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In February 2014, a group of scientists convened as part of the University of California Davis Cardiovascular Symposium to bring together experimental and mathematical modelling perspectives and discuss points of consensus and controversy on the topic of sodium in the heart. This paper summarizes the topics of presentation and discussion from the symposium, with a focus on the role of aberrant sodium channels and abnormal sodium homeostasis in cardiac arrhythmias and pharmacotherapy from the subcellular scale to the whole heart. Two following papers focus on Na(+) channel structure, function and regulation, and Na(+)/Ca(2+) exchange and Na(+)/K(+) ATPase. The UC Davis Cardiovascular Symposium is a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The focus on Na(+) in the 2014 symposium stemmed from the multitude of recent studies that point to the importance of maintaining Na(+) homeostasis in the heart, as disruption of homeostatic processes are increasingly identified in cardiac disease states. Understanding how disruption in cardiac Na(+)-based processes leads to derangement in multiple cardiac components at the level of the cell and to then connect these perturbations to emergent behaviour in the heart to cause disease is a critical area of research. The ubiquity of disruption of Na(+) channels and Na(+) homeostasis in cardiac disorders of excitability and mechanics emphasizes the importance of a fundamental understanding of the associated mechanisms and disease processes to ultimately reveal new targets for human therapy., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

A Ca(2+) ion-dependent inactivation (CDI) of L-type Ca(2+) channels (LCC) is vital in limiting and shaping local Ca(2+) ion signalling in a variety of excitable cell types. However, under physiological conditions the unitary LCC properties that underlie macroscopic inactivation are unclear. Towards this end, we have probed the gating kinetics of individual cardiac LCCs recorded with a physiological Ca(2+) ion concentration (2 mM) permeating the channel, and in the absence of channel agonists. Upon depolarization the ensemble-averaged LCC current decayed with a fast and a slow exponential component. We analysed the unitary behaviour responsible for this biphasic decay by means of a novel kinetic dissection of LCC gating parameters. We found that inactivation was caused by a rapid decrease in the frequency of LCC reopening, and a slower decline in mean open time of the LCC. In contrast, with barium ions permeating the channel ensemble-averaged currents displayed only a single, slow exponential decay and little time dependence of the LCC open time. Our results demonstrate that the fast and slow phases of macroscopic inactivation reflect the distinct time courses for the decline in the frequency of LCC reopening and the open dwell time, both of which are modulated by Ca(2+) influx. Analysis of the evolution of CDI in individual LCC episodes was employed to examine the stochastic nature of the underlying molecular switch, and revealed that influx on the order of a thousand Ca(2+) ions may be sufficient to trigger CDI. This is the first study to characterize both the unitary kinetics and the stoichiometry of CDI of LCCs with a physiological Ca(2+) concentration. These novel findings may provide a basis for understanding the mechanisms regulating unitary LCC gating, which is a pivotal element in the local control of Ca(2+)-dependent signalling processes.

Ryanodine receptors (RyR2s) are ion channels in the sarcoplasmic reticulum (SR) that are responsible for Ca2+ release in rat ventricular myocytes. Localization of RyR2s is therefore crucial for our understanding of contraction and other Ca2+-dependent intracellular processes. Recent results (e.g. circular waves and Ca2+ sparks in perinuclear area) raised questions about the classical views of RyR2 distribution and organization within ventricular cells. A Ca2+ spark is a fluorescent signal reflecting the activation of a small group of RyR2s. Frequency and spatio-temporal characteristics of Ca2+ sparks depend on the state of cytoplasmic and intraluminal macromolecular complexes regulating cardiac RyR2 function. We employed electron microscopy, confocal imaging of spontaneous Ca2+ sparks and immunofluorescence to visualize the distribution of RyR2s in ventricular myocytes and to evaluate the local involvement of the macromolecular complexes in regulation of functional activity of the RyR2 group. An electron microscopy study revealed that the axial tubules of the transverse-axial tubular system probably do not have junctions with the network SR (nSR). The nSR was found to be wrapped around intermyofibrillar mitochondria and contained structures similar to feet of the junctional cleft. Treatment of ventricular myocytes with antibodies against RyR2 showed that in addition to the junctional SR, a small number of RyR2s can be localized at the middle of the sarcomere and in the zone of perinuclear mitochondria. Recordings of spontaneous Ca2+ sparks showed the existence of functional groups of RyR2s in these intracellular compartments. We found that within the sarcomere about 20% of Ca2+ sparks were not colocalized with the zone of the junctional or corbular SR (Z-line zone). The spatio-temporal characteristics of sparks found in the Z-line and A-band zones were very similar, whereas sparks from the zone of the perinuclear mitochondria were about 25% longer. Analysis of the initiation sites of Ca2+ sparks within the same junctional SR cluster suggested that 18-25 RyR2s are in the functional group producing a spark. Because of the similarity of the spatio-temporal characteristics of sarcomeric sparks and ultrastructural characteristics of nSR, we suggest that the functional groups of RyR2s in the middle of the sarcomere are macromolecular complexes of approximately 20 RyR2s with regulatory proteins. Our data allowed us to conclude that a significant number of functional RyR2s is located in the middle of the sarcomere and in the zone of perinuclear mitochondria. These RyR2s could contribute to excitation-contraction coupling, mitochondrial and nuclear signalling, and Ca2+-dependent gene regulation, but their existence raises many additional questions.

Excitation-contraction coupling in the heart depends on the positive feedback process of Ca2+-induced Ca2+ release (CICR). While CICR provides for robust triggering of Ca2+ sparks, the mechanisms underlying their termination remain unknown. At present, it is unclear how a cluster of Ca2+ release channels (ryanodine receptors or RyRs) can be made to turn off when their activity is sustained by the Ca2+ release itself. We use a novel experimental approach to investigate indirectly this issue by exploring restitution of Ca2+ sparks. We exploit the fact that ryanodine can bind, nearly irreversibly, to an RyR subunit (monomer) and increase the open probability of the homotetrameric channel. By applying low concentrations of ryanodine to rat ventricular myocytes, we observe repeated activations of individual Ca2+ spark sites. Examination of these repetitive Ca2+ sparks reveals that spark amplitude recovers with a time constant of 91 ms whereas the sigmoidal recovery of triggering probability lags behind amplitude recovery by approximately 80 ms. We conclude that restitution of Ca2+ sparks depends on local refilling of SR stores after depletion and may also depend on another time-dependent process such as recovery from inactivation or a slow conformational change after rebinding of Ca2+ to SR regulatory proteins.

The transcript of the Na+-Ca2+ exchanger gene NCX1 undergoes alternative splicing to produce tissue-specific isoforms. The cloned NCX1 isoforms were expressed in Xenopus oocytes and studied using a two-electrode voltage clamp method to measure Na+-Ca2+ exchanger activity. The cardiac isoform (NCX1.1) expressed in oocytes was less sensitive to depolarizing voltages and to activation by [Ca2+]i than the renal isoform (NCX1.3). The cardiac isoform of NCX1 is more sensitive to activation by protein kinase A (PKA) than the renal isoform which may be explained by preferential phosphorylation. The cardiac isoform of NCX1 is phosphorylated to a greater extent than the renal isoform. The action of PKA phosphorylation which increases the activity of the cardiac isoform of the Na+-Ca2+ exchanger in oocytes was confirmed in adult rat ventricular cardiomyocytes by measuring Na+-dependent Ca2+ flux. We conclude that alternative splicing of NCX1 confers distinct functional characteristics to tissue-specific isoforms of the Na+-Ca2+ exchanger.

