人胎儿鼻咽上皮细胞的背景氯电流

采用膜片钳和图像分析技术,研究人胎儿鼻咽上皮细胞背景电流的特性及其与容积激活性氯电流的关系。在等张溶液中,可记录到一背景电流,该电流呈微弱的外向整流性,无明显时间依赖性失活,其翻转电位为(?0.73±1.7)mV(n=21),接近氯离子平衡电位(?0.9mV)。细胞外高张刺激(440mOsmol/L)明显抑制此电流(59.6±7.1)%,而低张刺激(160mOsmol/L)则诱发细胞产生容积激活性氯电流。氯通道阻断剂tamoxifen和5-硝基-2-(3-苯丙胺基)苯甲酸[5-nitro-2-(3-phenylpropylamino)benzoicacid,NPPB]显著地抑制背景电流并使细胞基础容积增大。上述结果表明,人胎儿鼻咽上皮细胞的背景Cl?电流是背景电流的重要成分,此Cl?电流与容积激活性氯电流及细胞基础容积调节有关。     

The Resting Potential of Mouse Leydig Cells: Role of an Electrogenic Na+/K+ Pump

Resting potentials (Vm) were measured in mouse Leydig cells, using the whole-cell patch-clamp technique. In contrast to conventional microelectrode measurements, where a biphasic potential was observed, we recorded a stable Vm around -32.2 +/- 1.2 mV (mean +/- SEM, n = 159), at 25 degrees C, and an input resistance larger than 2.7 x 109 W. Although Vm is sensitive to changes in the extracellular concentrations of potassium and chloride, the relationship between Vm and these ions' concentrations cannot be described by either the Goldman-Hodgkin-Katz or the Nernst equation. Perifusing cells with potassium-free solution or 10?3 M ouabain induced a marked depolarization averaging 20.1 +/- 3.2 mV (n = 9) and 23.1 +/- 2.8 mV, (n = 7), respectively. Removal of potassium or addition of ouabain with the cell voltage-clamped at its Vm, resulted in an inwardly directed current, due to inhibition of the Na+K+ATPase. The pump current increased with temperature with a Q10 coefficient of 2.3 and had an average value of -6.5 +/- 0.4 pA (n = 21) at 25 degrees C. Vm also varied strongly with temperature, reaching values as low as -9.2 +/- 1.2 mV (n = 22) at 15 degrees C. Taking the pump current at 25 degrees C and a minimum estimate for the membrane input resistance, we can see that the Na+K+ATPase could directly contribute with 17.7 mV to the Vm of Leydig cells, which is a major fraction of the ?32.2 +/- 1.2 mV (n = 159) observed.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

A dual action of taurine on the delayed rectifier K+ current in embryonic chick cardiomyocytes

Summary Effects of taurine on the delayed rectifier K⁺ channel in isolated 10-day-old embryonic chick ventricular cardiomyocytes were examined at different intracellular Ca²⁺ concentrations ([Ca]_i), using whole-cell voltage and current clamp techniques. Experiments were performed at room temperature (22°C). Test pulses were applied between -20 to +90m V from a holding potential of -40mV. When [Ca]_i was pCa 7, addition of 10 and 20 mM taurine to the bath solution reduced the delayed rectifier K⁺ current (I_K) at +90mV by 17.4 ± 2.8% (n = 5, P < 0.01) and 25.5 ± 2.6% (n = 5, P < 0.001), respectively. In contrast, when [Ca]_i was pCa 10, I_K at +90 mV was enhanced by 19.1 ± 3.1% (n = 7, P < 0.01) at 10mM taurine, and by 29.3 ± 2.4% (n = 7, P < 0.001) at 20mM taurine. The voltage of half-maximum activation (V_1/2) was shifted in a hyperpolarizing direction; at pCa 7, the value was +0.2 ± 2.2mV (n = 5) in control and -10.6 ± 1.8mV (n = 5) in 20mM taurine. At pCa 10, the V_1/2 value was +18.5 ± 4.6mV (n = 5) in control and +6.6 ± 5.2mV (n = 5) in taurine (20mM). Taurine decreased the action potential duration (APD) at pCa 10, but at pCa 7 did not affect it. In addition, taurine enhanced the transient outward current in a concentration-dependent manner. These results indicate that taurine modulates the delayed rectifier K⁺ channel, an effect dependent on [Ca]_i and capable of regulating APD.     

