Inactivation of the Ca channel of Aplysia neurons was studied in the absence of potassium current in cells that were cesium-loaded with the aid of the ionophore nystatin. Inactivation was substantially decreased by methods that limited Ca entry. Depolarizations commensurate with the equilibrium potential of Ca resulted in minimal inactivation. Replacement of extracellular Ca by Ba also decreased inactivation. It is concluded that inactivation of the Ca channel is a function of the extent of Ca entry rather than membrane potential, thus differing fundamentally from the purely voltage-dependent mechanism for sodium inactivation.The importance of transient changes in levels of intracellular free Ca in various cell functions has led to increased interest in the properties of the Ca channel that is activated by membrane depolarization. Voltage-dependent Ca entry has been demonstrated in muscle fibers (1), nerve cell bodies (2), axons (3), presynaptic terminals (4), and dendrites (5). Rapid increases in intracellular Ca concentration function in such diverse processes as excitation-contraction coupling (6), synaptic transmission (7), and the activation of a Ca-mediated potassium conductance (8).Voltage-clamp analyses of Ca current (ICa) and the behavior of the Ca channel have been hampered by the presence of other simultaneous ionic currents, especially that of potassium ('K), that sum algebraically with ICa' A number of experimental procedures have been devised to circumvent this problem in molluscan neurons. Use of the calcium-sensitive molecules arsenazo III (9) and aequorin (10-12) allows monitoring of changes in intracellular Ca levels and indirect measurement of Ca entry. Direct measurement of ICa has been made possible by minimizing competing currents with internal perfusion (13-15) and nystatin-mediated loading to replace intracellular K (16, 17). In the present study the ionophore nystatin was used to substitute the impermeant ion Cs+ for intracellular K+, thereby minimizing IK during depolarization.In previous voltage-clamp studies, inactivation of the Ca channel has been observed both as a relaxation of ICa during a single depolarizing pulse (14,15,18) and as a decreased peak amplitude ICa during the second of two closely spaced depolarizing pulses (17). In two studies on molluscan neurons (15, 19), of which only one used a method for minimizing 'K (15), it was suggested that the mechanism of Ca inactivation is similar to that for Na inactivation. The experiments of the present report offer quite a different conclusion: that Ca inactivation results from the extent of Ca entry by a process not directly dependent upon membrane potential and therefore differs fundamentally from the purely voltage-dependent Na channel inactivation in the Hodgkin-Huxley formulation (20). Some of these experiments have appeared in abstract form (21). MATERIALS AND METHODSExperiments were performed on single identified neurons of Aplysia californica that were kept in a self-contained seawater *system. The visceral ganglion...

SUMMARY1. The intracellular potassium in giant neurones ofAplysia californica was replaced with caesium by a method utilizing the ionophore nystatin. Because caesium ions have low permeability through potassium channels, outward currents during voltageclamp depolarization were strongly curtailed after the caesium loading procedure and the subsequent wash-out of the ionophore.2. The calcium current elicited by a test voltage-clamp depolarization (pulse 2) was depressed following the entry of calcium elicited by a prior depolarization (pulse 1).3. The percentage depression of the test current was a linear function of the pulse 1 current-time integral, and thus appears to be related linearly to the amount of calcium carried into the cell during pulse 1. This linear relation was maintained when calcium entry was varied by changes in external calcium concentration, by altered pulse 1 amplitude and altered pulse 1 duration. Depression was substantially reduced by injection of EGTA, and by substitution of barium for extracellular calcium.4. The calcium current was unaffected by prior hyperpolarization ofthe membrane or by prior depolarizations to about Eca. Depression of the current was not altered by addition of extracellular 50 mm-TEA or by a strong hyperpolarization between the conditioning and test pulses. 5. The rate relaxation of the inward current during a given depolarization depended on the rate of entry and accumulation of free calcium. Relaxation under a given command potential became slower when calcium was partially replaced with magnesium so as to produce a smaller calcium current; or when accumulation of intracellular free calcium was retarded by injected EGTA or by barium substitution for extracellular calcium.6. Evidence is considered that accumulation of calcium ions at the cytoplasmic surface of the membrane leads to inactivation through an action upon the calcium conductance. Reduced driving force and intracellular surface-charge neutralization do not adequately account for the observed depression of the calcium current resulting from intracellular accumulation of calcium ions.