1. We examined [Ca2+]i and L-type Ca2+ channel current (ICa) in single cardiac myocytes to determine how the intracellular protein phospholamban (PLB) influences excitation-contraction (E-C) coupling in heart. Wild type (WT) and PLB-deficient (KO) mice were used. Cells were patch clamped in whole-cell mode while [Ca2+]i was imaged simultaneously using the Ca2+ indicator fluo-3 and a confocal microscope. 2. Although ICa was similar in magnitude, the decay of ICa was faster in KO than in WT cells and the [Ca2+]i transient was larger and decayed faster. Furthermore, the E-C coupling 'gain' (measured as delta[Ca2+]i/ICa) was larger in KO cells than in WT cells. 3. Spontaneous Ca2+ sparks were three times more frequent and larger in KO cells than in WT myocytes but, surprisingly, the time constants of decay were similar. 4. SR Ca2+ content was significantly greater in KO than in WT cells. When the SR Ca2+ content in KO cells was reduced to that in WT cells, Ca2+ sparks in these 'modified' (KO') cells decayed faster. E-C coupling gain, [Ca2+]i transient amplitude and the kinetics of decay of ICa were similar in KO' and WT cells. 5. We conclude that SR Ca2+ content influences (1) ICa, (2) the amplitude and kinetics of Ca2+ sparks and [Ca2+]i transients, (3) the sensitivity of the RyRs to triggering by [Ca2+]i, (4) the amount of Ca2+ released, (5) the magnitude of the E-C coupling 'gain' function, and (6) the rate of Ca2+ re-uptake by the SR Ca(2+)-ATPase. In KO cells, the larger [Ca2+]i transients and Ca2+ sparks speed up ICa inactivation. Finally, we conclude that PLB plays an important regulatory role in E-C coupling by modulating SR Ca(2+)-ATPase activity, which establishes the SR Ca2+ content and consequently influences the characteristics of local and global Ca2+ signalling.

1. We examined the effects of the immunosupressant drug FK506 on excitation-contraction coupling in isolated rat ventricular myocytes. [Ca2+]i transients were recorded using the intracellular Ca2+ indicators fluo-3 and indo-1 while action potentials (APs) or membrane currents were recorded using patch-type microelectrodes in the whole cell mode. 2. FK506 (25 microM) rapidly and reversibly increased the magnitude of the [Ca2+]i transient in intact cells without changing resting [Ca2+]i or the kinetics of the [Ca2+]i transient, a finding consistent with previous reports that investigated the actions of FK506 on the sarcoplasmic reticulum Ca2+ release channel. 3. The 36% increase in the [Ca2+]i transient produced by FK506 was accompanied by a 293% increase in AP duration (by 293%). Importantly, the addition of FK506 had no effect on the [Ca2+]i transient when the depolarizing duration was controlled in voltage clamp experiments. The increased AP duration could be explained by a marked inward shift in the net membrane current that was observed in these experiments. 4. The net inward current change was not directly responsible for a change in Ca2+ influx, since no change in L-type Ca2+ current (ICa) was observed. Instead, FK506 inhibited both the transient outward K+ current (Ito) and the delayed rectifier K+ current (IK). 5. We conclude that FK506 increases the [Ca2+]i transient during normal contractions by an indirect action: it prolongs the action potential. This action does not appear to depend on the established action of FK506 on the ryanodine receptor. Instead, the inhibition of outward K+ currents prolongs the AP which secondarily increases Ca2+ influx and/or decreases Ca2+ efflux.

1. Brief localized elevations in myoplasmic [Ca2+] (Ca2+ sparks) in individual sarcomeres of voltage-clamped frog skeletal muscle fibres were examined by laser scanning confocal microscopy. 2. Fibres held at 0 mV were briefly repolarized to -90 mV (repriming pulse) to restore only a small fraction of sarcoplasmic reticulum (SR) calcium release. Subsequent depolarization to 0 mV (test pulse) caused the appearance of small numbers of Ca2+ sparks at different sarcomeres from pulse to pulse. Increasing the repriming time resulted in an increase in the frequency of occurrence of the Ca2+ sparks. 3. The amplitude and spatio-temporal extent of the Ca2+ sparks were independent of the repriming time and test pulse voltage. Ca2+ sparks recorded during small depolarizations of fibres held at -90 mV had a similar amplitude and spatio-temporal extent as those recorded after brief repriming of the same fibre held at 0 mV. 4. We conclude that stereotyped Ca2+ sparks underlie calcium release at all voltages and all extents of repriming. The amplitude of Ca2+ release is thus graded by the frequency but not by the amplitude or spatio-temporal extent of the individual SR Ca2+ release events.

1. We sought to evaluate the contribution of the sarcoplasmic reticulum (SR) Ca2+ pump (vs. diffusion) to the kinetics of [Ca24]i decline during Ca2+ sparks, which are due to spontaneous local SR Ca2+ release, in isolated rat ventricular myocytes measured using fluo-3 and laser scanning confocal microscopy. 2. Resting Ca2+ sparks were compared before (control) and after the SR Ca2(+)-ATPase was either completely blocked by 5 microM thapsigargin (TG) or stimulated by isoprenaline. Na(+)-Ca2+ exchange was blocked using Na(+)-free, Ca(2+)-free solution (0 Na+, O Ca2+) and conditions were arranged so that the SR Ca2+ content was the same under all conditions when Ca2+ sparks were measured. 3. The control Ca2+ spark amplitude (281 +/- 13 nM) was not changed by TG (270 +/- 21 nM) or isoprenaline (302 +/- 10 nM). However, the time constant of [Ca2+]i decline was significantly slower in the presence of TG (29.3 +/- 4.3 ms) compared with control (21.6 +/- 1.5 ms) and faster with isoprenaline (14.5 +/- 0.9 ms), but in all cases was much faster than the global [Ca2+]i decline during a control twitch (177 +/- 10 ms). 4. The spatial spread of Ca2+ during the Ca2+ spark was also influenced by the SR Ca2+ pump. The apparent 'space constant' of the Ca2+ sparks was longest when the SR Ca2+ pump was blocked, intermediate in control and shortest with isoprenaline. 5. We conclude that while Ca2+ diffusion from the source of Ca2+ release is the dominant process in local [Ca2+]i decline during the Ca2+ spark, Ca2+ transport by the SR contributes significantly to both the kinetics and spatial distribution of [Ca2+]i during the Ca2+ spark.

1. The effects of the serine/threonine protein phosphatases (PP) type 1 and 2A on L-type Ca2+ current (ICa) and the intracellular [Ca2+]i transient were examined in rat ventricular myocytes. ICa was measured under voltage clamp using patch-type microelectrodes in the whole-cell mode with the cells in a steady state of sarcoplasmic reticulum (SR) Ca2+ loading. [Ca2+]i transients were measured simultaneously using the fluorescent Ca2+ indicator indo-1 (50 microM) which was added to the pipette filling solution along with PP-1 or PP-2A (4 units ml-1). 2. PP-1 had no effect on the ICa-V relationship but decreased the [Ca2+]i-voltage relationship (by 43% at 0 mV). PP-2A decreased both ICa-V (by 26% at 0 mV) and the [Ca2+]i transient-voltage (by 65% at 0 mV). Excitation-contraction coupling gain, defined as (delta [Ca2+]i/ICa), was decreased to 43% of control by PP-1 and to 29% of control by PP-2A at-28 mV. 3. Diastolic [Ca2+]i (i.e.[Ca2+]i measured immediately before each voltage clamp pulse) was not altered by PP-1 or PP-2A and neither phosphatase changed steady-state SR Ca2+ content, as measured with caffeine. 4. We conclude that the reduced [Ca2+]i transient following the application of PP-1 was due to reduced SR Ca2+ release channel activity. The effects of PP-2A, while more broadly based, were still consistent with a decrease in SR Ca2+ release channel activity. 5. Our experiments, combined with recent experiments by others, suggest that the basal state of contractility in heart is dynamically regulated by dephosphorylation and phosphorylation of the SR Ca2+ release channel.

