Transmembrane Calcium Flux Research Articles

Interleukin 2-induced lymphocyte proliferation is independent of increases in cytosolic-free calcium concentrations.

Activation of lymphocytes by mitogenic lectins initiates a sequence of events that culminates in DNA synthesis and cell proliferation. The mitogenic effects of lectins on T lymphocytes leads to the production of a group of lymphokines including the interleukins. The binding of interleukin 2 (IL 2) to its receptor results in activation of the cell leading to DNA synthesis. An increase in cytosolic-free Ca++ ([Ca++]i) is associated with activation of lymphocytes by mitogenic lectins and also appears to be a prerequisite for induction of DNA synthesis and cell proliferation. We have determined whether the proliferative response triggered by IL 2 binding to its receptor is associated with or requires an increase in [Ca++]i. Using human and murine IL 2-sensitive cell lines, we have demonstrated that the IL 2-induced proliferative response, in contrast to that induced by mitogens such as phytohemagglutinin or concanavalin A, is not accompanied by an increase in [Ca++]i as monitored by the fluorescent indicator quin-2. Furthermore, IL 2-dependent triggering of lymphoblasts occurs in the presence of extremely low extracellular calcium concentrations that prevent transmembrane calcium flux. Activation of IL 2 receptor-bearing T cells, therefore, does not appear to be associated with or to require an increase in [Ca++]i as part of the activation and signaling process. The critical step requiring calcium flux in cell signaling by mitogenic lectins must therefore occur elsewhere in the activation cascade.

Increase in cytosolic free calcium concentration is an intracellular messenger for the production of interleukin 2 but not for expression of the interleukin 2 receptor.

Activation of lymphocytes by mitogenic lectins results in the production of a group of soluble factors, the lymphokines. Proliferation of activated T cells requires interaction of one of these lymphokines, interleukin 2 (IL 2), with its receptor. The induction of IL 2 receptor expression and IL 2 production may involve different activation signals; some mitogens or antigens may activate both, whereas others may activate only one. An increase in cytosolic free calcium concentrations [( Ca++]i) is one of the signals involved in cellular activation by lectins. By using the fluorescent indicator quin-2, we have demonstrated that increases in [Ca++]i accompany phytohemagglutinin induced proliferative responses of human T lymphocytes. Preventing the increase in [Ca++]i also prevents proliferation. We demonstrate that an increase in [Ca++]i is not required for the expression of the IL 2 receptor, which is expressed even in the presence of extremely low external calcium concentrations. In contrast, IL 2 production requires an increase in [Ca++]i and does not occur in the absence of extracellular free calcium. IL 2 production appears to be the critical step requiring transmembrane calcium flux. In the absence of transmembrane calcium flux and subsequent IL 2 production, lectins are not able to trigger DNA synthesis and cell proliferation.

Therapeutic application of calcium-channel antagonists for pulmonary hypertension

Calcium-channel antagonists may provide an effective approach to the treatment of pulmonary hypertensive disorders. Biochemical evidence suggests that pulmonary vasoconstriction results from the transmembrane flux of calcium into vascular smooth muscle; accordingly, the pulmonary pressor responses in experimental hypoxic pulmonary hypertension can be attenuated by verapamil and nifedipine. In patients with chronic obstructive lung disease, nifedipine decreases pulmonary artery pressures and pulmonary vascular resistance in proportion to the severity of hypoxemia before treatment. However, little pulmonary vasodilator effect is seen when hypoxemia is corrected by inhalation of oxygen, and systemic arterial oxygen desaturation can occur after nifedipine in patients breathing room air; most importantly, long-term studies in patients with chronic lung disease are lacking. In selected patients with primary pulmonary hypertension and other obliterative diseases of the pulmonary vasculature, nifedipine produces shortand long-term hemodynamic improvement at rest and during exercise, and these benefits are frequently paralleled by amelioration of dyspnea and fatigue. However, in patients in whom right ventricular function has been severely compromised by chronic pressure overload, both verapamil and nifedipine may exert notable depressant effects on right ventricular performance, despite the decrease in right ventricular afterload that would be expected to accompany a decrease in pulmonary vascular resistance. These negative inotropic actions may result in serious deleterious clinical reactions. Although calcium-channel antagonists represent a promising approach to the management of patients with pulmonary hypertension, the long-term efficacy and safety of these drugs in this disorder remain to be established.

