Slow inactivation of L-type calcium current distorts the measurement of L- and T-type calcium current in Purkinje myocytes.

We have examined slow inactivation of L-type calcium current in canine Purkinje myocytes with the whole cell patch clamp technique. Slow inactivation is voltage dependent. It is negligible at -50 mV but can inactivate more than half of available iCaL at -10 mV. There are two major consequences of this slow inactivation. First, standard protocols for the measurement of T-type current can dramatically overestimate its contribution to total calcium current, and second, the position and steepness of the inactivation versus voltage curve for iCaL will depend on the method of measurement. Given the widespread attempts to identify calcium current components and characterize them biophysically, an important first step should be to determine the extent of slow inactivation of calcium current in each preparation.

Expression and characterization of a canine hippocampal inwardly rectifying K+ current in Xenopus oocytes.

1. An inwardly rectifying potassium current expressed in Xenopus laevis oocytes injected with canine hippocampal poly(A)+ RNA was investigated with the two-microelectrode voltage clamp technique. 2. Xenopus oocytes injected with canine hippocampal poly(A)+ RNA expressed a current activated by hyperpolarization. This current contained an instantaneous and a time-dependent component. Both components were inwardly rectifying and could be blocked by extracellular Cs+ or Ba2+. 3. The expressed current was carried mainly by K+. Its reversal potential measured in different [K+]os could be fitted by the Nernst equation with a slope of -50.7 per tenfold change in [K+]o. Extracellular Cl- and Na+ made minimal contributions to the current. 4. The activation of the expressed current depended on both voltage and [K+]o. Activation started near EK and the activation curve shifted along the voltage axis in parallel with EK when [K+]o was altered. 5. The activation time constants of the expressed current also depended on both voltage and [K+]o. The voltage dependence of the time constants was bell-shaped and the peak value was at a potential 30-50 mV more negative than EK. The voltage dependence of the time constants shifted along the voltage axis when EK was changed. 6. The poly(A)+ RNA extracted from canine hippocampus was fractionated in a 10-31% linear sucrose gradient. The size of the mRNA required to express the inwardly rectifying current was estimated to be around 4 kb. 7. In conclusion, the expressed current is an inwardly rectifying potassium current. The canine hippocampal mRNA should be an excellent source for expression-cloning of the inward rectifier channel.

Cooling/heating module for tissue chambers and solutions: theoretical considerations and practical design.

We provide a theoretical framework for the estimation of the performance of a modular cooling/heating device for tissue baths. The framework can be adapted to other designs using Peltier elements for cooling and heating. The design employs a Peltier as a heat pump and a flat heat pipe to transport heat to or from a 'remote' site. In the cooling mode heat from the hot side of the Peltier is removed by a heat sink cooled by a fan. The small cross section of the heat pipe permits cooling/heating of tissue chambers on microscope stages or in locations where it would be impractical to mount a Peltier element. The faces of the heat pipe can be used to pre-cool/heat solutions using a simple capillary heat exchanger.

Background K+ current in isolated canine cardiac Purkinje myocytes.

The current-voltage (I-V) relation of the background current, IK1, was studied in isolated canine cardiac Purkinje myocytes using the whole-cell, patch-clamp technique. Since Ba2+ and Cs+ block IK1, these cations were used to separate the I-V relation of IK1 from that of the whole cell. The I-V relation of IK1 was measured as the difference between the I-V relations of the cell in normal Tyrode (control solution) and in the presence of either Ba2+ (1 mM) or Cs+ (10 mM). Our results indicate that IK1 is an inwardly rectifying K+ current whose conductance depends on extracellular potassium concentration. In different [K+]0's the I-V relations of IK1 exhibit crossover. In addition the I-V relation of IK1 contains a region of negative slope (even when that of the whole cell does not). We also examined the relationship between the resting potential of the myocyte, Vm, and [K+]0 and found that it exhibits the characteristic anomalous behavior first reported in Purkinje strands (Weidmann, S., 1956, Elektrophysiologie der Herzmuskelfaser, Med. Verlag H. Huber), where lowering [K+]0 below 4 mM results in a depolarization.

Properties of an electrogenic sodium-potassium pump in isolated canine Purkinje myocytes.