1. The regulation of the cardiac Ca2+ release channel-ryanodine receptor (RyR) by exogenous acid phosphatase (AcPh) and purified Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) was studied in swine and rabbit sarcoplasmic reticulum (SR) vesicles using [3H]ryanodine binding and planar bilayer reconstitution experiments. 2. Addition of AcPh (1-20 U ml-1) to a standard incubation medium increased [3H]ryanodine binding in a Ca(2+)-dependent manner. Stimulation was only readily apparent in media containing micromolar Ca2+ concentrations. 3. Scatchard analysis of [3H]ryanodine binding curves revealed that AcPh enhanced binding by increasing the affinity of the receptor for [3H]ryanodine without recruiting additional receptor sites (Kd, 9.8 +/- 0.85 and 3.9 +/- 0.65 nM; Bmax (the maximal receptor density), 1.45 +/- 0.14 and 1.47 +/- 0.12 pmol mg-1 for control and AcPh, respectively). The failure of AcPh to increase Bmax suggested that the number of receptors that were 'dormant' due to phosphorylation in the SR preparation was very small. 4. At the single channel level, AcPh increased the open probability (Po) of RyR channels by increasing the opening rate and inducing the appearance of a longer open state while having no effect on single channel conductance. Thus AcPh acted directly on RyR channels or a closely associated regulatory protein. 5. CaMKII decreased both [3H]ryanodine binding and Po of RyRs when added to medium supplemented with micromolar levels of Ca2+ and calmodulin (CaM). Addition of a synthetic peptide inhibitor of CaMKII, or replacement of ATP with the non-hydrolysable ATP analogue adenylyl[beta, gamma-methylene]-diphosphate (AMP-PCP), prevented CaMKII inhibition of RyRs, suggesting that CaMKII acted specifically through a phosphorylation mechanism. 6. The inhibition of RyR channel activity by CaMKII was reversed by the addition of AcPh. Thus we showed that an in vitro phosphorylation-dephosphorylation mechanism effectively regulates RyRs. 7. The results suggest that intracellular signalling pathways that lead to activation of CaMKII may reduce efflux of Ca2+ from the SR by inhibition of RyR channel activity. The Ca2+ dependence of CaMKII inhibition suggests that the role of the phosphorylation mechanism is to modulate the RyR response to Ca2+.

1. Na(+)-Ca2+ exchange current was activated in giant excised patches of guinea-pig cardiac sarcolemma by raising the intracellular sodium concentration ([Na+]i). When the pHi was simultaneously acidified to 6.4, the current was transient, dropping by 80% in 30 s. 2. Pre-exposure to a pHi of 6.4 for 15 s reduced the peak Na(+)-Ca2+ exchange current without altering the decay rate or steady-state current. Recovery from proton inhibition was seen when [Na+]i was removed for 9 s. 3. A mathematical model of Na(+)-Ca2+ exchange function reproduced the experimental results. In addition, two model-dependent predictions were seen experimentally. (i) [Na+]i-dependent 'inactivation' of Na(+)-Ca2+ exchange may arise from pHi effects. We observed experimentally that pre-exposure to acidic pHi can remove the transient current component attributed to [Na+]i-dependent 'inactivation'. (ii) self-exchange should be inhibited by acidification. This has been observed by other investigators. 4. We have hypothesized that there are two components to inhibition of the Na(+)-Ca2+ exchanger by intracellular protons, and that one is enhanced by increased [Na+]i (Doering & Lederer, 1993b). This hypothesis is supported by the data presented here and by a model of Na(+)-Ca2+ exchange behaviour in which binding of intracellular sodium to the exchanger enhances the affinity of the exchanger for inhibitory intracellular protons.

1. We recorded cardiac sodium-calcium exchange current (INa-Ca) in giant excised membrane patches obtained from cardiac myocytes of the adult guinea-pig. 2. Rapid changes in ion concentrations on the cytoplasmic side of the excised membrane patch were produced using a modified oil-gate bath. 3. Sodium-calcium exchange current was activated by step increases in sodium concentration on the cytoplasmic side of the membrane ([Na+]i), which led to an increase in outward INa-Ca to a new steady-state level. The [Na+]i required to half-maximally activate the sodium-calcium exchange current (K1/2) was 21 mM. 4. Step increases in cytoplasmic calcium concentration ([Ca2+]i) stimulated the [Na+]i-activated INa-Ca up to 1 microM [Ca2+]i, then inhibited the exchange current at very high [Ca2+]i (1 mM). 5. A step decrease in cytoplasmic pH from 7.2 to 6.4 (increase in [H+]i) produced a biphasic but monotonic decrease in INa-Ca. Alkalinization of cytoplasmic pH from 7.2 to 8.0 caused a large, biphasic increase in INa-Ca. 6. When INa-Ca was activated by a step increase in [Na+]i and [H+]i was simultaneously increased, the outward current rose to a peak and then declined to a low steady level. The peak current seen was always less than the maximum current produced by an identical elevation of [Na+]i at constant pHi. This reduction in peak outward current reflected a rapid 'primary' inhibition of the sodium-calcium exchange by protons. The decay of the sodium-calcium exchange current following the peak was slow and corresponded to the time course of the onset of a 'secondary' proton block. 7. Rapid primary inhibition of the sodium-calcium exchanger could also be produced by cytoplasmic acidification in the absence of cytoplasmic sodium. The primary blockade was revealed when a subsequent increase in [Na+]i activated INa-Ca and a smaller peak outward current was observed. Secondary inhibition of the sodium-calcium exchanger was not, however, produced by cytoplasmic acidification in the absence of cytoplasmic sodium. Regardless of the duration of exposure to elevated [H+]i, the 'secondary' block by protons was still seen on activation of INa-Ca by increased [Na+]i as a gradual reduction of outward current amplitude. 8. Treatment of the sodium-calcium exchanger with the proteolytic enzyme alpha-chymotrypsin largely removed its sensitivity to protons. 9. We conclude that the action of alpha-chymotrypsin on the monomeric sodium-calcium exchange protein is in part to remove a proton-sensitive regulatory component(s) or render the regulation ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)

1. L-type Ca2+ currents and Ca2+ channel gating currents were studied in isolated guinea-pig ventricular heart cells using the whole-cell patch-clamp technique, in order to investigate the mechanism of Ca(2+)-dependent inactivation. The effect of altering the intracellular Ca2+ concentration ([Ca2+]i) on these currents was studied through photorelease of intracellular Ca2+ ions using the photolabile Ca2+ chelators DM-nitrophen and nitr-5. 2. We found that step increases in [Ca2+]i produced by photorelease could either increase or decrease the L-type Ca2+ current. Specifically, Ca2+ photorelease from DM-nitrophen almost exclusively caused inactivation of the Ca2+ current. In contrast, Ca2+ photorelease from nitr-5 had a biphasic effect: a small, rapid inactivation of the Ca2+ current was followed by a slow potentiation. These two Ca(2+)-dependent processes seemed to differ in their Ca2+ dependence, as small Ca2+ photoreleases elicited potentiation without a preceding inactivation, whereas larger photoreleases elicited both inactivation and potentiation. 3. The mechanism of the Ca(2+)-dependent inactivation of Ca2+ channels was explored by comparing the effects of voltage and photoreleased Ca2+ on the Ca2+ current and the Ca2+ channel gating current. Voltage was found to reduce both the Ca2+ current and the gating current proportionally. However, Ca2+ photorelease from intracellular DM-nitrophen inactivated the Ca2+ current without having any effect on the gating current. 4. The dephosphorylation hypothesis for Ca(2+)-dependent inactivation was tested by applying isoprenaline to the cells before eliciting a maximal rise of [Ca2+]i (maximal flash intensity, zero external [Na+]i). Isoprenaline could completely prevent Ca(2+)-dependent inactivation under these conditions, even when [Ca2+]i rose so high as to cause an irreversible contracture of the cell. 5. We concluded from these experiments that voltage and Ca2+ ions inactivate the L-type Ca2+ channel through separate, independent mechanisms. In addition, we found that Ca(2+)-dependent inactivation does not result in the immobilization of gating charge, and apparently closes the Ca2+ permeation pathway through a mechanism that does not involve the voltage-sensing region of the channel. Furthermore, we found that Ca(2+)-dependent inactivation is entirely sensitive to beta-adrenergic stimulation. These facts suggest that either Ca(2+)-dependent inactivation results from Ca(2+)-dependent dephosphorylation of the Ca2+ channel, or that Ca(2+)-dependent inactivation is modulated by protein kinase A.