The effect of altering the external calcium concentration and a calcium channel blocker, verapamil, on microvascular leaky sites and dextran clearance in the hamster cheek pouch

The role of calcium in contractile mechanisms has been well documented. Since the formation of intraendothelial gaps may be due to a contractile process, these studies were initiated to describe the effects of altering the external calcium concentration and a calcium blocker, verapamil, on the development of leaky sites and clearance of dextran in the hamster cheek pouch under normal and histamine-stimulated conditions. Adult Syrian hamsters were anesthetized and tracheostomized, and the femoral vein was cannulated for injecting the FITC-Dextran-70K. In all hamsters, a removable plastic chamber was placed in the cheek pouch to observe and collect suffusate from the microvasculature. In one series of experiments, suffusion was begun with either “normal” calcium (1.5 m M) or a calcium-free, Ringer's bicarbonate (pH 7.4, 36°) buffer. After a 30-min period, fluorescein-labeled dextran (FITC-Dextran-70K) was given intravenously and the number of leaky sites and dextran clearance determined for 30–45 min. Two 5-min periods of stimulation with histamine (10 −7 and 10 −6 M) followed by a 30-min equilibration were completed while the above measurements were repeated. Then, the suffusate was switched to the opposite calcium buffer and the same sequence repeated. A similar protocol was followed using either the “normal” buffer or a high (4.5 m M)-calcium buffer. In another series of experiments, similar measurements were completed with and without verapamil (10 −4 M) using a “normal” calcium buffer and histamine-stimulated conditions. In the control, nonstimulated state, there was a significant increase in the leaky sites while suffusing with low-calcium buffer; however, there were no changes in leaky sites while suffusing with the high-calcium buffer. In the histamine-stimulated state, the increase in the number of leaky sites and dextran clearance was attenuated with the low-calcium buffer and potentiated with the high-calcium buffer. While suffusing with the verapamil buffer, there was an increase in the control number of leaky sites and in the histamine-stimulated state there was an attenuation of the histamine-stimulated response. These results indicate that external calcium influences and modulates microvascular leaky sites in normal and stimulated states and that the formation of leaky sites and dextran clearance after histamine stimulation require the transmembrane flux of calcium. Also, the formation of leaky sites was coupled directly to the clearance of dextran.

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin.

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.

Role of calcium in airway smooth muscle contraction and mast cell secretion.

The principal pathological features of asthma, including tracheobronchial smooth muscle contraction and mast cell mediator synthesis and release, are calcium-dependent processes. Calcium plays an integral role in transmitting signals at the cell surface to the enzymatic machinery of the cell interior; its role as the agent for "excitation-contraction coupling" of airway smooth muscle and for "stimulus-secretion coupling" of mast cells is reviewed. A rise in intracellular calcium ion concentration triggers cellular activation. In smooth muscle, calcium bound to calmodulin stimulates the myosin light chain kinase which is important in the regulation of actin-myosin interaction. In mast cells, calcium may bind to calmodulin or to a calmodulinlike regulatory protein, and it also stimulates enzymes important in the synthesis of newly generated mediators including prostaglandins and leukotrienes. The regulatory role of cyclic AMP in both cell systems is discussed, especially as it pertains to calcium metabolism. By interfering with transmembrane calcium fluxes, the calcium channel blocking drugs have the potential for significantly modifying bronchoconstriction and airway inflammation in asthma and related bronchospastic disorders. Some of the in vitro studies of calcium channel blockers in these two cell systems are reviewed. Finally a speculation about the role of abnormal sensitivity to calcium in airway smooth muscle as a potential cause of airway hyperreactivity is entertained.

Nifedipine with and without propranolol in the treatment of myocardial ischemia: effect on ventricular arrhythmias and recovery of regional wall function.

