Biomedical subjects

J L Kenyon

Publications and source records attributed to J L Kenyon.

49 records · Page 3Linked to original sources

PubMed1990

Transient potassium currents in avian sensory neurons.

1. We used the patch-clamp technique to study voltage-activated transient potassium currents in freshly dispersed and cultured chick dorsal root ganglion (DRG) cells. Whole-cell and cell-attached patch currents were recorded under conditions appropriate for recording potassium currents. 2. In whole-cell experiments, 100-ms depolarizations from normal resting potentials (-50 to -70 mV) elicited sustained outward currents that inactivated over a time scale of seconds. We attribute this behavior to a component of delayed rectifier current. After conditioning hyperpolarizations to potentials negative to -80 mV, depolarizations elicited transient outward current components that inactivated with time constants in the range of 8-26 ms. We attribute this behavior to a transient outward current component. 3. Conditioning hyperpolarizations increased the rate of activation of the net outward current implying that the removal of inactivation of the transient outward current allows it to contribute to early outward current during depolarizations from negative potentials. 4. Transient current was more prominent on the day the cells were dispersed and decreased with time in culture. 5. In cell-attached patches, single channels mediating outward currents were observed that were inactive at resting potentials but were active transiently during depolarizations to potentials positive to -30 mV. The probability of channels being open increased rapidly (peaking within approximately 6 ms) and then declined with a time constant in the range of 13-30 ms. With sodium as the main extracellular cation, single-channel conductances ranged from 18 to 32 pS. With potassium as the main extracellular cation, the single-channel conductance was approximately 43 pS, and the channel current reversed near 0 mV, as expected for a potassium current. 6. We conclude that the transient potassium channels mediate the component of transient outward current seen in the whole-cell experiments. This current is a relatively small component of the net current during depolarizations from normal resting potentials, but it can contribute significant outward current early in depolarizations from hyperpolarized potentials.

Animals↗

PubMed1987

Theory of the kinetic analysis of patch-clamp data.

This paper describes a theory of the kinetic analysis of patch-clamp data. We assume that channel gating is a Markov process that can be described by a model consisting of n kinetic states and n(n - 1) rate constants at each voltage, and that patch-clamp data describe the occupancy of x different conductance levels over time. In general, all the kinetic information in a set of patch-clamp data is found in either two-dimensional dwell time histograms describing the frequency of observation of sequential dwell times of durations tau 1 and tau 2 (Fredkin, D. R., M. Montal, and J. A. Rice, 1985, Proceedings of the Berkeley Conference in Honor of Jerzy Neyman and Jack Kiefer, vol. 1, 269-289) or in three-point joint probability functions describing the probability that a channel is in a given conductance at time t, and at time t + tau 1, and at time t + tau 1 + tau 2. For the special case of channels with a single open state plus multiple closed states, one-dimensional analyses provide all of the kinetic information. Stationary patch-clamp data have information that can be used to determine H rate constants, where H = n(n - 1) - G and G is the number of intraconductance rate constants. Thus, to calculate H rate constants, G rate constants must be fixed. In general there are multiple sets of G rate constants that can be fixed to allow the calculation of H rate constants although not every set of G rate constants will work. Arbitrary assignment of the G intraconductance rate constants equal to zero always provides a solution and the calculation of H rate constants. Nonstationary patch-clamp data have information for the determination of H rate constants at a reference voltage plus n(n - 1) rate constants at all test voltages. Thus, nonstationary data have extra information about the voltage dependencies of rate constants that can be used to rule out kinetic models that cannot be disqualified on the basis of stationary data.

Electric Conductivity↗

PubMed1987

Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers.

We have used the two-microelectrode voltage-clamp technique to investigate the components of membrane current that contribute to the formation of the early part of the plateau phase of the action potential of calf cardiac Purkinje fibers. 3,4-Diaminopyridine (50 microM) reduced the net transient outward current elicited by depolarizations to potentials positive to -30 mV but had no consistent effect on contraction. We attribute this effect to the blockade of a voltage-activated transient potassium current component. Ryanodine (1 microM), an inhibitor of sarcoplasmic reticulum calcium release and intracellular calcium oscillations in Purkinje fibers (Sutko, J.L., and J.L. Kenyon. 1983. Journal of General Physiology. 82:385-404), had complex effects on membrane currents as it abolished phasic contractions. At early times during a depolarization (5-30 ms), ryanodine reduced the net outward current. We attribute this effect to the loss of a component of calcium-activated potassium current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. At later times during a depolarization (50-200 ms), ryanodine increased the net outward current. This effect was not seen in low-sodium solutions and we could not observe a reversal potential over a voltage range of -100 to +75 mV. These data suggest that the effect of ryanodine on the late membrane current is attributable to the loss of sodium-calcium exchange current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. Neither effect of ryanodine was dependent on chloride ions, which suggests that chloride ions do not carry the ryanodine-sensitive current components. Strontium (2.7 mM replacing calcium) and caffeine (10 mM), two other treatments that interfere with sarcoplasmic reticulum function, had effects in common with ryanodine. This supports the hypothesis that the effects of ryanodine may be attributed to the inhibition of sarcoplasmic reticulum calcium release.

