Biomedical subjects
N Sperelakis
Publications and source records attributed to N Sperelakis.
Regulation of the calcium slow channel by cyclic GMP dependent protein kinase in chick heart cells.
In order to assess the interaction between the cAMP-dependent and the cGMP-dependent phosphorylation pathways on the slow Ca2+ current (ICa(L)), whole-cell voltage-clamp experiments were conducted on embryonic chick heart cells. Addition of 8Br-cGMP to the bath solution reduced the basal (unstimulated) ICa(L). Intracellular application of the catalytic subunit of PK-A (PK-A(cat); 1.5 microM) via the patch pipette rapidly potentiated ICa(L) by 215 +/- 16%) (n = 4); subsequent addition of 1 mM 8Br-cGMP to the bath reduced the amplitude of ICa(L) towards the initial control values (123 +/- 29%). Intracellular application of PK-G (25 nM pre-activated by 10(-7) M cGMP), rapidly inhibited the basal ICa(L) by 64 +/- 6% (n = 8). Heat-denatured PK-G was ineffective. Subsequent additions of relatively high concentrations of 8Br-cAMP (1 mM) or isoproterenol (ISO, 1-10 microM) did not significantly remove the PK-G blockade of ICa(L). The results of the present study suggest that: (a) 8Br-cGMP can inhibit the basal or stimulated (by PK-A(cat)) ICa(L) in embryonic chick myocardial cells. (b) PK-G applied intracellularly inhibits the basal ICa(L).
Potassium currents in rat colonic smooth muscle cells and changes during development and aging.
In a previous study on freshly isolated single smooth muscle cells from the circular layer of the rat distal colon, we reported that the L-type Ca2+ current density increased during development and gradually declined with further aging [ZI Xiong, N Sperelakis, N Noffsinger, C Fenoglio-Preiser (1993) Am J Physiol 265: C617-C625]. Since K+ current plays a key role in controlling excitability of the cells and hence the motility of the colon, in the present study the voltage-gated K+ channel currents, (IK) were investigated using the whole-cell voltage-clamp technique in colonic myocytes from rats of different ages. A Ca(2+)-sensitive K+ current [IK(Ca)] and two kinds of Ca(2+)-insensitive outward K+ currents were identified and characterized. IK(Ca) was recorded at potentials more positive than -40 mV in Ca(2+)-containing bath solution, and was blocked by Ca2+ channel antagonists and tetraethylammonium ion (TEA+). After removing Ca2+ from the bath solution and using a high ethylenebis(oxonitrilo)tetraacetate (EGTA, 4 mM) concentration in the pipette, two types of Ca(2+)-insensitive IK were recorded. The first and faster component was usually activated at potentials more positive than -50 mV, and was more sensitive to 4-aminopyridine (4-AP). In contrast, the second and slower (delayed) component was activated at potentials more positive than -30 mV, and was more sensitive to TEA. The total density of the Ca(2+)-insensitive IK component decreased dramatically during the neonatal period: from 32.2 +/- 3.2 pA/pF in 3-day-old rats to 17.8 +/- 2.6 pA/pF in 40-day-old rats; there was no further decline during aging (up to 480 days).(ABSTRACT TRUNCATED AT 250 WORDS)
Long openings of calcium channels in fetal rat ventricular cardiomyocytes.
Whole-cell and single-channel Ca2+ currents (ICa) of single fetal (12-18 days) and neonatal (1-10 d) rat ventricular myocytes were recorded using patch clamp techniques. Whole-cell ICa density increased markedly during the fetal period and remained almost constant during the neonatal period. In cell-attached patch recordings (with 110 mM Ba2+ in the pipette), the L-type Ca2+ channels, observed on both fetal d-12 and neonatal d 5, had the same conductance (23 pS). On fetal d-12, many relatively long openings were observed in addition to brief openings, whereas on neonatal d-5, long openings were less observed and brief openings dominated. Therefore, long openings of the Ca2+ channels occur in early development in rat heart cells, similar to that reported for chick hearts [7, 8]. The increase of the whole-cell current amplitude observed during development may be due to an increase in available channel number.
Dioctanoyl-glycerol inhibits L-type calcium current in embryonic chick cardiomyocytes independent of protein kinase C activation.
