was recorded simultaneously with intracellular and extracellular recording electrodes. When the extracellular electrode was moved along the fibre surface, outward and inward currents of variable amplitude were recorded. Inward currents coincided with the fast rising phase of the intracellularly recorded action potential; up to four inward current peaks could be detected in single fibres investigated over 3.4--8 mm of their length. The distance between inward current peaks was generally 1--2 mm, but greater distances were also observed. Composite action potentials could be shown to be due to inward currents arising in separate areas of the slow fibre membrane. It is concluded that after denervation Na-channels are incorporated into spatially limited areas of the membrane of slow muscle fibres.     

Ionic mechanisms of pacemaker activity in cardiac Purkinje fibers.

Rhythmic activity in cardiac Purkinje fibers can be analyzed by using the voltage clamp technique to study pacemaker currents. In normally polarized preparations, pacemaker activity can be generated by two distinct ionic mechanisms. The standard pacemaker potential (phase 4 depolarization) involves a slow potassium current, IK2. Following action potential repolarization, the IK2 channels slowly deactivate and thus unmask a steady background inward current. The resulting net inward current causes the slow pacemaker depolarization. Epinephrine accelerates the diastolic depolarization by promoting more complete and more rapid deactivation of IK2 over the pacemaker range of potentials. The catecholamine acts rather selectively on the voltage dependence of the gating mechanism, without altering the basic character of the pacemaker process. The nature of the pacemaker depolarization is altered by intoxication with high concentrations of cardiac glycosides or aglycones. These compounds promote spontaneous impulses in Purkinje fibers by a mechanism that supersedes the ordinary IK2 pacemaker. The digitalis-induced depolarization is generated by a transient inward current that is either absent or very small in untreated preparations. The transient inward current is largely carried by sodium ions. Its unusual time course probably reflects an underlying subcellular event, the oscillatory release of calcium ions from intracellular stores.     

Calcium currents in squid giant axon.

Voltage-clamp experiments were carried out on intracellularly perfused squid giant axons in a Na-free solution of 100 mM CaCl2+sucrose. The internal solution was 25 mM CsF+sucrose or 100 mM RbF+50mM tetraethylammonium chloride+sucrose. Depolarizing voltage clamp steps produced small inward currents; at large depolarizations the inward current reversed into an outward current. Tetrodotoxin completely blocked the inward current and part of the outward current. No inward current was seen with 100 mM MgCl2+sucrose as internal solution. It is concluded that the inward current is carried by Ca ions moving through the sodium channel. The reversal potential of the tetrodotoxin-sensitive current was +54mV with 25 mM CsF+sucrose inside and +10 mV with 100 mM RbF+50 mM tetraethylammonium chloride+sucrose inside. From the reversal potentials measured with varying external and internal solutions the relative permeabilities of the sodium channel for Ca, Cs and Na were calculated by means of the constant field equations. The results of the voltage-clamp experiments are compared with measurements of the Ca entry in intact axons.     

Calcium channel currents in isolated smooth muscle cells from human bronchus

An electrophysiological study was carried out on smooth muscle cells that were enzymatically dissociated from bundles of muscle fibers dissected out of human bronchi obtained at thoracotomy. These cells that retain the contractile properties of intact bundles were voltage-clamped by means of the whole-cell patch-clamp technique. Upon voltage steps from a holding potential of -60 mV to more positive levels, the initial inward current was followed by large outward currents that inactivated slowly. These were subsequently reduced by substituting Cs+ for K+ in the internal solution and by using Ba2+ instead of Ca2+ as a charge carrier in the external solution. Under these conditions, the inward current did not completely inactivate in the course of 300-ms voltage steps. Inward current measured after leak subtraction was activated at a membrane potential of -25.8 +/- 5 mV, was maximum at +18 +/- 4 mV, and had an apparent reversal potential of +52.5 +/- 5.5 mV (n = 5). The potential at which steady-state inactivation was half-maximum was -28 mV (n = 5). This inward current was identified as a calcium current on the following basis: 1) it was not altered by 10 microM tetrodotoxin (TTX) or by lowering to 10 mM external Na+ concentration; 2) it was blocked by 2.5 mM Co2+ or 1 microM PN 200-110; 3) it was enhanced by 1 microM BAY K 8644, which in addition suppressed the PN 200-110 blockade.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic and charge-displacement currents evoked by temperature jumps in Xenopus oocytes

Membrane currents were recorded in voltage-clamped oocytes of Xenopus laevis. Currents were produced in response to temperature jumps imposed by a heating lamp. Responses were larger when the animal (pigmented) hemisphere of the oocyte was illuminated as compared to the vegetal hemisphere; they arose because of a thermal effect as they were attenuated by removal of infrared wavelengths. The temperature jump responses comprised two distinct components: (i) a slow maintained current, which inverted direction at a membrane potential of about -25 mV and, (ii) a fast transient current, which at all potentials examined (-160 to +30 mV), was inward at the onset of a light flash and outward at the offset. The slow component probably arises through temperature-dependent changes in the 'leakage' current of the oocyte, and measurements of reversal potentials in solutions of different ionic composition indicated that currents carried by Na+ and H+ ions contribute to the response. In contrast, the fast component was not altered by changes in composition of the bathing solution. This observation, together with the finding that the charge movements associated with the on and off transients were of similar magnitude, suggest that the fast current may arise because of the displacement of charges across the plasma membrane.