Sudden reperfusion of ischemic myocardium has been related to an extension of damage, due to sudden increases in transmembrane calcium fluxes. When the calcium slow channel blocker nifedipine (1 microgram kg-1 min-1 i.v.) was infused just prior to release of a 30 min partial obstruction of the left anterior descending coronary artery (LAD) in anesthetized pigs, recovery of regional function was better (75%) than in untreated animals (50%). The number of reperfusion arrhythmias was also significantly lower in the treated animals. In another series of experiments, ventricular fibrillation occurred in 80% of the untreated animals within 10 min after the complete acute occlusion of the LAD. Animals pretreated with propranolol (0.5 mg kg-1 i.v. bolus + infusion) or nifedipine (0.05 microgram kg-1 min-1 i.c.) showed a similar high incidence of ventricular fibrillation. However, when the administration of these drugs was combined, 60% of the animals survived three periods (10 min) of intermittent myocardial ischemia. When the continuous infusion of nifedipine was preceded by a bolus of 2.0 microgram kg-1, sufficient to abolish systolic wall thickening completely, the incidence of ventricular fibrillation was also reduced. Recovery of regional function was nearly complete in these animals.

Hypoxia, calcium, and contracture as mediators of myocardial enzyme release.

During calcium-free perfusion, anoxic contracture of myocardial cells causes cells to separate at intercalated disks and leads to an energy-independent enzyme release in the absence of active transmembrane calcium fluxes. It is proposed that contracture mediates membrane damage and enzyme release in cells sensitized to the calcium paradox.

Clinical pharmacology of slow channel blocking agents

ATIONAL and effective pharmacotherapy requires an understanding of the patient’s disease process as well as an appreciation of both the pharmacodynamics and the pharmacokinetics of the drug(s) selected for use. Although the slow channel antagonist drugs have been widely used outside the United States for the past decade, the body of data contributing to our understanding of the clinical pharmacology of these agents is only now beginning to expand. Clinical studies that have shown the dramatic effectiveness of the slow channel blockers in control of supraventricular tachyarrhythmiasif2 and in relief of symptoms associated with vasospastic coronary artery disease3-’ have been largely empiric, and therefore, have demonstrated the potential usefulness of these new drugs largely in qualitative fashion. Further elaboration of the clinical value of the slow channel antagonist compounds will depend heavily on data from which rational dosing schedules can be derived, on which appropriate dosing intervals may be based, and with which predictable drug toxicity may be avoided. The purpose of this article is to examine the information currently available pertaining to the clinical pharmacology of the three slow channel antagonists undergoing advanced clinical evaluation in this country-verapamil, nifedipine, and diltiazem-and which have received preliminary FDA approval for marketing. Other compounds with similar effects on transmembrane calcium flux, such as perhexilene and lidoflazine, have not been studied in this country to the extent the former group has and are not so far along in the lengthy process leading to clinical availability. VERAPAMIL Actions

Does Pituitary and Adrenocortical Hormone Release in Man Depend on, Transmembrane Calcium Flux?

Does Pituitary and Adrenocortical Hormone Release in Man Depend on, Transmembrane Calcium Flux?

Ca-calmodulin mediates the DNA-synthetic response of calcium-deprived liver cells to the tumor promoter TPA

Incubation in low (0.02 mM)-calcium medium inhibited the proliferation of T51B rat liver cells. Addition of TPA (12- O-tetradecanoyl-phorbol-13-acetate; 50 ng/ml) or calcium (1.25 mM) caused the arrested, calcium-deprived cells to initiate DNA synthesis within 2 h and eventually to resume proliferating. The rapid DNA-synthetic response to TPA addition appeared to depend on the small amount of calcium remaining in the calcium-deficient medium, because it was blocked by correspondingly small amounts of the specific calcium chelator EGTA or La 3+, which displaces calcium from the cell surface and inhibits transmembrane calcium fluxes. The common mediation of TPA and calcium actions through Ca-calmodulin complexes was indicated by the following observations: The DNA-synthetic responses to both agents were blocked by the supposedly specific Ca-calmodulin blocker trifluoperazine; the trifluoperazine-induced block was overcome by pure calmodulin; and the responses to both TPA and calcium were blocked by anti-calmodulin antibodies.