1. In isolated rat heart ventricular myocytes exposed to 2 mM-cyanide in the presence of 10 mM-2-deoxyglucose (complete metabolic blockade), there is a time-dependent increase in ATP-sensitive potassium (KATP) channel activity. The increase in KATP channel activity accompanies the decline of twitch amplitude. Channel activation and decline of the twitch amplitude precede the development of a 'rigor' contracture. 2. We measured KATP channel activity in permeabilized cells using the open-cell attached (O-C-A) patch configuration (by establishing a cell-attached patch and then permeabilizing the cell by exposure to saponin). The apparent ATP dependence of KATP channel activity could be described by a sigmoid curve with ki, ATP (ATP concentration required for half-maximum inhibition of channel activity) = 122 microM and H (Hill coefficient) = 1.225. 3. In the O-C-A patch configuration, 10 mM-creatine phosphate (CrP) decreased the apparent ki, ATP from 122 microM to about 10 microM, and the maximal activity (in zero ATP) was decreased to about 30% of the maximal activity in the absence of CrP. 4. In isolated inside-out (I-O) patches, ATP inhibited KATP channel activity at much lower [ATP] than in the O-C-A patch configuration (ki, ATP = 25 microM, H = 2). CrP was without effect on I-O patches. 5. These results are consistent with the hypothesis that the difference in the ATP dependence of KATP channel activity in the O-C-A and I-O patch configurations arises because of ATP consumption in the O-C-A patch configuration. The results suggest that hydrolysis of ATP to ADP by endogenous ATPases leads to the development of gradients of [ATP] and [ADP] between the bath and the 'inside' of the open cell. By re-phosphorylating ADP, CrP is able to dissipate these gradients, revealing the 'true' ATP dependence of channel activity, which is the same as that in the I-O patch configuration. 6. In order to estimate the contribution of KATP channel activity to the rat cardiac action potential at different [ATP] we have made the following measurements. Using electrodes of resistance 2-8 M omega the density of KATP channels was 10.3 +/- 0.1 channels per patch (n = 162).(ABSTRACT TRUNCATED AT 400 WORDS)

1. Voltage-clamp experiments were carried out in calf Purkinje fibres to determine the basis of transient depolarizations (TDs) associated with digitalis-induced arrhythmias. 2. Under the influence of strophanthidin, depolarizing clamp pulses were followed by a transient inward current (TI) which was small or absent in untreated preparations. The TI also appeared in the wake of a train of action potentials. 8. The TI can help generate spontaneous depolarizations in preparations showing the low voltage oscillations which often occur with advanced digitalis toxicity. 8. The TI can help generate spontaneous depolarizations in preparations showing the 'low voltage oscillation' which often occur with advanced digitais toxicity. It was designated TI because its magnitude and timing were appropriate to account for the TD. 3. Longitudinal voltage non-uniformity during the TI was determined with two voltage-recording micro-electrodes. Although the non-uniformity was not severe, the TI wave form was observed when the voltage difference signal was used to measure membrane current density. 4. Over the diastolic range of potential, the strophanthidin-induced TI appeared superimposed upon the normal pace-maker mechanism, the decay of a potassium current, iK2. The TI could be dissociated from iK2, however, by means of its unusual kinetic properties. 5. TIs could also be recorded at holding potentials positive to -55 mV, i.e. outside the range where iK2 deactivation occurs. 6. The TI amplitude showed a slow and strongly sigmoid dependence on the duration of the preceding depolarizing pulse. Stronger depolarizing reduced the TI amplitude, while slowing and exaggerating the sigmoid time-dependence. 7. Two clamp pulses in close succession gave additive effects in evoking a subsequent TI. This finding and the sigmoid time-dependence fit with previous observations that TDs are most prominent following a series of closely spaced action potentials.

1. Calcium current (ICa) was recorded in single rat heart cells at two periods during development: (1) at 2-7 days post-partum (neonatal), and (2) at 6-8 weeks (adult). 2. We measured both transient and steady-state components of ICa and could describe ICa in terms of the steady-state activation (d infinity) and inactivation (f infinity) parameters, the channel reversal potential (Echannel) and a relative conductance parameter, gr. 3. In adult single cells, the application of ryanodine (10 microM), an agent known to alter the function of the sarcoplasmic reticulum (SR), abolished contraction rapidly and increased ICa. Ryanodine also produced a 13 mV shift in f infinity towards more positive potentials and altered its slope, while producing a small increase in gr but no effect on d infinity. In neonatal single cells, ryanodine (10 microM) had no significant effect on contraction, ICa, d infinity, f infinity, or gr. Caffeine (10 mM), a less specific agent widely used to investigate sarcoplasmic reticulum function, had actions similar to those of ryanodine. 4. In adult myocytes, when EGTA (10 or 20 mM) or bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 10 mM) were included in the pipette solution, contractions were rapidly abolished, while a small (4 mV) shift of f infinity to more positive potentials was seen. A large additional shift of f infinity was observed when ryanodine (10 microM) was added to the superfusion solution in the continued presence of EGTA or BAPTA. The alterations of ICa in EGTA (or BAPTA) plus ryanodine were the same as those seen in ryanodine alone. In neonatal cells, in contrast, when EGTA or BAPTA were included in the pipette solution we observed only a small effect on f infinity and the application of ryanodine had no effect. 5. Electron micrographs of our preparations show that the dissociated adult cells have sharp sarcolemmal borders, fully developed sarcomeres with T-tubules and sarcoplasmic reticulum membranes. In contrast, the neonatal cells that we use have few of these intracellular structures. Our observations in these preparations are consistent with the work of others (e.g. Penefsky, 1974; Hirakow & Gotoh, 1975; Ishikawa & Yamada, 1975; Legato, 1975; Hoerter, Mazet & Vassort, 1981). 6. Our data suggest that fully developed sarcoplasmic reticulum in rat heart cells can affect ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Mammalian Purkinje fibres and ventricular muscle are significantly affected by exposure to low K solutions (Eisner & Lederer, 1979). Such exposure produces two classes of effects. ;Early' effects, developing over tens of seconds include (in ventricular muscle) a more negative resting potential and a lengthening of the action potential. In Purkinje fibres the principal ;early' effect is a decrease in slope conductance. ;Late' effects develop over minutes. In ventricular muscle such effects include a shortening of the action potential, an increase in twitch and tonic tension, and the development of transient depolarizations and aftercontractions. The late effects in Purkinje fibres are the increase in twitch tension and voltage dependent tonic tension, the development of transient depolarizations and the underlying oscillatory transient inward currents, the appearance of aftercontractions accompanying the transient depolarizations or transient inward currents, and the development of a slow ;creep' in both current and tension.2. The rate of development of early effects is consistent with the time taken to change the bathing K concentration, K(o). However the time course of onset of the late effects (including the positive inotropy) is too slow to be explained by the time taken to change K(o).3. The late effects of reducing K(o) from 4 to 0 mM can be prevented by including appropriate concentrations of the activator cations of the Na pump (Tl, Rb, Cs, NH(4) or Li) in the 0 K(o) bathing solution. Similarly the late effects of 0 K(o), once established, can be reversed by adding these cations to the 0 K(o) superfusing solution.4. The order of potency of these cations to remove the effects of 0 K(o) was found to be: Tl > K approximately Rb > NH(4) approximately Cs > Li. This is similar to the order of efficacy shown to activate the external K site of the Na pump in nerve and other tissue (Rang & Ritchie, 1968).5. Strophanthidin (10(-5)M) produces qualitatively similar electrical and mechanical effects as those seen in 0 K(o). However, the effects of strophanthidin are not reversed by the activator cations. Furthermore, in the presence of strophanthidin (10(-5)M), these cations do not reverse the effects of 0 K(o).6. In voltage-clamped Purkinje fibres, returning to a solution of 4 mM-K(o) after exposure to 0 K(o) produces a transient increase in outward current. Similarly, during exposure to 0 K(o) the addition of activator cations also produces a transient increase of outward current. The ability of these ions to develop this outward transient current is correlated with their ability to remove the inotropic and arrhythmogenic effects of 0 K(o).7. The transient outward current produced by activator cations in 0 K(o) is blocked by strophanthidin (10(-5)M). We conclude that the outward current transient reflects activation of an electrogenic Na pump. Furthermore, we find that, as in other tissues, the activator cations can substitute for K(o) in activating the Na-K pump.8. The reversal of inotropic and arrhythmogenic effects of 0 K(o) by activator cations indicates that such effects result from Na pump blockade. No additional explanation (e.g. Ca/K exchange) need be invoked.