Action Potentials↗

PubMed1986

Effects of amrinone and milrinone on calcium influx into the myocardium.

We have examined the effects of milrinone on calcium influx into cardiac cells by testing its effects on three mechanisms of calcium entry: the slow inward calcium current, sodium-calcium exchange diffusion, and the passive calcium permeability of sarcolemmal membranes. Milrinone increased the magnitude of the slow inward calcium current in voltage-clamped calf cardiac Purkinje fiber. High concentrations of this agent also caused a small, inconsistent inhibition of calcium uptake mediated by sodium-calcium exchange in bovine cardiac sarcolemma membranes, but did not alter the time course of passive efflux of calcium from these vesicles. In conclusion, milrinone and its analogue amrinone appear to increase the entry of calcium into myocardial cells, primarily by increasing the influx of this cation during the slow inward calcium current. A small and sodium-calcium exchange may also influence calcium influx, particularly at high drug concentrations. The relationship existing between these effects and the inotropic responses produced by amrinone and milrinone is discussed.

Aminopyridines↗

PubMed1985

Phenothiazine suppression of transient depolarizations in rabbit ventricular cells.

We have examined the effects of trifluperazine and fluphenazine on action potentials and transient depolarizations of rabbit ventricular cells. Isolated myocytes were prepared by perfusing rabbit hearts with low calcium enzyme-containing solutions, and action potentials were stimulated at 1 Hz and recorded using patch-type pipettes. In normal saline, 10 microM trifluperazine or fluphenazine shifted the action potential plateau to more negative potentials and increased the rate of phase 2 repolarization. Transient diastolic depolarizations appeared in solutions containing 50 nM isoproterenol plus 1 microM strophanthidin. These transient depolarizations were abolished by the addition of 10 microM trifluperazine or fluphenazine. In addition, spontaneous transient depolarizations were occasionally observed, and these too were abolished by the phenothiazines. Because these compounds are potent inhibitors of calmodulin, these data raise the possibility that calcium-calmodulin-regulated processes are important in the generation of arrhythmogenic transient depolarizations.

Action Potentials↗

PubMed1985

Ryanodine: a modifier of sarcoplasmic reticulum calcium release in striated muscle.

We have proposed that the naturally occurring alkaloid ryanodine reduces the release of calcium from the sarcoplasmic reticulum (SR) in cardiac muscle cells. We summarize the data that support this hypothesis and discuss possible mechanisms for 1) the differences in sensitivity to ryanodine displayed by intact skeletal and cardiac muscle preparations vs. that of skinned cardiac cells and isolated SR membranes, 2) the ability of ryanodine to cause either an increase or a decrease in calcium accumulation by isolated skeletal muscle SR vesicles depending on experimental conditions, and 3) the positive inotropic effects produced by ryanodine in cardiac muscle preparations under certain experimental circumstances. In addition, we also show how ryanodine can be used to evaluate the contributions made by SR calcium release to cellular events in striated muscle.

Alkaloids↗

PubMed1984

Arterial pressure responses to increasing interstitial potassium in hindlimb muscle of dogs.

Static contraction of hindlimb skeletal muscle is known to increase reflexly arterial pressure and heart rate. Potassium is known to be released by the working muscle and is thought to activate the afferents responsible for the reflex cardiovascular responses to muscular contraction. However, it is not known whether potassium, at interstitial concentrations within the range observed during static contraction, is capable of stimulating these afferents. Thus we injected potassium into the gracilis artery of chloralose-anesthetized dogs while we measured interstitial potassium concentrations in the gracilis muscle with potassium-selective electrodes. In 16 dogs, we found that potassium injections, which increased interstitial potassium concentrations by 4.7 +/- 0.3 mM, increased mean arterial pressure by 18 +/- 3 mmHg and heart rate by 12 +/- 8 beats/min; cutting the obturator nerve abolished these increases. These heart rate and blood pressure responses were of short duration (20 +/- 7 s), even though interstitial potassium remained elevated for a period of several minutes. In 5 of the 16 dogs, static contraction of the gracilis muscle for 60 s increased interstitial potassium concentration by 4.3 +/- 0.3 mM. Our data are consistent with the hypothesis that potassium plays a role in causing the reflex cardiovascular responses to static muscular contraction.

Animals↗

PubMed1983

Ryanodine modification of cardiac muscle responses to potassium-free solutions. Evidence for inhibition of sarcoplasmic reticulum calcium release.