1. Purkinje myocytes were isolated from canine Purkinje strands by collagenase exposure and gentle trituration. The myocytes were studied by a switched single-micro-electrode voltage-clamp technique at 37 degrees C in Tyrode solution containing 8 mM-K+ and 2 mM-Ca2+. 2. The dose-response relation for the cardiotonic steroid dihydroouabain (DHO) was obtained by measuring the change in membrane current caused by application of concentrations of 1-100 microM. The KD obtained in fourteen experiments was 3.7 +/- 1.1 microM (mean +/- S.E. of mean). 3. We employed 100 microM-DHO (a concentration more than 25-fold greater than the KD) to estimate the resting pump current (Ip) in the isolated myocytes. A value of 0.27 +/- 0.02 microA microF-1 (mean +/- S.E. of mean, n = 32) was obtained. 4. Myocytes were also exposed to K+-free solution for a period of 200 s. On return to K+-containing Tyrode solution there was a slowly decaying outward current. The time constant of decay of this pump current transient was 87 +/- 8 s (mean +/- S.E. of mean, n = 8). The integral beneath this transient was used to obtain a second estimate of the resting pump current. In four preparations where exposures in DHO and in K+-free solutions were employed the ratio Ip, DHO/Ip, K-free was 1.76 +/- 0.15 (mean +/- S.E. of mean). 5. From the magnitude of resting pump current, in the presence of total pump blockade the Na+ activity should rise at a rate of 1.3 mM min-1. 6. Reducing [K+]o from 8 to 1 mM reduced Ip by more than 40% initially. Ip then slowly increased over the next 30 min. These results suggest that the steady-state inward background current is not greatly altered by changes in [K+]o, and that [Na+]i rises to a new level. The changes in Ip obtained at early times following reduction of [K+]o to 1 or 0.5 mM (t less than 1.75 min) were used to estimate the Km for external K+; a value of 0.8 mM was obtained. 7. The results suggest that the properties of the Na+-K+ pump in isolated canine Purkinje myocytes are similar to those in canine Purkinje strands. This argues against major distortions of measured pump properties in the canine Purkinje strand and for the physiological state of the Na+-K+ pump in the isolated Purkinje myocyte.

Gating of delayed rectification in acutely isolated canine cardiac Purkinje myocytes. Evidence for a single voltage-gated conductance.

Studies of time-dependent, plateau outward current (delayed rectification) in the heart are complicated by the accumulation and depletion of K+ ions in intercellular clefts. To minimize this problem, we studied delayed rectification in acutely isolated (enzymic solution, gentle agitation) canine cardiac Purkinje myocytes using the single microelectrode voltage-clamp technique. We found a sigmoidal voltage-dependence for activation of outward plateau current, with maximal activation occurring at potentials near -10 mV. The activation and deactivation of plateau outward current was adequately described as the sum of a fast and slow exponential component. A comparison of the time course of activation of plateau outward current and the "envelope" of tail currents suggests that a single voltage-gated conductance with one open and two closed states can account for delayed rectification in Purkinje myocytes. These results differ from those previously obtained with intact sheep Purkinje fibers in which two time-dependent conductances were postulated to account for delayed rectification (Noble, D., and R. W. Tsien, 1969, J. Physiol. (Lond.), 200:205-231).

Versatile temperature controlled tissue bath for studies of isolated cells using an inverted microscope.

The design of a small, inexpensive temperature controlled bath (0.25 ml volume) for electrophysiological studies of isolated cells is described. The design creates a uniform temperature (+/- 1 degree C) from ambient temperature to approximately 37 degrees C over a bath chamber area of 0.6 X 1 cm for flow rates from zero to 2 ml/min. Access for microelectrodes and unobstructed preparation viewing with an inverted microscope is afforded by a finely stranded heater sandwiched between two glass cover slips and located beneath the bath chamber. Battery operation of the feedback temperature controller and solution pre-heater minimizes electrical interference.

Microprocessor controlled trituration device for the dissociation of cardiac and other tissues.

The construction of an inexpensive microprocessor controlled piston/cylinder used in the dissociation of canine cardiac Purkinje fibres is described. The microprocessor, interfaced to a linear actuator, controls the motion of a piston using the higher level language "Tiny BASIC" and two machine language routines. Pressure changes generated by the piston are transferred to the lumen of a pipette. Movement of enzymatically-treated tissues through the pipette orifice facilitates dissociation into single cells. With this device, specific agitation protocols for different quantities and types of tissues are possible.