Tension was measured in voltage clamped sheep cardiac Purkinje fibres while simultaneously measuring the intracellular Na activity (aiNa) with a recessed-tip, Na-selective micro-electrode. Inhibiting the Na-K pump either by exposing the preparation to a K-free solution or by adding the cardioactive steroid strophanthidin increased both aiNa and twitch tension and resulted in the development of tonic tension, after-contractions and a transient inward current (ITI). The increase of twitch tension was present at lower aiNa than that required to produce the other phenomena. The relationship between the magnitude of the twitch tension and aiNa was always non-linear. Twitch tension increased steeply with aiNa at first but the relationship flattened off at higher aiNa and tension eventually decreased. Over the steep range, the relationship between tension and aiNa could be represented as: twitch tension = b (aiNa)y where y had a mean value of 3.2. Changing membrane potential or [Ca2+]o changed b but had little effect on y. Mn (2 mmol/l) greatly decreased twitch tension but, at least initially, had little effect on tonic tension. The steep relationship between twitch tension and aiNa was seen, irrespective of whether the Na-K pump was inhibited either by exposure to K-free solution or to strophanthidin and whether the relationship was measured either when aiNa was increasing or after it had reached a steady state. The steep dependence of twitch tension on aiNa observed in the present work means that manoeuvres which produce even small changes of aiNa will have significant effects on contraction.

1. Intracellular Na activity (aiNa) was measured in sheep cardiac Purkinje fibres using a recessed-tip Na+-sensitive micro-electrode. The membrane potentials was controlled with a two-micro-electrode voltage clamp. Tension was measured simultaneously. 2. Removing external K produced a rise of aiNa and both twitch and tonic tension. On adding 4-10 mM-[Rb]0 to reactivate the Na-K pump aiNa and tension declined. An electrogenic Na pump current transient accompanied the fall of aiNa. 3. The half-time of decay of the electrogenic Na pump current transient was similar to that of aiNa, (mean tNa0.5/tI0.5 = 0.97 +/- 0.03 (S.E.M.; n = 28)). Following the re-activation of the Na-K pump, the electrogenic Na pump current transient was linearly related to aiNa. 4. The duration of exposure to K-free, Rb-free solutions was varied to change the level of aiNa. On subsequently re-activating the Na-K pump with 10 mM-[Rb]0, the ratio of the charge extruded to the total change of aiNa was constant. It is concluded that the fraction of Na extruded electrogenically is unaffected by changes of aiNa. About 26% of the total Na extrusion appeared as charge transfer. 5. The relationship between tonic tension and aiNa was usually different during Na-K pump inhibition in a K-free, Rb-free solution compared with the relationship during Na-K pump re-activation. In general, a given aiNa was associated with a greater level of tonic tension during Na-K pump inhibition compared with that during pump re-activation. A similar hysteresis was often seen between twitch tension and aiNa.

1. Twitch shortening of isolated rat ventricular myocytes was measured on exposure to complete metabolic blockade (2 mM-cyanide in the presence of 10 mM-2-deoxyglucose). Under these conditions twitch shortening declines to undetectable levels over 1-15 min. This 'early' contractile failure is followed by the development of a maintained contracture. 2. Contractures induced by caffeine (20 mM) were similar in amplitude before and after 'early' contractile failure. This result suggests that 'early' contractile failure is not due to depletion of Ca2+ from the sarcoplasmic reticulum. 3. The action potential shortened as the twitch magnitude declined during 'early' contractile failure, raising the possibility of a causal link. Voltage-clamp experiments show that an enormous increase in K+ conductance (greater than 20-fold) occurs during the period of 'early' contractile failure, and presumably underlies the action potential shortening. 4. If the K+ conductance changes are inhibited by replacement of intracellular K+ with N-methyl glucosamine and inclusion of 2 mM-tolbutamide in intra- and extracellular solutions, good voltage control can be achieved. Under these conditions, 'early' contractile failure did not occur on exposure to complete metabolic blockade and neither Ca2+ current nor the twitch were completely abolished until a maintained contracture had begun to occur. 5. Injection of ATP following 'early' contractile failure could partially restore the twitch and prolong the foreshortened action potential. 6. These results are consistent with the hypothesis that 'early' contractile failure occurring under non-voltage-clamped conditions is due principally to failure of activation of the Ca2+ current because of the shortening of the action potential. Although a decline in the availability of Ca2+ current also occurs, action potential shortening results mainly from increased conductance through ATP-sensitive K+ channels which are activated by a fall of intracellular [ATP]. Contractile failure arises not because of a primary alteration, or defect, in the coupling of excitation to contraction, but because the cell membrane is effectively clamped at a potential close to the K+ equilibrium potential.

1. The Na pump is examined in sheep cardiac Purkinje fibres using a two micro-electrode voltage clamp technique.2. After reducing the external K concentration, [K](o), to zero for 2 min or more, subsequent addition of an ;activator cation' (known to activate the Na pump in other preparations) produces a transient increase of outward current. This outward current transient is abolished by 10(-5)M-strophanthidin (cf. Gadsby & Cranefield, 1979a).3. It is concluded that this transient increase of outward current is a result of a transient stimulation of the sodium pump by the raised [Na](i) following exposure to 0-K(o). Although this current transient may reflect the activity of an electrogenic Na pump, it is difficult to use K as the activator cation to establish this point. This is due to the extracellular K depletion that occurs during Na pump reactivation and the subsequent change that this K depletion produces in the current-voltage relationship of the Purkinje fibre.4. Rb(o) or Cs(o) have been used instead of K(o) to reactivate the Na pump when examining the transient increase of outward current. On adding either of these cations after exposing a preparation to a solution without such ;activator cations', the outward current transient is relatively voltage independent over a wide range of potentials (-90 to +10 mV). It is concluded that, following the addition of Rb(o) or Cs(o), the transient increase of outward current is a direct measure of the transient increase of the electrogenic Na pump current.5. Increasing [Rb](o) or [Cs](o) over the range of 0-40 mM increases the rate of decay of the electrogenic Na pump current transient. Using a simple model (cf. Rang & Ritchie, 1968), it is shown that the decay rate constant of the electrogenic Na pump current transient is a good measure of the degree of activation of the external site of the Na pump. At a given concentration of activator cation, Rb(o) produces a greater activation of the Na pump than does Cs(o). The K(0.5) for Rb(o) is 6.3 mM and for Cs(o) is 14.2 mM. Li(o) activates the Na pump more weakly than Rb(o) and Cs(o).6. The coupling ratio of the Na pump is shown to be independent of Rb(o) or Cs(o) over the range 2-40 mM. Furthermore, consistent with the results of Gadsby & Cranefield (1979a), the coupling ratio is independent of Na(i) over the range considered.7. The Q(10) for the electrogenic Na pump current transient varies between 1.6 and 2.3 over the range of temperature 26-46 degrees C.8. A maximum Na pump current of about 0.78 muA cm(-2) is obtained. Assuming a coupling ratio of 3Na/2K, the rate of Na ion transport into the cell is estimated to be about 23 p-mole cm(-2) sec(-1). Assuming a Na pump turnover of 150 sec(-1), we estimate that there are about 1000 Na pump sites per mum(2) of cell surface.9. We conclude that the electrogenic Na pump current transient provides a good measure of the activity of the Na pump when Rb or Cs are used as ;activator cations'. This measure can be used in the intact preparation to investigate the relationship between Na pump rate and other cellular events such as the regulation of tension (Eisner & Lederer, 1980).