To test whether ryanodine blocks the release of calcium from the sarcoplasmic reticulum in cardiac muscle, we examined its effects on the aftercontractions and transient depolarizations or transient inward currents developed by guinea pig papillary muscles and voltage-clamped calf cardiac Purkinje fibers in potassium-free solutions. Ryanodine (0.1-1.0 microM) abolished or prevented aftercontractions and transient depolarizations by the papillary muscles without affecting any of the other sequelae of potassium removal. In the presence of 4.7 mM potassium and at a stimulation rate of 1 Hz, ryanodine had only a small variable effect on papillary muscle force development and action potential characteristics. In calf Purkinje fibers, ryanodine (1 nM-1 microM) completely blocked the aftercontractions and transient inward currents without altering the steady state current-voltage relationship. Ryanodine also abolished the twitch in potassium-free solutions, but it enhanced the tonic force during depolarizing voltage-clamp steps. This latter effect was dependent on the combination of ryanodine and potassium-free solutions. The slow inward current was not blocked by 1 microM ryanodine, but ryanodine did appear to abolish an outward current that remained in the presence of 0.5 mM 4-aminopyridine. Our observations are consistent with the hypothesis that ryanodine, by inhibiting the release of calcium from the sarcoplasmic reticulum, prevents the oscillations in intracellular calcium that activate the transient inward currents and aftercontractions associated with calcium overload states.

Action Potentials↗

PubMed1982

Delays in inactivation development and activation kinetics in myxicola giant axons.

Na inactivation was studied in Myxicola (two-pulse procedure, 6-ms gap between conditioning and test pulses). Inactivation developed with an initial delay (range 130-817 microseconds) followed by a simple exponential decline (time constant tau c). Delays (deviations from a simple exponential) are seen only for brief conditioning pulses were gNa is slightly activated. Hodgkin-Huxley kinetics with series resistance, Rs, predict deviations from a simple exponential only for conditioning pulses that substantially activate gNa. Reducing INa fivefold (Tris substitution) had no effect on either tau c or delay. Delay in not generated by Rs or by contamination from activation development. The slowest time constant in Na tails is approximately 1 ms (Goldman and Hahin, 1978) and the gap was 6 ms. Shortening the gap to 2 ms had no effect on either tau c or delay. Delay is a true property of the channel. Delay decreased with more positive conditioning potentials, and also decreased approximately proportionally with time to peak gNa during the conditioning pulse, as expected for sequentially coupled activation and inactivation. In a few cases the difference between Na current values for brief conditioning pulses and the tau c exponential could be measured. Difference values decayed exponentially with time constant tau m. The inactivation time course is described by a model that assumes a process with the kinetics of gNa activation as a precursor to inactivation.

Animals↗

PubMed1979

Internally perfused Myxicola giant axons showing long-term survival.

An improved method for internally perfusing the Myxicola giant axon based on removing the axoplasm by dispersing it in KCl-KF salt solutions is described. Proteolytic enzymes are not introduced. With this improved method perfused preparations show long-term stability of their electrical properties and the ability to generate action potentials for many hours. Mean initial values for resting membrane potential, action potential amplitude, and peak inward current were -68 mV, 118 mV, and 3.62 mA/cm2, respectively. Mean resting membrane resistance was 75% of that in intact axons. In one series of voltage clamp experiments, perfused preparations remained excitable for a mean period of 5 1/2 h, but this period could exceed 10 h. 4 min are needed for exchange of internal solutions. At least 50 mM KF is required both in the axoplasm liquefying solution and in the standard perfusate to obtain stable preparations.

Action Potentials↗

PubMed1979

Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers.

In voltage clamp studies of cardiac Purkinje fibers, a large early outward current is consistently observed during depolarizations to voltages more positive than -20 mV. After the outward peak of the current, the total membrane current declines slowly. Dudel et al. (1967. Pfluegers Arch. Eur. J. Physiol. 294:197--212) reduced the extracellular chloride concentration and found that the outward peak and the decline of the current were abolished. They concluded that the total membrane current at these voltages was largely determined by a time- and voltage-dependent change in the membrane chloride conductance. We reinvestigated the chloride sensitivity of this current, taking care to minimize possible sources of error. When the extracellular chloride concentration was reduced to 8.6% of control, the principal effect was a 20% decrease in the peak amplitude of the outward current. This implies that the membrane chloride conductance is not the major determinant of the total current at these voltages. The reversal potential of current tails obtained after a short conditioning depolarization was not changed by alterations in the extracellular chloride or potassium concentrations. We suspect that the tail currents contain both inward and outward components, and that the apparent reversal potential of the net tail current largely reflects the kinetics of the outward component, so that this experiment does not rule out potassium as a possible charge carrier. The possibility that potassium carries much of the early outward current was further investigated using tetraethylammonium, which blocks potassium currents in nerve and skeletal muscle. This drug substantially reduced the early outward current, which suggests that much of the early outward current is carried by potassium ions.

Action Potentials↗

PubMed1979

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.

Action Potentials↗

PubMed1977

Effects of low-chloride solutions on action potentials of sheep cardiac Purkinje fibers.

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode's solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode's solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode's solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.

Action Potentials↗