Current- and Voltage-Clamped Studies on Myxicola Giant Axons : Effect of tetrodotoxin

A new dissection procedure for preparing Myxicola giant axons for observation under voltage clamp is described. Preparation time is generally 40–45 min. 65–70% of the preparations attempted may be brought through the entire procedure, including insertion of the long internal electrode, and support an initial action potential amplitude of 100 mv or greater. Mean values for axon diameter, resting membrane potential, action potential amplitude, maximum peak inward transient current, and resting membrane resistance are 560 µ, —66.5 mv, 112 mv, 0.87 ma/cm² and 1.22 KΩ cm ² respectively. Cut branches do not seem to be a problem in this preparation. Behavior under voltage clamp is reasonably stable over several hours. Reductions in maximum inward transient current of 10% and in steady-state current of 5–10% are expected in the absence of any particular treatment. Tetrodotoxin blocks the action potential and both the inward and outward transient current, but has no effect on either the resting membrane potential or the steady-state current. This selective action of tetrodotoxin on the transient current is taken as an indication that this current component is probably carried by Na.     

Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures

The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures. The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps. In all protoplasts, a voltage- and time-dependent outward rectifying current was present. The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course. The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration. The outward current did not show inactivation. In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well. The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course. The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.     

Differences between Pacemaker and Nonpacemaker Neurons of Aplysia on Voltage Clamping

The responses of pacemaker and nonpacemaker Aplysia neurons to voltage clamp commands of less than 200 msec duration are essentially identical. For moderate depolarizing commands there is an early inward transient current followed by a late outward current and an outward tail current when the membrane is clamped back to resting potential. On long (1–2 sec) commands in pacemakers there is a marked sag in the late current and an inward tail current. E_tail, the potential of the membrane at which there is no net current flow under the conditions prevailing at the end of the clamp, shifts from about -9.0 mv on short commands to +5.0 mv on long commands. In contrast there is no marked sag of the late current or inward tail current on long commands in nonpacemakers, and E_tail is near -9.0 mv for both short and long commands. The current sag and shift in E_tail can be ascribed to a decreased conductance (presumably to K⁺) at the end of the long as compared to the short command in half of the pacemaker neurons. In the remaining cells the essential difference from nonpacemakers appears to be either a greater restricted extracellular space or a more active potential-dependent electrogenic Na⁺ pump in pacemakers.     

Whole cell K and Cl currents in dissociated eccrine secretory coil cells during stimulation

Using the whole-cell voltage clamp (to determine the membrane current) and current clamp (to determine membrane potential) methods in conjunction with the nystatin-perforation technique, we studied the effect of methacholine (MCh) and other secretagogues on whole cell K and Cl currents in dissociated rhesus palm eccrine sweat clear cells. Application of MCh by local superfusion induced a net outward current (at a holding potential of ?60 mV and a clamp voltage of 0 mV), and a transient hyperpolarization by 5.6 mV, suggesting the stimulation of K currents. The net outward current gradually changed to the inward (presumably Cl) currents over the next 1 to 2 min of continuous MCh stimulation. During this time the membrane potential also changed from hyperpolarization to depolarization. The inward currents were increasingly more activated than outward (presumably K) currents during repeated MCh stimulations so that a net inward current (at ?60 mV) was observed after the fourth or fifth MCh stimulation. Ionomycin (10 μm) also activated both inward and outward current. The observed effect of MCh was abolished by reducing extracellular [Ca] to below 1 nm (Ca-free + 1 mm EGTA in the bath). MCh-activated outward currents were inhibited by 5 mm Ba and by 0.1 mm quinidine, although these agents also suppressed the inward currents. Bi-ionic potential measurements indicated that the contribution of Na to the membrane potential was negligible both before and after MCh or ISO (isoproterenol) stimulations and that the observed membrane current was carried mainly by K and Cl. MCh increased the bi-ionic potential by step changes in external K and Cl concentrations, further supporting that MCh-induced outward and inward currents represent K and Cl currents, respectively. Stimulation with ISO or FK (forskolin) resulted in a depolarization by about 55 mV and a net inward (most likely Cl) current independent of external Ca. CT-cAMP mimicked the effects of FK and ISO. The bi-ionic potential, produced by step changes in the external Cl concentration, increased during ISO stimulation, whereas that of K decreased. This indicates that the ISO-induced inward current is due to Cl current and that K currents were unchanged or slightly decreased during stimulation with ISO or 10 μm FK. Both myoepithelial and dark cells responded only to MCh (but not to FK) with a marked depolarization of the membrane potential due to activation of Cl, but not K, currents. We conclude that MCh stimulates Ca-dependent K and Cl currents, whereas ISO stimulates cAMP-dependent Cl currents in eccrine clear cells.     