L-type Ca2+ currents in ventricular myocytes from neonatal and adult rats

Postnatal changes in the slow Ca2+ current (I(Ca)(L)) were investigated in freshly isolated ventricular myocytes from neonatal (1-7 days old) and adult (2-4 months old) rats, using whole-cell voltage clamp and single-channel recordings. The membrane capacitance (mean+/-SEM) averaged 23.2+/-0.5 pF in neonates (n = 163) and 140+/-4.1 pF in adults (n = 143). I(Ca)(L) was measured as the peak inward current at a test potential of +10 mV (or +20 mV) by applying a 300-ms pulse from a holding potential of -40 mV; 1.8 mM Ca2+ was used as charge carrier. The basal ICa(L) density was 6.7+/-0.2 pA/pF in neonatal and 7.8+/-0.2 pA/pF in adult cells (p < 0.05). The time course of inactivation of the fast component (at +10 ms) was significantly longer in the neonatal (10.7+/-1.4 ms) than in the adult (6.6+/-0.4 ms) cells (p < 0.05). Ryanodine (10+/-M) significantly increased this value to 18.0+/-1.9 in neonate (n = 8) and to 17.7+/-2.0 in adult (n = 9). For steady-state inactivation, the half-inactivation potential (Vh) was not changed in either group. For steady-state activation, Vh was 5.1 mV in the neonatal (n = 6) and -7.9 mV in the adult cells (n = 7). Single-channel recordings revealed that long openings (mode-2 behavior) were occasionally observed in the neonatal cells (11 events from 1080 traces/11 cells), but not in the adult cells (400 traces/4 cells). Slope conductance was 24 pS in both the neonatal and adult cells. Results in rat ventricular myocytes suggest the following: (i) the peak Ca2+ current density is already well developed in the neonatal period (being about 85% of the adult value); (ii) the fast component of inactivation is slower in neonates than in adults; and (iii) naturally occurring long openings are occasionally observed in the neonatal stage but not in the adult. Thus, the L-type Ca2+ channels of the neonate were slightly lower in density, were inactivated more slowly, and occasionally exhibited mode-2 behavior as compared with those of the adult.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Whole-cell potassium current in rabbit corneal epithelium activated by fenamates

Summary Rabbit corneal epithelium contains a large-conductance, potassium-selective channel, which is a major contributor to the whole-cell current. In perforated-patch recordings of the macroscopic current, the isolated cells studied had resting voltages of –41±20 mV and capacitances of 5.8±2.6 pF (mean + sd for n=255). Activation of the channels was weakly voltage dependent. They opened at about –100 mV and reached an open probability of about 0.2 at +100 mV. The current was blocked by millimolar concentrations of external Ba²⁺ and quinidine. Diltiazem also blocked when applied to the external surface of the membrane. Nonsteroidal anti-inflammatory agents of the fenamate group were powerful activators of the channel at submillimolar concentrations when applied either to the inside or the outside of the channels. The mechanism of action which leads to his activation is not yet known.We are grateful to Erika Wohlfiel for secretarial help, Helen Hendrickson for cell preparation, and Joan Rae for software development. This work was supported by NIH grants EY06005 and EY03282 and an unrestricted award from Research to Prevent Blindness.     

Membrane properties of isolated mudpuppy taste cells

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.     