Slow inactivation of a tetrodotoxin-sensitive current in canine cardiac Purkinje fibers.

We used the two-microelectrode voltage clamp technique and tetrodotoxin (TTX) to investigate the possible occurrence of slow inactivation of sodium channels in canine cardiac Purkinje fibers under physiologic conditions. The increase in net outward current during prolonged (5-20 s) step depolarizations (range -70 to +5 mV) following the application of TTX is time dependent, being maximal immediately following depolarization, and declining thereafter towards a steady value. To eliminate the possibility that this time-dependent current was due to inadequate voltage control of these multicellular preparations early during square clamp pulses, we also used slowly depolarizing voltage clamp ramps (range 5-100 mV/s) to ensure control of membrane potential. TTX-sensitive current also was observed with these voltage ramps; the time dependence of this current was demonstrated by the reduction of the peak current magnitude as the ramp speed was reduced. Reducing the holding potential within the voltage range of sodium channel inactivation also decreased the TTX-sensitive current observed with identical speed ramps. These results suggest that the TTX-sensitive time-dependent current is a direct measure of slow inactivation of canine cardiac sodium channels. This current may play an important role in modulating the action potential duration.

Secretion of acetylcholine in response to graded depolarization of motor nerve terminals.

1. Depolarizing current pulses were used to elicit transmitter secretion from motor nerve terminals in mouse phrenic nerve-diaphragm preparations exposed to solutions containing tetrodotoxin and 4-aminopyridine. 2. The amplitude of end-plate potentials (e.p.p.s.) elicited by depolarizing pulses was depressed for several milliseconds following a hyperpolarizing pulse, and potentiated following a subthreshold depolarizing pulse. Recovery followed an exponential time-course with time constant of 1.5 to 2.5 ms. 3. The time constant of nerve terminals, calculated from strength-duration curves obtained with depolarizing current pulses that elicited transmitter secretion, ranged from 1.5 to 2.56 ms. 4. It was concluded that the nerve terminal time constant would explain the effects of conditioning polarizations if there were a presynaptic "threshold potential" for end-plate potentials. The decrease in latency of e.p.p.s. as stimulus strength was increased could be explained in the same way. 5. The amplitude of e.p.p.s. increased as depolarizing pulses were lengthened from 2 to 10 ms. When a brief depolarizing pulse was superimposed on a longer subthreshold pulse, e.p.p. amplitude could not be related to the maximum depolarization of the presynaptic terminal. Transmitter secretion appeared to depend on both the level and duration of the depolarization.

Phasic secretion of acetylcholine at a mammalian neuromuscular junction.

1. The transient increase in secretion of quanta of acetylcholine (phasic secretion) produced by an action potential or brief depolarizing current pulse in mouse phrenic nerve terminals was examined. 2. Following an activating stimulus, there was a brief delay (minimum latency) followed by a sigmoidal increase in secretion which then decayed exponentially. 3. The minimum latency, rise time and rate of decay of phasic secretion, whether elicited by action potentials or electrotonic depolarization, were all extremely sensitive to temperature, with Q10s as high as 4 at temperatures of 5-15 degrees C. Arrhenius plots of results showed a change in slope with temperature, the change appearing most marked at 20-25 degrees C. 4. Phasic secretion in response to action potentials prolonged by inhibitors of K conductance (4-aminopyridine, uranyl, tetraethylammonium or Zn ions) showed an increase in minimum latency but no other change in time course. 5. Depolarizing pulses of varying width (0.2-2 msec) applied to nerve terminals (in the presence of tetrodotoxin and 4-aminopyridine) affected minimum latency, but had no great effect on the time course of phasic secretion. 6. Neither an increase in extracellular K ion concentration nor a decrease in pH had any effect on the time course of phasic secretion nor was any change produced by ethanol or octanol. 7. Variations in extracellular Ca concentration, substitution of Sr ions for Ca ions and repetitive stimulation, while producing changes in the magnitude of secretion, produced no change in the time course of phasic secretion.