Diacylglycerol analogs and phorbol esters are used as protein kinase C (PKC) activators to investigate the effect of PKC on L-type calcium current [ICa(L)] in cardiomyocytes. 1-2-dioctanoyl-sn-glycerol (diC8) is a potent analog of diacylglycerol (DAG) which produces positive inotropic effects in guinea-pig atria cardiomyocytes via PKC activation. DiC8 effect on ICa(L), recorded (at 25 degrees C) in whole-cell voltage-clamp, was measured in 17-day-old embryonic chick cardiomyocytes in culture. ICa(L) was recorded in Na+, K(+)-free solution (external) and Ca(2+)-, K(+)-free solution (pipette), with depolarizing steps (to +10 mV) applied from a holding potential of -40 mV. Perfusion with different concentrations of diC8 (from 0.1 to 100 microM) inhibited ICa(L) in a dose-dependent manner, with half-maximal inhibition occurring at 12.5 microM. The effect of diC8 occurred rapidly, the effect beginning within 2 min and being completed within 5 min. In order to determine if the inhibitory effect of diC8 on ICa(L) was through activation of PKC, 25 microM diC8 was applied after pre-incubation of the cardiomyocytes with the PK inhibitors staurosporine (1 microM) or H-7 (50 microM). The effect of diC8 was not prevented by staurosporine or H-7. To further rule out the involvement of PKC in the action of diC8, experiments were performed using another analog of DAG, 1-oleyl-2-acetyl-glycerol (OAG, 50 microM) and Angiotensin-II (A-II, 0.1 microM). OAG failed to produce any effect on ICa(L). A-II, believed to act by activation of PKC did not affect ICa(L) within a test period of 8 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Modulation of L-type Ca2+ current by isoprenaline, carbachol and phorbol ester in cultured rat aortic vascular smooth muscle (A7r5) cells.
1. Effects of isoprenaline (ISO), carbachol and phorbol ester (a stimulator of protein kinase C) on L-type Ca2+ channels in single cultured rat aortic vascular smooth muscle (A7r5) cells were examined using whole-cell voltage clamp (at room temperature 22 degrees C). 2. With 20 mmol/l Ca2+ in the bath solution and 10 mmol/l EGTA in the pipette solution, a slow ICa (L-type) current was observed in the A7r5 cell line, which was blocked by nifedipine (2 mumol/l). 3. ISO (5 mumol/l) inhibited ICa by 18.3 +/- 2.2% (P < 0.001), and carbachol (1 mumol/l) also decreased ICa by 15.0 +/- 3.2% (P < 0.01). 8-Br-cAMP (1 mmol/l) and 8-Br-cGMP (1 mmol/l) both inhibited ICa by 30.1 +/- 2.8% (P < 0.001) and 18.8 +/- 3.8% (P < 0.01), respectively. 4. Phorbol ester, 4-beta-phorbol-12, 13-dibutyrate (PDB), at 0.1-1 mumol/l, had almost no effect on ICa in most cells, but slightly potentiated (or slightly enhanced) the inhibitory effects of ISO. 5. Time decay (inactivation) of ICa consisted of two exponentials. Both the fast and slow time constants were slightly prolonged by ISO (5 mumol/l), and by carbachol (1 mumol/l); PDB (1 mumol/l) slightly shortened the fast time constant only. The half-maximum voltages of inactivation were not significantly affected by any of the agents. 6. These results suggest that the L-type ICa current is modulated by cyclic nucleotides (cAMP and cGMP) and by PK-C stimulation, and thereby contribute to regulation of contraction of the vascular smooth muscle cells.
Ca2+ currents in human colonic smooth muscle cells.
Voltage-gated Ca2+ currents were investigated in single smooth muscle cells freshly isolated from the circular layer of the human colon (ascending and descending portions) using the whole cell voltage-clamp technique. Tissue samples were obtained at the time of therapeutic surgery. In physiological salt solution (containing 2 mM Ca2+), an inward current was observed when the cell membrane was depolarized in the presence of tetrodotoxin. This current disappeared when Ca2+ was removed from the bath solution and was inhibited when Ca2+ channel blockers were applied, indicating that the inward current was a Ca2+ current (ICa). Changing the holding potential (HP) from -100 mV to more positive potentials (e.g., -60 and -40 mV) markedly decreased the amplitude of ICa. The voltage dependence of steady-state activation and inactivation was represented by Boltzmann distributions; there was a substantial amount of overlap (window current) between -60 and -10 mV. A fast-inactivating ICa component followed by a slow-inactivating ICa component was observed in some cells from both ascending and descending colons. The fast ICa component was observed only when cells were held at -80 or -100 mV, and had a more negative threshold potential (-70 to -60 mV). This component was sensitive to low concentrations of Ni2+ (30 microM) but was resistant to nifedipine (10-20 microM). In contrast, the slow (sustained) ICa component was observed at all HPs (-40 to -100 mV) and had a more positive threshold potential (about -40 mV). This component was insensitive to low concentration of Ni2+ but was sensitive to nifedipine and BAY K 8644.(ABSTRACT TRUNCATED AT 250 WORDS)
cGMP-dependent protein kinase regulation of the L-type Ca2+ current in rat ventricular myocytes.