Characterization of the calcium-sensitive voltage-gated delayed rectifier potassium channel in isolated guinea pig hepatocytes

The voltage-dependent K+ channel was examined in enzymatically isolated guinea pig hepatocytes using whole-cell, excised outside-out and inside- out configurations of the patch-clamp technique. The resting membrane potential in isolated hepatocytes was -25.3 +/- 4.9 mV (n = 40). Under the whole-cell voltage-clamp, the time-dependent delayed rectifier outward current was observed at membrane potentials positive to -20 mV at physiological temperature (37 degrees C). The reversal potential of the current, as determined from tail current measurements, shifted by approximately 57 mV per 10-fold change in the external K+ concentration. In addition, the current did not appear when K+ was replaced with Cs+ in the internal and external solutions, indicating that the current was carried by K+ ions. The envelope test of the tails demonstrated that the growth of the tail current followed that of the current activation. The ratio between the activated current and the tail amplitude was constant during the depolarizing step. The time course of growth and deactivation of the tail current were best described by a double exponential function. The current was suppressed in Ca(2+)-free, 5 mM EGTA internal or external solution (pCa > 9). The activation curve (P infinity curve) was not shifted by changing the internal Ca2+ concentration ([Ca2+]i). The current was inhibited by bath application of 4-aminopyridine or apamin. alpha 1-Adrenergic stimulation with noradrenaline enhanced the current but beta-adrenergic stimulation with isoproterenol had no effect on the current. In single- channel recordings from outside-out patches, unitary current activity was observed by depolarizing voltage-clamp steps whose slope conductance was 9.5 +/- 2.2 pS (n = 10). The open time distribution was best described by a single exponential function with the mean open lifetime of 18.5 +/- 2.6 ms (n = 14), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 2.0 +/- 0.3 ms (n = 14) and that for the slow component of 47.7 +/- 5.9 ms (n = 14). Ensemble averaged current exhibited delayed rectifier nature which was consistent with whole-cell measurements. In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The concentration of Ca2+ at the half-maximal activation was 0.031 microM. These results suggest that guinea pig hepatocytes possess voltage-gated delayed rectifier K+ channels which are modified by intracellular Ca2+.     

Calcium channel currents in isolated smooth muscle cells from human bronchus

An electrophysiological study was carried out on smooth muscle cells that were enzymatically dissociated from bundles of muscle fibers dissected out of human bronchi obtained at thoracotomy. These cells that retain the contractile properties of intact bundles were voltage-clamped by means of the whole-cell patch-clamp technique. Upon voltage steps from a holding potential of -60 mV to more positive levels, the initial inward current was followed by large outward currents that inactivated slowly. These were subsequently reduced by substituting Cs+ for K+ in the internal solution and by using Ba2+ instead of Ca2+ as a charge carrier in the external solution. Under these conditions, the inward current did not completely inactivate in the course of 300-ms voltage steps. Inward current measured after leak subtraction was activated at a membrane potential of -25.8 +/- 5 mV, was maximum at +18 +/- 4 mV, and had an apparent reversal potential of +52.5 +/- 5.5 mV (n = 5). The potential at which steady-state inactivation was half-maximum was -28 mV (n = 5). This inward current was identified as a calcium current on the following basis: 1) it was not altered by 10 microM tetrodotoxin (TTX) or by lowering to 10 mM external Na+ concentration; 2) it was blocked by 2.5 mM Co2+ or 1 microM PN 200-110; 3) it was enhanced by 1 microM BAY K 8644, which in addition suppressed the PN 200-110 blockade.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-dependent inactivation of the potassium current of embryonic chick hepatocytes

The whole-cell patch electrode voltage clamp technique was used to study the inactivation properties of the delayed rectifying potassium current of single cultured embryonic chick hepatocytes at 20 degrees C. The potassium current activates maximally within 250-500 ms of membrane depolarization, after which it decays with a monoexponential time course. Both steady-state activation and inactivation are voltage dependent. Steady-state inactivation declines from 100% at -5 mV to 0 near -70 mV. with half inactivation at -41 mV. At the resting potential (EM) of these cells (-21.5 +/- 6.0 mV, n = 36) 6-18% of the IK channels are not inactivated and less than 5% are open. Development and removal of inactivation follow single exponential time courses. The inactivation time constant attains a maximum of around 30 s at -35 mV and is sharply voltage dependent at the EM of these cells. Measurement of EM under current clamp shows random oscillations of 5-10 mV amplitude. We suggest that the voltage- and time-dependent properties of IK, in tandem with a time- and voltage-independent, non-selective current also seen here, would provide the mechanism for a fluctuating EM.     