4-Aminoquinoline-induced ‘giant’ miniature endplate potentials at mammalian neuromuscular junctions

4-Aminoquinoline (4-AQ) in concentrations around 200 micrometers induces, within minutes of its application to isolated mouse or rat neuromuscular junctions, the appearance of a population of miniature endplate potentials (m.e.p.ps) with a larger than normal amplitude, so-called giant m.e.p.ps (g.m.e.p.ps). With amplitudes 2-12 times the modal value of m.e.p.p. amplitude, the population of g.m.e.p.ps varied between 15 and 45% of the total population of m.e.p.ps. There was no increase in the frequency of m.e.p.ps but a positive correlation between the frequency of g.m.e.p.ps and the total frequency of m.e.p.ps. In many instances the rise time and decay time of g.m.e.p.ps were prolonged compared to normal. Elevated extracellular calcium concentrations increased the frequency of m.e.p.ps but had no effect on g.m.e.p.p. frequency. High extracellular potassium concentrations markedly increased m.e.p.p. frequency but failed to influence g.m.e.p.p. frequency. Similar observations were made with ethanol 0.1 M, ouabain 200 micrometers or black widow spider venom. Botulinum toxin type A markedly reduced total m.e.p.p. frequency but 4-AQ still induced g.m.e.p.ps. Nerve stimulation failed to release quanta corresponding to the g.m.e.p.ps. G.m.e.p.ps seemed to originate from quantal acetylcholine release from the nerve terminal since they were abolished by surgical denervation and by the addition of d-tubocurarine to the medium. Blockade of voltage-sensitive calcium or sodium channels by, respectively, manganese ions or tetrodotoxin failed to affect the appearance and the frequency of g.m.e.p.ps. The electrophysiological findings and a statistical analysis of the characteristics of the m.e.p.ps indicate that they belong to two populations. One population is accelerated by the depolarization-release coupling mechanism responsible for evoked transmitter release and is characterized by an amplitude distribution and a process in time that indicate that they correspond to releases occurring at 'active zones' in the nerve terminal. The second population of m.e.p.ps is uninfluenced by nerve terminal depolarization and transmembrane calcium fluxes. This population apparently originates from sites dispersed in the nerve terminal membrane and outside the 'active zones'. 4-AQ increases the frequency of this second m.e.p.p. population without affecting the first population.

Modulation of natural killer cell cytotoxicity by transmembrane calcium flux and calmodulin activity

Modulation of natural killer cell cytotoxicity by transmembrane calcium flux and calmodulin activity

Electrically induced Ca 2+ transport across the membrane of Paramecium caudatum measured by means of flow-through technique

Transmembrane calcium fluxes related to excitation were studied in Paramecium caudatum. Radioactive ( 45Ca 2+) or inactive solution was flowed through a dense suspension of unlabelled or labelled cells, and radioactivity was monitored in the solution. The organisms were electrically stimulated by means of extracellular electrodes. As a result of stimulation Ca 2+ uptake and release was measured. The uptake response dropped with increasing number of successive stimulation periods and increased with growing stimulus amplitude and duration. Maximum uptake was obtained at 20 V/cm and at least 60 s duration and for temperatures between 10 and 15°C. A Ca 2+ influx of 700 pmol/1000 cells upon 1 min stimulation was measured at 15°C. This corresponds to an increment of the intraciliary [Ca 2+] of about 5 · 10 -4 M . Ca 2+ release was dependent on the stimulus amplitude in a similar manner as was Ca 2+ uptake. Photographic recordings of the swimming behaviour of the organisms were used to interpret the flux data. At temperatures up to 15°C the cells swam backward perpendicular to the applied electric field of 10 to 20 V/cm. At 25°C they showed forward spiralling movement. For the first time evidence was brought for stimulated Ca 2+ influx in Paramecium at physiological temperatures. It is concluded from the results that a strong active Ca 2+ extrusion from the intraciliary space counteracts the influx. The Ca 2+ pump rate must be at least 8 · 10 12 calcium ions per s per cm 2 ciliary surface.

The effect of verapamil on mechanical performance of acutely ischemic and reperfused myocardium in the conscious dog.