Effects of ACh and adenosine mediated by Kir3.1 and Kir3.4 on ferret ventricular cells

The inotropic effects of ACh and adenosine on ferret ventricular cells were investigated with the action potential-clamp technique. Under current clamp, both agonists resulted in action potential shortening and a decrease in contraction. Under action potential clamp, both agonists failed to decrease contraction substantially. In the absence of agonist, application of the short action potential waveform (recorded previously in the presence of agonist) also resulted in a decrease in contraction. Under action potential clamp, application of ACh resulted in a Ba(2+)-sensitive outward current with the characteristics of muscarinic K+ current (I(K,ACh)); the presence of the muscarinic K+ channel was confirmed by PCR and immunocytochemistry. In the absence of agonist, on application of the short ACh action potential waveform, the decrease in contraction was accompanied by loss of the inward Na(+)/Ca(2+) exchange current (I(NaCa)). ACh also inhibited the background inward K+ current (I(K,1)). It is concluded that ACh activates I(K,ACh), inhibits I(K,1), and indirectly inhibits I(NaCa); this results in action potential shortening, decrease in contraction, and, as a result of the inhibition of I(K,1), minimum decrease in excitability.     

4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers

We have studied the effects of the potassium-blocking agent 4-aminopyridine (4-AP) on the action potential and membrane currents of the sheep cardiac Purkinje fiber. 4-AP slowed the rate of phase 1 repolarization and shifted the plateau of the action potential to less negative potentials. In the presence of 4-AP, the substitution of sodium methylsulfate or methanesulfonate for the NaCl of Tyrode's solution further slowed the rate of phase 1 repolarization, even though chloride replacement has no effect on the untreated preparation. In voltage clamp experiments, 4-AP rapidly and reversibly reduced the early peak of outward current that is seen when the Purkinje fiber membrane is voltage-clamped to potentials positive to -20 mV. In addition, 4-AP reduced the steady outward current seen at the end of clamp steps positive to -40 mV. 4-AP did not appear to change the slow inward current observed over the range of -60 to -40 mV, nor did it greatly change the current tails that have been used as a measure of the slow inward conductance at more positive potentials. 4-AP did not block the inward rectifying potassium currents, IK1 and IK2. A phasic outward current component that was insensitive to 4-AP was reduced by chloride replacement. We conclude that the early outward current has two components: a chloride-sensitive component plus a 4-AP-sensitive component. Since a portion of the steady-state current was sensitive to 4-AP, the early outward current either does not fully inactivate or 4-AP blocks a component of time-independent background current.     

Current clamp and voltage clamp study of the inhibitory action of DNP on membrane electrical properties of frog auricular heart muscle.

Current clamp studies showed that after 10 minutes under DNP 10(-4) M the membrane potential does not change significantly while an important shortening of the action potential duration and a diminished amplitude are observed. Voltage clamp studies have been performed on the slow inward and delayed outward currents. DNP 10(-4) M induced a marked decrease of the slow inward current related to the reduction in both conductance and driving force, and a decrease in the amplitude of the delayed current. The decrease of the slow inward current seems to be mainly responsible for the suppression of the plateau of the action potential during metabolic inhibition.     

Role of Na-Ca exchange in the action potential changes caused by drive in cardiac myocytes exposed to different Ca2+ loads.

The role of Na-Ca exchange in the membrane potential changes caused by repetitive activity ("drive") was studied in guinea pig single ventricular myocytes exposed to different [Ca2+]o. The following results were obtained. (i) In 5.4 mM [Ca2+]o, the action potentials (APs) gradually shortened during drive, and the outward current during a train of depolarizing voltage clamp steps gradually increased. (ii) The APs shortened more and were followed by a decaying voltage tail during drive in the presence of 5 mM caffeine; the outward current became larger and there was an inward tail current on repolarization during a train of depolarizing steps. (iii) These effects outlasted drive so that immediately after a train of APs, currents were already bigger and, after a train of steps, APs were already shorter. (iv) In 0.54 mM [Ca2+]o, the above effects were much smaller. (v) In high [Ca2+]o APs were shorter and outward currents larger than in low [Ca2+]o. (vi) In 10.8 mM [Ca2+]o, both outward and inward currents during long steps were exaggerated by prior drive, even with steps (+80 and +120 mV) at which there was no apparent inward current identifiable as I(Ca). (vii) In 0.54 mM [Ca2+]o, the time-dependent outward current was small and prior drive slightly increased it. (viii) During long steps, caffeine markedly increased outward and inward tail currents, and these effects were greatly decreased by low [Ca2+]o. (ix) After drive in the presence of caffeine, Ni2+ decreased the outward and inward tail currents. It is concluded that in the presence of high [Ca2+]o drive activates outward and inward Na-Ca exchange currents. During drive, the outward current participates in the plateau shortening and the inward tail current in the voltage tail after the action potential.     