1. Under the influence of strophantidin, Purkinje fibres exhibit transient inward current (TI) which contributes to arrhythmogenic activity. Voltage-clamp experiments were carried out to study the role of Ca ions in this phenomenon. 2. The amplitude of TI varied directly with the extracellular Ca concentration, CaO. Magnesium ions had an antagonistic effect. 3. TI was closely associated with a phasic increase in force ("aftercontraction"). Like TI, the aftercontraction was evoked by a preceding action potential or by the break of a strong depolarizing pulse. 4. TI and the aftercontraction displayed similar wave forms although peak current preceded peak force by 50--100 msec. Both transients were enhanced by increasing the strength or duration of the preceding depolarization pulse. Both events were slowed as the potential level following the pulse was displaced in the negative direction. 5. TI and the aftercontraction could be evoked in the absence of cardiotonic steroids by strongly elevating CaO. 6. Additional experiments were carried out to test the hypothesis the TI reflects an influx of Ca2+ ions. Moninhibited TI but the developed and removal of the inbibition lagged far behind the effects on the slow inward current. 7. TI could be suppressed and eventually inverted by varying the membrane potential in the positive direction. The inversion potential averaged -5mV and was not consistent with a Ca-specific pathway. The aftercontraction was more closely related to the phasic conductance change underlying the current than to thecurrent flow itself. 8. The results are consistent with the idea that an oscillatory release of Ca from an intracellular store is the primary event underlying both the aftercontraction and the conductance change which generates TI. Digitalis intoxication or very high CaO may promote such events by elevating intracellular Ca levels.

1. We have investigated the electrical and mechanical effects of reducing the bathing K concentration, K0, over the range from 4-0 mM in guinea-pig papillary muscle and in sheep Purkinje fibres. 2. In papillary muscle, reducing K0 to zero produces a negative shift in the resting potential and an initial increase in action potential duration. An increase of twitch tension ensues, followed by a reduction in actin potential duration and, eventually, an increase in tonic tension. This increase in tonic tension is often accompanied by a decrease of twitch tension. Finally, transient depolarizations and after contractions are produced. 3. In voltage clamped Purkinje fibres, K0 reduction decreases the slope conductance at the more negative potentials and reduces the pace-maker current, iK2. Twitch tension increases rapidly and voltage dependent tonic tension develops. After even very short exposures to very low K0 (1 mM and below), an oscillatory transient inward current and accompanying aftercontraction can be seen. The oscillatory transient inward current and aftercontraction are similar to those described for cardiotonic steroid intoxication by Kass, Lederer, Tsien & Weingart (1978). 4. Prolonged exposure to 0 K0 leads to the development of a slow current 'creep'. This current is activated by depolarization and has a reversal potential of -6.7 +/- 3.5 mV. The development of this creep current is accompanied by an increasing 'creep' in tonic tension with the same time course. On repolarization both the current creep and creep in tension recover with time courses still similar to each other. 5. Fluctuations appear in both the tension and current records during exposure to low K0. The tension and current fluctuations have similar principal frequencies (about 1 Hz). 6. Ca0 removal, substituting Ba0 for Ca0 or adding Mn0 (2 mM) can each remove the transient inward current, aftercontraction, fluctuations of current and tension, and creep current as well as the increase of twitch and tonic tension. 7. Replacing Ca0 by Sr0 leads to an increased inotropic effect of low K0 with altered kinetics and appears to abolish the transient inward current, aftercontraction and fluctuations of current and tension. 8. It is concluded that Ca1 plays a central role in the inotropic and arrhythmogenic effects of low K0. Possible mechanisms of Ca1 control are discussed in light of the results that have been presented.

1. We have measured the ATP dependence of KATP channel activity, and the effect of various metabolites on this relationship, in inside-out membrane patches isolated from rat ventricular myocytes. 2. The inhibition of KATP channel activity by ATP could be described as a sigmoid function of [ATP] with a Hill coefficient (HATP) of 2 and a half-maximal inhibition at an ATP concentration (Ki, ATP) of 25 microM, in the presence of 0 mM, or 0.5 mM, total [Mg2+]. The non-hydrolysable ATP analogue, AMP-PNP, also inhibited the channel with Ki, AMP-PNP = 60 microM and HAMP-PNP = 2. 3. Acidosis caused a small, but significant, increase in Ki, ATP from 25 microM at pH 7.25 to 50 microM at pH 6.25, but phosphate and lactate were without effect (at 20 mM) on channel activity. 4. In the absence of ATP or Mg2+, ADP3- inhibited channel activity with Ki, ADP = 275 microM, and HADP = 1.2. Other purine and pyrimidine triphosphates, diphosphates and monophosphates also inhibited the channel with apparent order of inhibitory effectiveness ATP greater than AMP-PNP greater than ADP greater than CTP greater than GDP = AMP = ITP. 5. In the absence of Mg2+, but in the presence of 40 microM-ATP, channel inhibition by GTP, ITP, CTP, GDP, ADP or AMP was additive with inhibition by ATP. 6. In the presence of 0.5 mM-Mg2+ and 40 microM-ATP, inhibition by GTP, GMP and AMP was still additive with inhibition by ATP. The diphosphates ADP and GDP, however, paradoxically increased channel activity in the presence of ATP. This increase in channel activity appeared to result from a competitive increase in Ki, ATP, MgADP did not appear to cause any inhibition of channel activity. 7. We conclude that, in cardiac tissue, KATP channels are regulated by [ATP], and that this regulation is sensitive to other intracellular nucleotides, Mg2+, and pH, but not to phosphate or lactate. A simple, interactive two binding-site model is consistent with the nucleotide-dependent regulation that we observe.

Ouabain-sensitive Na efflux, membrane potential and membrane current were measured in single, perfused muscle cells taken from the giant barnacle, Balanus nubilus. This preparation permits control of the intracellular and extracellular solution composition as well as control of the membrane potential while measuring ion fluxes across the plasma membrane. The addition of ouabain (10(-4) M) to the extracellular solution produces a rapid depolarization of membrane potential (1-4 mV) and a simultaneous and proportional reduction of Na efflux (10-40 pmol/s). Ouabain-induced changes in membrane potential or Na efflux do not depend on the presence of extracellular Na. Under voltage control, the application of ouabain (10(-4) M) produces a rapid monotonic fall in outward current (1-3 microA) and a simultaneous fall in Na efflux (10-40 pmol/s). The fraction of ouabain-dependent Na efflux that appears as outward current is constant in any given preparation as the Na-pump turnover rate varies. Over a limited range, changes in membrane potential do not affect ouabain-sensitive Na efflux. The ouabain-sensitive Na efflux and membrane current are not altered by the presence of 50 mM-internal tetraethylammonium (TEA) ions. We conclude that the Na pump is electrogenic in barnacle muscle and that 49 +/- 10% of the extruded Na+ leaves the intracellular compartment as uncompensated charge. We find that the transport stoicheiometry of Na:K, calculated from the ouabain-dependent changes in membrane current and Na efflux, is between 3:2 and slightly more than 2:1.