The effect of verapamil, an inhibitor of transmembrane calcium flux, was studied in intact conscious dogs with myocardial ischemia produced by inflating a balloon cuff implanted on the left anterior descending coronary artery. Six dogs received a continuous infusion of verapamil (10 microgram/kg per min) beginning prior to coronary occlusion, and six received normal saline infusions. Systolic ejection shortening (SES) was measured from subendocardial ultrasonic crystals implanted in the central ischemic zone (IZ) and border zone (BZ), and in a nonischemic control zone (CZ). Hearts were paced at a constant heart rate with periodic introduction of closely coupled extrasystoles. SES was measured both for normally paced beats and during postextrasystolic potentiation (PESP). Regional myocardial blood flow was measured by injecting radioactive microspheres before, during, and after coronary occlusion. There were no significant differences between verapamil-treated dogs and saline control dogs in mean aortic pressure, heart rate, left ventricular end-diastolic pressure or dP/dt, cardiac output, or regional myocardial blood flow in IZ, BZ, or CZ. Differences in mechanical performance between two groups were noted, however. In the IZ, SES was abolished completely for normally paced beats in both groups but was significantly preserved for PESP beats in the verapamil-treated animals. In the BZ, SES was significantly reduced for normally paced beats only in the saline controls, and PESP responses were preserved to a significantly greater degree in the verapamil-treated animals. These results indicate that verapamil pretreatment exerts beneficial effects upon mechanical performance of ischemic myocardium. Since no changes in systemic hemodynamics or regional myocardial blood flow were observed, the effect may be due to the calcium-antagonistic properties of the agent.

Calcium, Calcium Antagonists, and Cardiovascular Disease

Why are cardiologists so interested in “calcium antagonists?” Firstly, active coronary vasomotor changes are now recognized to play an important role in certain anginal syndromes. Coronary spasm superimposed on atherosclerotic coronary artery disease is probably the most important contributing factor to angina at rest. Secondly, knowledge regarding the mechanism of contraction of vascular smooth muscle is rapidly advancing and the important role played by transmembrane calcium flux in this process is being defined. Thirdly, a diverse group of pharmacologic agents that block transmembrane calcium flux are clinically available and are being widely tested for their efficacy in a number of disorders, especially Prinzmetal's or variant angina. These drugs may also have intracellular actions as well. Because of the intense interest of both basic and clinical scientists in the calcium antagonists, a conference of investigators from very diverse fields was held in Ixtapa, Mexico, November 8–10, 1979 to exchange ideas and present new information. This publication represents the proceedings of this symposium. Some of the highlights are reviewed below.

Cardiac interaction between parathyroid hormone, beta-adrenoceptor agents, and verapamil in the guinea-pig in vitro.

SUMMARY1. The present study examines the modulation, by parathyroid hormone, of the changes in myocardial contractile force induced by isoprenaline, propranolol isomers, verapamil, and nadolol.2. Cardiac contractile force was estimated by the use of guinea‐pig isolated auricles. Synthetic bovine 1‐34 parathyroid hormone (sPTH) alone did not modify contractile force; conversely, sPTH significantly inhibited the cardiode‐pressant effect of l‐propranolol, d, l‐propranolol, d‐propranolol, and verapamil. These results suggested an action of sPTH independent of β‐adrenoceptors. Against an hypothesis of a single, non‐β‐adrenoceptor mechanism of sPTH action on the heart are the following observations: (i) when β‐adrenoceptors were blocked with nadolol, sPTH no longer inhibited the cardiodepressant effect of propranolol, (ii) sPTH reduced the inotropic effects of isoprenaline. Our conclusion, therefore, is that sPTH probably affects cardiac muscle contraction by at least two mechanisms, one of which involves non‐adrenergic transmembrane calcium flux and the second β‐adrenoceptors.3. When these studies were extended into the clinical pharmacological field, it was found that plasma ultrafiltrates from severely hyperparathyroid patients in chronic renal failure inhibited like sPTH the cardiodepressant action of propranolol. No such effect was seen with ultrafiltrates from parathyroidectomized patients. Accordingly, high PTH levels may inhibit the action of cardiotropic drugs administered to hyperparathyroid patients, and may be one factor in the cardiomyopathy seen in such patients.