Regulation of L-type Ca2+ channel current [ICa(L)] by cGMP-dependent protein kinase (PK-G) was investigated in ventricular myocytes from 2- to 21-day-old rats using whole-cell voltage clamp with internal perfusion. ICa(L) was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Stimulated ICa(L) (by 2 mumol/L isoproterenol) was inhibited to the basal level by internal perfusion with 50 nmol/L PK-G (activated by 8Br-cGMP, 0.1 mumol/L). When ICa(L) was enhanced by Bay K8644 (1 mumol/L), the enhanced basal ICa(L) was also reduced by PK-G. Basal ICa(L) (nonstimulated through the cAMP/cAMP-dependent protein kinase [PK-A] pathway) was also inhibited to various degrees (large, medium, or small) by internal application of PK-G (25 nmol/L). The average inhibition was 42.1% (n = 36), and there were no differences in the inhibition during development. The inhibition by PK-G was blocked by the PK-G substrate peptide (cG-PKI, 300 mumol/L) and by heat inactivation of the PK-G. Relatively specific PK-G inhibitors (eg, cG-PKI and H-8) sometimes reversed the inhibition (5 of 25 cells), whereas isoproterenol stimulated ICa(L) (7 of 8 cells). When a holding potential of -80 mV was used, the inhibition produced by PK-G was much less. The inhibitory effects of PK-G were not mediated by activating phosphodiesterase or protein phosphatase but most likely by a direct phosphorylation of the Ca2+ channel or associated regulatory protein. The inhibitory effect of PK-G may be explained by a balance between activities of PK-A and PK-G in regulating the slow Ca2+ channels at two separate sites.
Oxytocin induces an inward current in pregnant rat myometrial cells.
The effects of oxytocin (OT) on holding current were studied in uterine smooth muscle cells freshly isolated from the longitudinal layer of 18-20 day pregnant rats, using the nystatin method of whole-cell voltage clamp. As we previously reported, the voltage-dependent Ca2+ current (L type) was partially inhibited by OT (about 30% inhibition at 1 microM). When the cells were held at the holding potential (HP) of -60 mV and the holding current was monitored, OT induced an inward current. The amplitude of this OT-induced current was 72 +/- 26 pA (n = 27). When the cell was held at more positive potentials (HP 0 or +40 mV), the OT-induced current reversed direction, becoming outward. This current usually was long lasting (74% of cells responding to OT); a transient current was observed in 26% of the cells. In the absence of either Na+ or Ca2+ in the bath solution, OT induced an inward current (at HP -60 mV). However, the OT-induced current was absent when both of these ions were omitted from the bath. These results suggest that OT induces an inward current through receptor-activated nonselective cation channels. The resulting increase of intracellular Ca2+ may contribute to the inhibition of voltage-dependent Ca2+ current produced by OT. This OT-induced current may also play an important role for membrane depolarization and accompanying contraction produced by OT in pregnant rat myometrium.
Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus.
Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L-type) Ca2+ channels and fast (T-type) Ca2+ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by TTX (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current, and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na+ channels, and it is suggested that the fast Na+ current may be involved in spread of excitation. The Ca2+ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 of 12 mM) and nifedipine (K0.5 of 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of ICa(s). Figure 11 summarizes the possible mechanisms by which uterine contractility can be modulated. In contrast to vascular smooth muscle, neither ISO nor adenosine, which produce elevation of cyclic AMP, affected ICa and INa. Therefore, no arrow can be drawn between cA-PK/cG-PK and the Ca2+ slow channel.(ABSTRACT TRUNCATED AT 400 WORDS)
Taurine inhibition of fast Na+ current in embryonic chick ventricular myocytes.