The actions of extracellular sodium (Nao) on membrane potential, membrane current, membrane conductance and Ca efflux were examined in single muscle cells from the giant barnacle, Balanus nubilus. The intracellular compartment was perfused to facilitate the control of intracellular constituents including calcium ions (Ca2i+). Changing Nao has no large effect on Ca efflux when free intracellular calcium activity, [Ca2+]i, is low (about 0.1 microM). However, increasing [Ca2+]i leads to the development of Nao-dependent Ca efflux as well as to an augmentation in Nao-independent Ca efflux. Reducing Nao (using Li+ as a substitute cation) leads to a depolarization of the membrane when [Ca2+]i is low (about 0.1 microM). Increasing [Ca2+]i causes the membrane to depolarize. With [Ca2+]i at about 10.0 microM, reduction of Nao produces a hyperpolarization of the membrane. Significant Nao-dependent inward current is seen when [Ca2+]i is high. This current is large with respect to the Nao-dependent changes in Ca efflux (about 1 microA per p-mole/sec). The Ca2i+-activated, Nao-dependent changes in Ca efflux and membrane current are not sensitive to La3o+. However, Lao3+ does inhibit a fraction of the Cai2+-activated changes in membrane current and Ca efflux which are not dependent on Nao. Over a limited range of membrane potential Ca efflux is not voltage-dependent. Possible relationships between the Nao-dependent changes in Ca efflux and Nao-dependent changes in membrane potential or current are discussed. We find that these changes cannot be readily interpreted in terms of a single transport mechanism.

The effects of lidocaine have been examined on the arrhythmogenic transient inward current (ITI) in voltage-clamped sheep cardiac Purkinje fibres. Tension and intracellular Na activity (aiNa) were measured simultaneously. The addition of lidocaine (200-300 microM) produced an immediate decrease of inward holding current and a gradual fall of aiNa. The relative magnitudes of the changes of current and aiNa were shown to be consistent with the outward shift of current representing principally a reduction of inward Na current. The Na pump was inhibited by reducing the external Rb concentration in a K-free solution. This produced an after-contraction and transient inward current (ITI) along with a rise of aiNa. The subsequent addition of lidocaine decreased the magnitude of ITI and the after-contraction while decreasing aiNa. Tetrodotoxin (TTX) had qualitatively similar effects to lidocaine on inward holding current, aiNa, ITI and the after-contraction. When aiNa was changed by (i) lidocaine, (ii) TTX or (iii) small changes of external Rb concentration, a hysteresis was seen in the relationship between aiNa and ITI or after-contraction. The hysteresis was similar to that previously found between aiNa and contraction (Eisner, Lederer & Vaughan-Jones, 1981). Despite this hysteresis, neither lidocaine nor TTX affected the relationship between magnitudes of ITI and the after-contraction. It is suggested that the fall of aiNa is a major factor in the reduction of ITI by lidocaine. These results are discussed in relation to the anti-arrhythmic actions of lidocaine.

1. Sheep cardiac Purkinje fibres were studied under voltage clamp conditions to examine the relationship between the Na pump rate and twitch tension.2. The Na pump activity was measured following a period of decreased Na pump rate and increased internal Na activity, Na(i), by examining the electrogenic Na pump current transient produced by reactivating the Na pump (see Eisner & Lederer, 1980).3. Various concentrations of Cs or Rb were used to reactivate the Na pump in the absence of extracellular K, K(o). Results from such experiments showed that these ;activator cations' produced a monotonic increase in Na pump rate with increasing concentration while producing monotonic decreases in steady-state twitch tension. A given concentration of Rb was more potent than the same concentration of Cs in its effects on both Na pump rate and tension. Nevertheless, varying [Rb](o) or [Cs](o) produced the same relationship between Na pump activity and tension.4. After the preparation was exposed to low K(o) (below 4 mM), the Na pump was reactivated with 10 mM-Rb(o) (0-K(o)). The area under the resulting electrogenic Na pump current transient gives a measure of the increase of [Na](i) that occurred when the preparation was exposed to the test solution compared to the steady-state Na(i) in 10 mM-Rb(o). Increasing the duration of exposure to low K(o), further augments the twitch tension achieved at the end of the test period and the area under the electrogenic Na pump current transient. Similarly, for equal periods of exposure, the lower the [K](o) in the test solution, the greater the increase of twitch tension at the end of the test period and the greater the area under the electrogenic Na pump current transient.5. The relationship between tension and the area under the electrogenic Na pump current transient is the same for a variable duration exposure to 0-K(o) or for a constant exposure to various low K(o). It is concluded that a rise in [Na](i) is the rate limiting step linking Na pump activity and twitch tension.6. In an experiment similar to the one described in (4), the fibre was exposed to a test solution containing a variable concentration of one of the activator cations of the Na pump for a fixed period. The effects of different cations were compared with the effects of a test solution containing no activator cation. Increasing the concentration of a particular activator cation from zero reduced the twitch tension augmentation in the test solution and also reduced the area under the electrogenic Na pump current transient on subsequently reactivating the Na pump with 10 mM-Rb(o). It is concluded that the ability of a test solution to reduce the area of the electrogenic Na pump current transient reflects the ability of the test solution to activate the Na pump. Equivalent concentrations of the activator cations that are required to activate the Na pump are: 2 mM-K(o) = 2 mM-Rb(o) = 6 mM-Cs(o) = 6 mM-NH(4o) = 22 mM-Li(o). Tl(o) was more effective than K(o). The order of these cations to reduce twitch tension is found to be the same as that to reduce the area under the electrogenic Na pump current transient.7. We conclude that the effects of the activator cations on twitch tension are determined by their effects on the Na pump and thereby on [Na](i).

Intracellular Na activity (aiNa) was measured with recessed-tip, Na-selective micro-electrodes in voltage-clamped sheep cardiac Purkinje fibres. Tension was measured simultaneously. aiNa was increased reversibly either by exposing the preparation to K-free, Rb-free solution of by adding the cardioactive steroid strophanthidin. An increase of aiNa produced an increase of tonic tension which was larger at depolarized membrane potentials. At sufficiently negative membrane potentials, changes of aiNa (over the range 6-30 mM) had no effect on tonic tension. Therefore, both an increase of aiNa and a depolarization are required to increase tonic tension. It is concluded that either a low level of aiNa or a large negative membrane potential is sufficient to maintain a low intracellular Ca concentration. Tonic tension was measured as a function of aiNa. At a given membrane potential the relationship can be described empirically by an equation of the form: tonic tension = b(aiNa)y, where y is a constant and b depends on membrane potential. In five experiments y was found to be 3.7 +/- 0.7 (mean +/- S.E.M.) over a range of potentials from -60 to -10 mV. Tonic tension was measured as a function of membrane potential. At a given aiNa the relationship can be described approximately as: tonic tension = k exp (aV), where a is a constant and k depends on aiNa. In five experiments a was found to be 0.06 +/- 0.01 mV-1 (mean +/- S.E.M.). A depolarization of 10 mV increases tonic tension by the same amount as does an increase of aiNa that is equivalent to a 3.7 mV change of the Na equilibrium potential, ENa. Hence ENa is nearly 3 times more effective than membrane potential in controlling tonic tension. During a prolonged depolarization (several minutes) the initial increase of tonic tension decays gradually. This is associated with a fall of aiNa. The relationship between tonic tension and aiNa is similar to that seen when aiNa is increased by inhibiting the Na pump. It is concluded that the fall of aiNa is responsible for the decay of tonic tension. The changes of tonic tension reported in this paper are consistent with the effects of aiNa and membrane potential on a voltage-dependent Na-Ca exchange. The possibility that a voltage-dependent Ca channel contributes to tonic tension is also discussed.

1. Calcium currents (ICa) from neonatal rat ventricular heart muscle cells grown in primary culture were examined using the 'whole-cell' voltage-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Examination of ICa was limited to one calcium channel type, 'L' type (Nilius, Hess, Lansman & Tsien, 1985), by appropriate voltage protocols. 2. We measured transient and steady-state components of ICa, and could generally describe ICa in terms of the steady-state activation (d infinity) and inactivation (f infinity) parameters. 3. We observed that the reduction of ICa by the calcium channel antagonist D600 can be explained by both a shift of d infinity to more positive potentials as well as a slight reduction of ICa conductance. D600 did not significantly alter either the rate of inactivation of ICa or the voltage dependence of f infinity. 4. The calcium channel modulator BAY K8644 shifted both d infinity and f infinity to more negative potentials. Additionally, BAY K8644 increased the rate of inactivation at potentials between +5 and +55 mV. Furthermore, BAY K8644 also increased ICa conductance, a change consistent with a promotion of 'mode 2' calcium channel activity (Hess, Lansman & Tsien, 1984). 5. We conclude that, as predicted by d infinity and f infinity, there is a significant steady-state component of ICa ('window current') at plateau potentials in neonatal rat heart cells. Modulation of the steady-state and transient components of ICa by various agents can be attributed both to specific alterations in d infinity and f infinity and to more complicated alterations in the mode of calcium channel activity.