SUMMARY1. The Ca current seen in response to depolarization was investigated in Paramecium caudatum under voltage clamp. Inactivation of the current was measured with the double pulse method; a fixed test pulse of an amplitude sufficient to evoke maximal inward current was preceded by a conditioning pulse of variable amplitude (0-120 mV).2. The amplitude of the current recorded during the test pulse was related to the potential of the conditioning pulse. Reduction of test pulse current was taken as an index of Ca current inactivation. The current recorded during a test pulse showed a progressive decrease to a minimum as the potential of the conditioning pulse approached + 10 to + 30 mV. Further increase in conditioning pulse amplitude was accompanied by a progressive restoration of the test pulse current. Conditioning pulses near the calcium equilibrium potential had only a slight inactivating effect on the test pulse current.3. Injection of a mixture of Cs and TEA which blocked late outward current had essentially no effect on the inward current or its inactivation.4. Elevation of external Ca from 0 5 to 5 mM was accompanied by increased inactivation of the test pulse current. The enhanced inactivation of the test pulse current was approximately proportional to the increase in current recorded during the conditioning pulse.5. Following injection of the Ca chelating agent, EGTA, the inactivation of the test pulse current was diminished; in addition, the transient inward current relaxed slightly more slowly, and the transient was followed by a steady net inward current.6. The time course of recovery from inactivation in the double pulse experiment approximated a single exponential having a time constant of 80-110 msec. Injection of EGTA shortened the time constant by as much as 50 %.7. It is concluded that interference with the entry of Ca or enhanced removal of intracellular free Ca2+ interferes with the process of Ca current inactivation, while enhanced entry of Ca promotes the process of inactivation. While the mechanism of inactivation is unknown, arguments are presented that the accumulation of intracellular Ca influences the Ca channel conductance.

Erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate the differentiation and proliferation of erythroid cells. To determine the cellular mechanism of action of these growth factors, we measured changes in intracellular free calcium concentration (jCaJ) in single human erythroid precursors in response to recombinant erythropoietin and GM-CSF. [CaJ in immature erythroblasts derived from cultured human cord blood erythroid progenitors was measured with fluorescence microscopy digital video imaging.When stimulated with erythropoietin, [CaJ in the majority of erythroblasts increased within 3 min, peaked at 5 min, and returned toward baseline at 10 min. The percentage of cells that responded to erythropoietin stimulation increased in a dose-dependent manner. Additional stimulation with GM-CSF in cells previously exposed to erythropoietin resulted in a second [Ca¢J increase. Immature erythroblasts treated with GM-CSF followed by erythropoietin responded similarly to each factor with a rise in [Ck]. The source of transient calcium is intracellular since erythroblasts were incubated in medium devoid of extracellular calcium. Our observations suggest that changes in [CaJ may be an intracellular signal that mediates the proliferative/differentiating effect of hematopoietic growth factors.

Myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) had lower peak cytosolic free Ca2+ concentration ([Ca2+]i) and reduced maximal extent of cell shortening during contraction, but Ca2+ entry via L-type Ca2+ channels was normal. In the current study using whole cell patch-clamp technique, reverse Na+/Ca2+ exchange current (INa/Ca; 3 Na+ out:1 Ca2+ in) was measured in myocytes in which Na+, K+, and Ca2+ currents were blocked or minimized. Steady-state outward currents measured under these conditions increased with depolarization or with elevation of extracellular Ca2+ concentration ([Ca2+]o) from 1.8 to 5.0 mM, but were inhibited by 5 mM Ni2+ or by reduction of [Ca2+]i to near zero. In addition, reduction of cytosolic free Na+ concentration or of [Ca2+]i also decreased the amplitude of the outward current. These characteristics indicate the outward current was INa/Ca operating in reverse mode. Reverse INa/Ca was significantly lower in MI myocytes, especially at more positive voltages. In addition, sarcoplasmic reticulum (SR)-releasable Ca2+ content as estimated by integrating forward INa/Ca during caffeine-induced SR Ca2+ release was also significantly lower in MI myocytes. Depressed Na+/Ca2+ exchange activity may contribute to abnormal [Ca2+]i dynamics in MI myocytes.