Inhibition of the positive inotropic effect of anthopleurin-A (AP-A) by dantrolene

The effect of dantrolene on the positive inotropic effects (PIE) of three cardiotonic agents was assessed on rat and rabbit atria. Dantrolene (10 −5M) had no effect on contractile tension or on the PIE to isoproterenol (10 −10−10 −7M) or ouabain (10 −6−10 −4). The dose-response curve for the PIE of anthopleurin-A (AP-A) was shifted to the right in rat and rabbit atria by dantrolene (10 −4−10 −6M). The maximum effect and the concentration of AP-A required for it remained the same. The results suggest that the PIE of AP-A involves intracellular translocation of calcium, unlike those of isoproterenol and ouabain which require increased transmembrane calcium flux. This conclusion is supported by the observation that the exchange and distribution of the labile calcium involved in excitation-contraction coupling was unaffected by AP-A (10 −8M), by dantrolene (10 −6M) or by the combination. Therefore, AP-A may produce its cardiotonic effect by a action at an intracellular site, a mechanism unlike that of isoproterenol or ouabain.

Stimulation of glucose transport in rat adipocytes by calcium

Glucose transport in rat adipocytes was studied by monitoring the conversion of [1-14C]-glucose to 14CO2 in a system where glucose transport was made rate-limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent which rapidly reoxidizes NADPH. Calcium increased both basal and insulin-stimulated apparent rates of glucose transport by approximately 40%. The maximum velocity of the apparent rate of glucose transport was increased by extracellular calcium both in the presence or absence of insulin. There was no change in the glucose concentration required for half-maximal rates of 14CO2 production. Calcium also enhanced the stimulation of apparent rates of glucose transport by insulin when examined over a range of hormone concentrations. Adipocyte cAMP concentrations were significantly lowered by calcium under conditions which led to increased apparent rates of glucose transport. In contrast, cobalt and nickel, antagonists of calcium action, elevated adipocyte cAMP levels and inhibited apparent rates of glucose transport. Agents which inhibit transmembrane calcium flux (verapamil, tetracaine, and procaine) inhibited apparent rates of glucose transport despite a reduction in adipocyte cAMP concentration.On the basis of the above data we suggest that calcium may increase apparent rates of glucose transport in rat adipocytes both by lowering intracellular cAMP concentration and by a further mechanism independent of changes in the level of cAMP. These results are consistent with the hypothesis that glucose transport in rat adipocytes may be controlled, in part, by a cAMP-induced phosphorylation mechanism.

Calcium current in molluscan neurones: measurement under conditions which maximize its visibility.

1. Membrane currents were studied in isolated somata of molluscan neurones from Archidoris monteryensis and Anisodoris nobilis. Under voltage clamp, inward current displayed a two phase time course, and in some cases a clear reversal potential difference could be shown for the fast and slow phases. The slower phase was carried predominantly by calcium ions. 2. The apparent magnitude of the slower phase was greatly influenced by conditions which altered potassium current flow. Blocking voltage-dependent potassium conductances, either by appropriate conditioning polarizations or by tetraethyl-ammonium (TEA) ion, enhanced the magnitude, while conditions which augmented potassium current made the slow phase disappear. 3. A fraction of the membrane potassium conductance was TEA insensitive. This fraction could be blocked by procedures which prevented internal levels of calcium from increasing during the voltage clamp pulse. Three such procedures were demonstrated; replacement of external calcium by magnesium, internal buffering by EGTA, and replacement of calcium by permeant barium. 4. Internal EGTA buffering or external barium in combination with external TEA produced an extreme change in membrane current as compared with the normal time course. Membrane current, when activated by pulses up to +50 mV, was net inward and showed only fractional inactivation over time courses running to several seconds. Pulses to voltages greater than +60 mV resulted in outward current. 5. It is concluded that under normal conditions the calcium conductance has the extended time course clearly evident under the modified conditions of paragraph 4 but that the calcium flux component is easily missed. 6. In agreement with several prior studies it is also concluded that a rise in internal calcium is causally related to a rise in potassium conductance. A transmembrane flux of calcium can be uncoupled from the gK increase by appropriate buffering of internal calcium. 7. The transient potassium current, IA, which bears a resemblance to calcium-dependent potassium transients in some muscle cells did not depend upon internal calcium but instead is a voltage-activated mechanism.