1. Non-linear capacitative current (charge movement) was studied in isolated guinea-pig and rat ventricular myocytes. Linear capacitance was subtracted using standard procedures. Most of the experiments were done with guinea-pig myocytes, while rat myocytes were used for comparison. 2. When a myocyte was held at -100 mV, depolarizing clamp steps produced a rapid outward current transient, which was followed by an inward current transient upon repolarization. This current was identified as the movement of charged particles in the cell membrane, rather than ionic movement across the membrane, for the following reasons: (1) the current saturated at membrane potentials positive to +20 mV; (2) the current was capacitative in nature, having no reversal potential; (3) in general, the charge moved during depolarization (Qon) approximated the charge moved during repolarization (Qoff). 3. Qoff was significantly less than Qon for a depolarization from -100 mV to 0 mV. However, the Qoff/Qon ratio approached unity if the cell was instead repolarized to -140 mV. This was interpreted as being due to the immobilization of a fraction of the charge during the depolarization, which recovered rapidly enough to be measured at -140 mV, but recovered too slowly at -100 mV. 4. Charge movement in these cells had a sigmoidal dependence on the membrane potential, which could be empirically described by the two-state Boltzmann equation Q = Qmax/(1 + exp[-(V-V*)/kappa]), where Q is the charge movement at potential V, Qmax is the maximum charge, V* is the membrane potential at Q = Qmax/2, and kappa is a slope factor. Qmax was 11.7 nC/microF, V* was -18 mV and kappa was 16 mV in guinea-pig myocytes held at -100 mV, while the values in rat myocytes were 10.9 nC/microF, -32 mV and 13 mV. 5. The charge movement could be partially immobilized by a prior depolarization. This effect developed over a broad voltage range, from -120 to +20 mV. The fraction of charge that could be immobilized by a 10 s pre-pulse to +20 mV was 59%. 6. The time course of decay of both Qon and Qoff could basically be described as a single-exponential process. The time constant was largest at -40 mV and decreased at both more positive and negative test potentials. A second, slower Qoff time constant, possibly representing remobilization of immobilized charge, could be seen under some conditions. 7. The temperature dependence of charge movement was studied between 11 and 35 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

Sheep cardiac Purkinje fibres were voltage clamped with a two-microelectrode technique. Under conditions that are known to elevate intracellular calcium (0 mM-external potassium), membrane currents were examined. In the above conditions, a brief depolarizing pulse leads to an oscillatory inward current (ITI) which peaks at about 300 ms after the repolarization. An after-contraction is also observed, the peak of which occurs about 80 ms after the peak of ITI. This result is in accord with the results of Kass, Lederer, Tsien & Weingart (1978a). We replaced external sodium with an isotonic CaCl2 solution to remove the sodium-calcium exchange mechanism as a possible current carrier for ITI. In the steady state under these conditions an oscillatory membrane current and after-contraction are seen following repolarization. This current was identified as ITI on the basis of its temporal relation to both the repolarization step and the after-contraction. In isotonic CaCl2, ITI has a reversal potential of -37 mV. Because of this fact ITI cannot be explained by an electrogenic sodium-calcium exchange mechanism alone. The reversal potential suggests that ITI arises from a channel which is permeable to both potassium and calcium. Fluctuations of membrane current and of tension were recorded in the steady state at different holding potentials. Power spectral analysis showed that the current fluctuations were at a minimum at a holding potential of -37 mV. Tension fluctuations were, however, relatively constant over the range of membrane potentials examined (-17 to -70 mV). The peak power of the current fluctuations occurred at about 1.5 Hz (at a holding potential of -70 mV). This peak shifted towards higher frequencies with increasing depolarization. A similar shift in frequency was observed for the tension fluctuations. Cross-correlations between membrane current and tension were calculated for various steady membrane potentials. This analysis shows that the current fluctuations are associated with the tension fluctuations, each with a principal period of about 0.5 s. This analysis also shows that at potentials more negative than the reversal potential of ITI, increasing tension is associated with increasing inward current and that the tension fluctuations follow current fluctuations by about 70 ms. At potentials positive to the reversal potential of ITI, increasing tension was associated with increasing outward current. This analysis therefore indicates that the fluctuations in membrane current reverse at a potential similar to the reversal potential of ITI.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The intracellular Ca2+ concentration [( Ca2+]i) was measured in voltage-clamped sheep cardiac Purkinje fibers while recording tension simultaneously. 2. When [Na+]i was elevated (by Na+-K+ pump inhibition) depolarization produced an increase of tonic tension. 3. Replacement of external Na+ by Li+ or choline produced a contracture which then relaxed spontaneously. Following this relaxation, depolarization either had no effect on tonic tension or produced a small decrease. 4. When external Na+ was replaced by Ca2+, depolarization (over the range -120 to -20 mV) produced a decrease of tonic tension and [Ca2+]i. Hyperpolarization increased tonic tension and [Ca2+]i. 5. An after-contraction and accompanying increase of [Ca2+]i were produced by repolarization in both Na+-free and Na+-containing solution. This eliminates the possibility that the stimulus for the after-contraction is the increase of [Ca2+]i during the depolarization and suggests that the stimulus may be the change of membrane potential. 6. The increase of [Ca2+]i on hyperpolarization seen in Na+-free solutions persisted in the presence of ryanodine. 7. These results show, in contrast to previous work, that in Na+-free solutions tonic tension is still sensitive to membrane potential. The results support the hypothesis that, in Na+-containing solutions, the increase of tonic tension on depolarization results from a voltage-dependent Na+-Ca2+ exchange. The reduction of tonic tension on depolarization in Na+-free solutions may be due to the decrease of the electrochemical gradient for Ca2+ to enter the cell.

1. Intracellular Na activity, aiNa, was measured in voltage-clamped sheep cardiac Purkinke fibres. 2. Increasing [Rb]0 from 0 to 4 mM in K-free solutions (at a fixed membrane potential) decreased aiNa. Further increases of [Rb]0 (up to 20 mM) had little or no effect. 3. Following exposure to Rb-free, K-free solution, the addition of a test concentration of Rb produced an exponential decrease of aiNa. The rate constant of decay of aiNa increased with increasing [Rb]0 over the measured range (0-20 mM). 4. The accompanying electrogenic Na pump current transient decayed with the same rate constant as aiNa over the range of [Rb]0 examined. During this decay the electrogenic Na pump current was a linear function of aiNa. Increasing [Rb]0 increased the steepness of the dependence of the electrogenic current on aiNa. 5. A constant fraction of the net Na efflux was electrogenic. This fraction was not affected by varying [Rb]0 over the range 0-20 mM. 6. Using a simple model, it is shown that the dependence of steady-state aiNa on [Rb]0 is half-saturated by less than 1 mM-[Rb]0. The rate constant of decay of aiNa and the slope of the relationship between electrogenic Na pump current and aiNa are, however, better fitted with a lower affinity for Rb (K0.5 = 4 mM-[Rb]0). 7. Depolarizing the membrane potential with the voltage clamp decreased aiNa; hyperpolarization increased it. These effects persisted in the presence of 10(-5) M-strophanthidin. An effect of membrane potential on the net passive Na influx can account for the observations. 8. The effects of membrane potential on the net passive Na influx were examined by measuring the maximum rate of rise of aiNa at different holding potentials after inhibiting the Na-K pump in a K-free, Rb-free solution. Depolarization decreased the Na influx. 9. Using the constant field equation, the net passive Na influx was used to estimate the apparent Na permeability coefficient, PNa. This was between 0.8 x 10(-8) and 1.5 x 10(-8) cm sec-1.