Cesium blockade of delayed outward currents and electrically induced pacemaker activity in mammalian ventricular myocardium

The effects of Cs+, 5-25 mM, were studied in cat and guinea pig papillary muscles using voltage clamp and current clamp techniques. In solutions containing normal K+, the major effects of Cs+ were depolarization of the resting potential and reduction of the delayed outward current (ixl) between -80 and -20 mV. Both inward and outward portions of the isochronal current voltage relation (l-s clamps) were reduced by extracellular Cs+. This resulted in a substantial reduction of inward rectification and, by subtraction from the normal I-V relationship, the definition of a Cs+-sensitive component of current. Under current clamp conditions, 5-10 mM Cs+ produced a dose-dependent slowing of repetitive firing induced by depolarization. At higher concentrations (25 mM) the resting potential was depolarized and repetitive activity could not be induced by further depolarization. However, release of hyperpolarizing pulses was followed by prolonged bursts of repetitive action potentials, suggesting partial reversal of blockade or participation of another pacemaker process. The experimental results and a numerical simulation show that under readily attainable conditions, reduction in an outward pacemaker current may slow pacemaker activity.     

Slow inward and outward currents of rat ventricular fibers under anoxia

Voltage and current clamp experiments were performed on rat ventricular strips under anoxia. 1. Under the influence of anoxia the membrane depolarized by 5 to 10 mV and the action potential amplitude decreased by 15 mV. The plateau disappeared and the duration of the action potential was shortened. 2. The slow inward current was reduced by 50 to 80% and its reversal potential became more negative by about 31 mV. The conductance of the slow inward channel decreased by 26%. 3. The net outward current was slightly depressed.     

Electrophysiological response of rat atrial myocytes to acidosis

The effect of acidosis on the electrical activity of isolated rat atrial myocytes was investigated using the patch-clamp technique. Reducing the pH of the bathing solution from 7.4 to 6.5 shortened the action potential. Acidosis had no significant effect on transient outward or inward rectifier currents but increased steady-state outward current. This increase was still present, although reduced, when intracellular Ca(2+) was buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); BAPTA also inhibited acidosis-induced shortening of the action potential. Ni(2+) (5 mM) had no significant effect on the acidosis-induced shortening of the action potential. Acidosis also increased inward current at -80 mV and depolarized the resting membrane potential. Acidosis activated an inwardly rectifying Cl(-) current that was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which also inhibited the acidosis-induced depolarization of the resting membrane potential. It is concluded that an acidosis-induced increase in steady-state outward K(+) current underlies the shortening of the action potential and that an acidosis-induced increase in inwardly rectifying Cl(-) current underlies the depolarization of the resting membrane potential during acidosis.     

Physiological implications of K accumulation in heart muscle

K+-selective microelectrodes in conjugation with the voltage clamp technique were used to examine the voltage and time dependence of K+ efflux and accumulation in cardiac muscle. K+ efflux per action potential is about 10 to 30 pmoles/cm2 per sec. Accumulation of K+ in the paracellular space plays an important role in regulation of action potential duration, so that the [K+]o prior to generation of an action potential determines the duration of following action potential. This regulation is brought about by the shift of inward rectifying K+ current along the voltage axis, so at higher [K+]o there is more outward current at plateau potentials. Monitoring [K+]o after a period of rapid beating provides quantitative data regarding Na-pump activity. The data suggest the Na-pump is electrogenic, making it difficult to assess the extent of K+ accumulation from the measurements of resting potential alone. These studies indicate that changes in [K+]o not only reflect outward membrane currents and Na-pump activity, but also play an important physiological regulatory role in determining the duration of the action potential.     