Effects of taurine on the fast Na+ current (INa) in 17-day-old embryonic chick ventricular myocytes were examined using the whole-cell voltage-clamp technique. The cells were spherical (10-15 microns diameter) and had a capacitance of 9.8 +/- 1.3 pF. The experiments were performed at room temperature (22 degrees C), and the holding potential was -90 mV. After the patch membrane was broken, peak INa initially increased, and then decreased and became stable within 3-5 min. The experiments on taurine were started after INa had stabilized. The characteristics of INa were as expected, including sensitivity to tetrodotoxin (10 microM). When added to the bath, taurine inhibited INa and shifted the reversal potential in the hyperpolarizing direction. At 10 mM, taurine inhibited INa by 38.2 +/- 4.3%, and shifted the reversal potential by 10.2 +/- 3.1 mV. The time to peak current was slowed: 0.83 +/- 0.20 ms (n = 11) in control, 1.03 +/- 0.18 ms (n = 9) in 10 mM taurine, and 1.10 +/- 0.19 ms (n = 10) in 20 mM taurine. These effects of taurine were not reversed by 30 min washout. At low concentrations, taurine actually enhanced INa in 3 of 8 cells at 1 mM, and in 4 of 10 cells at 5 mM; the reversal potential was still shifted in the hyperpolarizing direction by 5.7 +/- 1.6 mV. The time course of inactivation (fitted as a single exponential at test potential of -30 mV) was not affected: 1.1 +/- 0.5 ms in control 1.2 +/- 0.4 ms at 10 mM taurine.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of calcium slow channels in cardiac muscle and vascular smooth muscle cells.
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Taurine effects on ionic currents in myocardial cells.
The effects of taurine on the slow and fast Na+ currents and slow and fast Ca2+ currents in cultured single ventricular cells from young (3-day-old) and old (10, 17 day) chick embryos were studied using the whole-cell voltage clamp technique. In single 3-day cells that showed only a TTX-insensitive fast (transient) Na+ current (INa(f)), taurine (5 mM) rapidly increased the amplitude of this current. In single cells that showed only a typical slow (sustained) Na+ current (INa(s)), taurine (5 mM) induced a fast transient component. A slow Ca2+ current (ICa(s)) was also present in the 3-day-old embryonic chick cells, and taurine inhibited this current and activated a fast transient component (ICa(f)). Taurine had similar actions in 10-day-old embryonic heart cells. Thus, in embryonic chick heart cells, taurine stimulates the TTX-insensitive fast transient Na+ current and blocks the slow component. Taurine also activates a fast (transient) component of the Ca2+ current (ICa(f)). The activation of the TTX-insensitive INa(f) may increase Ca2+ influx via Na(+)-Ca2+ exchange. This may explain, in part, the positive inotropic effect of taurine in heart muscle, with relatively little effect on the Ca(2+)-dependent slow APs. Taurine (10 or 20 mM) added to the outside markedly inhibited TTX-sensitive INa, but slightly stimulated INa when added internally. ICa(s) and IK both were stimulated by external taurine at pCa 10 but inhibited at pCa 7. Taurine also inhibited IK in aortic VSM cells. In contrast, taurine induced or stimulated ICa(f). Elevation of [Ca]i was induced by taurine. The elevation may result from the enhancement of ICa(f) and possibly of Na(+)-Ca2+ exchange, resulting in a positive inotropic effect. It has been shown that in neurons, taurine increases the Cl current, resulting in hyperpolarization (Taber et al., 1986; Figure 12). Thus, taurine effects are complex, there being a number of actions on the membrane currents of cardiac cells, vascular smooth muscle cells, and neurons.
Kinetic and steady-state properties of Na+ channel and Ca2+ channel charge movements in ventricular myocytes of embryonic chick heart.
Nonlinear or asymmetric charge movement was recorded from single ventricular myocytes cultured from 17-d-old embryonic chick hearts using the whole-cell patch clamp method. The myocytes were exposed to the appropriate intracellular and extracellular solutions designed to block Na+, Ca2+, and K+ ionic currents. The linear components of the capacity and leakage currents during test voltage steps were eliminated by adding summed, hyperpolarizing control step currents. Upon depolarization from negative holding potentials the nonlinear charge movement was composed of two distinct and separable kinetic components. An early rapidly decaying component (decay time constant range: 0.12-0.50 ms) was significant at test potentials positive to -70 mV and displayed saturation above 0 mV (midpoint -35 mV; apparent valence 1.6 e-). The early ON charge was partially immobilized during brief (5 ms) depolarizing test steps and was more completely immobilized by the application of less negative holding potentials. A second slower-decaying component (decay time constant range: 0.88-3.7 ms) was activated at test potentials positive to -60 mV and showed saturation above +20 mV (midpoint -13 mV, apparent valence 1.9 e-). The second component of charge movement was immobilized by long duration (5 s) holding potentials, applied over a more positive voltage range than those that reduced the early component. The voltage dependencies for activation and inactivation of the Na+ and Ca2+ ionic currents were determined for myocytes in which these currents were not blocked. There was a positive correlation between the voltage dependence of activation and inactivation of the Na+ and Ca2+ ionic currents and the activation and immobilization of the fast and slow components of charge movement. These complementary kinetic and steady-state properties lead to the conclusion that the two components of charge movement are associated with the voltage-sensitive conformational changes that precede Na+ and Ca2+ channel openings.