Effect of reduced Na gradient on electrical activity in isolated bovine and feline ventricular myocytes

We have studied changes in electrical activity resulting from abrupt alterations of the Na gradient, using ventricular myocytes isolated from feline and bovine hearts. Attempting to investigate the ionic current possibly generated by Na-Ca exchange, we studied the effects of the changes in [Na]o in the presence of 20 mM CsCl to inhibit K currents. To facilitate the effect of Cs, we also used a K-free solution and a patch electrode filled with 150 mM cesium glutamate. The application of 20 mM Nao resulted in hyperpolarization and the action potential duration was reduced. Under voltage clamp, 20 of 45 mM Nao generated an outward current at all membrane potentials investigated. The initial part (100-200 ms) of this current was only partially inhibited by 5 mM NiCl2 which is known to fully block the Ca inward current. However, the outward current generated by the reduced [Na]o was fully inhibited by 20 mM MnCl2 (which presumably inhibits Na-Ca exchange). Our observations extend the work on multicellular cardiac preparations indicating that the outward current elicited by a sudden decrease in Na gradient could be generated by Na-Ca exchange. Although the characteristics of this outward current support certain concepts of the Na-Ca exchange in cardiac muscle, we cannot at present exclude a contribution of other membrane current(s).     

Single guard cell recordings in intact plants: light-induced hyperpolarization of the plasma membrane

Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit three physiological states. A minority of cells were classified as far-depolarized cells. These cells exhibited positive membrane potentials and were dominated by the activity of voltage-dependent anion channels. All other cells displayed both outward and inward rectifying K+-channel activity. These cells were either depolarized or hyperpolarized, with average membrane potentials of -41 mV (SD 16) and -112 mV (SD 19), respectively. Depolarized guard cells extrude K+ through outward rectifying channels, while K+ is taken up via inward rectifying channels in hyperpolarized cells. Upon a light/dark transition, guard cells that were hyperpolarized in the light switched to the depolarized state. The depolarization was accompanied by a 35 pA decrease in pump current and an increase in the conductance of inward rectifying channels. Both an increase in pump current and a decrease in the conductance of the inward rectifier were triggered by blue light, while red light was ineffective. From these studies we conclude that light modulates plasma membrane transport through large membrane potential changes, reversing the K+-efflux via outward rectifying channels to a K+-influx via inward rectifying channels.     

Electrophysiology of cardiac myocytes of Aplysia brasiliana

The electrical properties of Aplysia brasiliana myogenic heart were evaluated. Two distinct types of action potentials (APs) were recorded from intact hearts, an AP with a slow rising phase followed by a slow repolarizing phase and an AP with a 'fast' depolarizing phase followed by a plateau. Although these two APs differ in their rates of depolarization (2.2 x 0.3 V/s), both APs were abolished by the addition of Co2+, Mn2+ and nifedipine or by omitting Ca2+ from the external solution. These data suggest that a Ca2+ inward current is responsible for the generation of both types of APs. Two outward currents activated at -40 mV membrane potential were prominent in isolated cardiac myocytes: a fast activating, fast inactivating outward current similar to the A-type K+ current and a slow activating outward current with kinetics similar to the delayed rectifier K+ current were recorded under voltage clamp conditions. Based on the effects of 4-AP and TEA on the electrical properties of ventricular myocytes, we suggest that the fast kinetic outward current substantially attenuates the peak values of the APs and that the slow activating outward current is involved on membrane repolarization.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

Effects of conditioning polarization on the membrane ionic currents in rat myometrium

Summary Membrane ionic currents were measured in pregnant rat uterine smooth muscle under voltage clamp conditions by utilizing the double sucrose gap method, and the effects of conditioning pre-pulses on these currents were investigated. With depolarizing pulses, the early inward current was followed by a late outward current. Cobalt (1mm) abolished the inward current and did not affect the late outward currentper se, but produced changes in the current pattern, suggesting that the inward current overlaps with the initial part of the late outward current. After correction for this overlap, the inward current reached its maximum at about +10 mV and its reversal potential was estimated to be +62 mV. Tetraethylammonium (TEA) suppressed the outward currents and increased the apparent inward current. The increase in the inward current by TEA thus could be due to a suppression of the outward current. The reversal potential for the outward current was estimated to be –87 mV. Conditioning depolarization and hyperpolarization both produced a decrease in the inward current. Complete depolarization block occurred at a membrane potential of –20 mV. Conditioning hyperpolarization experiments in the presence of cobalt and/or TEA revealed that the decrease in the inward current caused by conditioning hyperpolarization was a result of an increase in the outward current overlapping with the inward current. It appears that a part of the potassium channel population is inactivated at the resting membrane potential and that this inactivation is removed by hyperpolarization.     

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.