Novel isoform of Ca2+ channel in rat fetal cardiomyocytes.
1. Single cardiomyocytes of 18-day-old rat fetuses were isolated to characterize the cardiac Ca2+ channels in the fetal period, using whole-cell voltage clamp (Na+, K(+)-free external solution and K(+)-free internal solution), and depolarizing test pulses from a holding potential (HP) of -87 mV were applied. 2. The Ca2+ current was completely blocked by 2 mM-CO2+, but not completely blocked by the dihydropyridine (DHP) Ca2+ antagonist nifedipine. Nifedipine (3 microM) decreased the amplitude of the current (at -7 mV) by 65.9 +/- 3.4% (n = 20). At a HP of -47 mV, nifedipine decreased the Ca2+ current to about the same degree. Diltiazem (1 microM) did not block the nifedipine-resistant current which remained. 3. Nitrendipine, another DHP Ca2+ antagonist, had effects on the Ca2+ current similar to those of nifedipine. 4. The DHP-resistant current was not blocked by T-type channel blockers (Ni2+, tetramethrine) or an N-type blocker (omega-conotoxin). 5. In conclusion, rat fetal cardiomyocytes may have a unique type of Ca2+ channel (ICa(fe)), which decreases in amplitude and becomes less prominent during subsequent development.
Fast Na+ channels in smooth muscle from pregnant rat uterus.
Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).
Oxytocin actions on voltage-dependent ionic channels in pregnant rat uterine smooth muscle cells.
The effects of oxytocin, a uterotonic polypeptide hormone, on the voltage-dependent slow calcium, fast sodium, and potassium channel currents were studied using whole-cell voltage clamp of freshly isolated cells from late pregnant (18-21 day) rat myometrium. The calcium current was rapidly inhibited by oxytocin (about 25% inhibition at 20 nM) in a dose-dependent manner, and this inhibitory effect was completely reversible by washout. However, inhibition was not observed when barium was used as the charge carrier. Sodium current and potassium current were not modified by oxytocin, thus sodium and potassium currents may not play important roles in oxytocin-induced augmentation of uterine contraction. It is concluded that oxytocin stimulates uterine contraction by mechanisms other than augmentation of the voltage-dependent calcium current, e.g., by release of Ca from sarcoplasmic reticulum (by inositol triphosphate) or by activation of a receptor-operated Ca channel. The inhibition of the slow calcium current may be induced by the elevation of [Ca]i.
Developmental changes in long-opening behavior of L-type Ca2+ channels in embryonic chick heart cells.
In the early (3-day) stage of development, long-lasting openings of the L-type Ca2+ channels (mode 2) occur in embryonic chick heart cells. Since mode-2 behavior is infrequently observed in adult heart cells of other species, in the present study, developmental change in behavior of the Ca2+ channel was examined in young (3-day) and old (17-day) embryonic chick heart cells. In the whole-cell voltage clamp, the L-type Ca2+ current carried by Ca2+ ions was smaller in amplitude and had a faster inactivation in 17-day cells than in 3-day cells. The peak current density was 8.1 +/- 0.2 microA/cm2 (mean +/- SEM, n = 5) and 5.1 +/- 0.3 microA/cm2 (n = 5) in 3-day and 17-day cells, respectively. When the charge carrier was Ba2+, the L-type Ca2+ channel current density was also smaller in 17-day cells (22.7 +/- 1.8 microA/cm2) than in 3-day cells (28.3 +/- 2.1 microA/cm2). In single-channel recordings, the mode-2 behavior was infrequent in 17-day cells compared with 3-day cells. High-open probability sweeps (with an open probability of greater than 0.25), reflecting mode-2 behavior, accounted for 20.2% and 3.7% in 3-day and 17-day cells, respectively. The ensemble-averaged currents in 17-day cells was 37% of that current in 3-day cells. In addition, decay of the averaged current appeared to be faster in 17-day cells than in 3-day cells. All data from the single-channel analysis agreed with the data from the whole-cell voltage clamp.(ABSTRACT TRUNCATED AT 250 WORDS)