2,4-dinitrophenol acutely inhibits rabbit atrial Ca2+ -sensitive Cl- current (I(TO2))

We investigated the effects of 2,4-dinitrophenol (DNP), the uncoupler of mitochondrial oxidative phosphorylation, on the Ca2+ -sensitive Cl- current component of the transient outward current (I(TO2)). Amphotericin B perforated-patch, whole-cell patch-clamp technique was employed (35 degrees C) using enzymatically isolated single rabbit atrial myocytes. We defined I(TO2) as the amplitude of the 2 mM 4-aminopyridine resistant transient outward current sensitive to anthracene-9-carboxylic acid (A9C). Between +5 and +45 mV, 0.2 mM A9C inhibited I(TO2) by approximately 70% (n = 13). Within 30 s after application of 0.2 mM DNP, both normal I(TO2) transients (n = 8) and the I(TO2) transients that remained after A9C treatment (n = 8) were inhibited completely. In cells expressing I(TO2) (70% of total), DNP also suppressed an A9C-insensitive slow outward current by approximately 40%, but the holding current at -80 mV was unaffected. There was a approximately 2 min latency between inhibitory effects of DNP and subsequent membrane current increase, presumably caused by activation of the ATP-sensitive K+ channels (n = 16). We conclude that DNP acutely inhibits I(TO2) via a mechanism presumably separate from metabolic inhibition.     

Sodium and calcium currents in dispersed mammalian septal neurons

Voltage-gated Na+ and Ca2+ conductances of freshly dissociated septal neurons were studied in the whole-cell configuration of the patch-clamp technique. All cells exhibited a large Na+ current with characteristic fast activation and inactivation time courses. Half-time to peak current at -20 mV was 0.44 +/- 0.18 ms and maximal activation of Na+ conductance occurred at 0 mV or more positive membrane potentials. The average value was 91 +/- 32 nS (approximately 11 mS cm-2). At all membrane voltages inactivation was well fitted by a single exponential that had a time constant of 0.44 +/- 0.09 ms at 0 mV. Recovery from inactivation was complete in approximately 900 ms at -80 mV but in only 50 ms at -120 mV. The decay of Na+ tail currents had a single time constant that at -80 mV was faster than 100 microseconds. Depolarization of septal neurons also elicited a Ca2+ current that peaked in approximately 6-8 ms. Maximal peak Ca2+ current was obtained at 20 mV, and with 10 mM external Ca2+ the amplitude was 0.35 +/- 0.22 nA. During a maintained depolarization this current partially inactivated in the course of 200-300 ms. The Ca2+ current was due to the activity of two types of conductances with different deactivation kinetics. At -80 mV the closing time constants of slow (SD) and fast (FD) deactivating channels were, respectively, 1.99 +/- 0.2 and 0.11 +/- 0.03 ms (25 degrees C). The two kinds of channels also differed in their activation voltage, inactivation time course, slope of the conductance-voltage curve, and resistance to intracellular dialysis. The proportion of SD and FD channels varied from cell to cell, which may explain the differential electrophysiological responses of intracellularly recorded septal neurons.     

Time-dependent outward current in guinea pig ventricular myocytes. Gating kinetics of the delayed rectifier

Several conflicting models have been used to characterize the gating behavior of the cardiac delayed rectifier. In this study, whole-cell delayed rectifier currents were measured in voltage-clamped guinea pig ventricular myocytes, and a minimal model which reproduced the observed kinetic behavior was identified. First, whole-cell potassium currents between -10 and +70 mV were recorded using external solutions designed to eliminate Na and Ca currents and two components of time-dependent outward current were found. One component was a La3(+)-sensitive current which inactivated and resembled the transient outward current described in other cell types; single-channel observations confirmed the presence of a transient outward current in these guinea pig ventricular cells (gamma = 9.9 pS, [K]o = 4.5 mM). Analysis of envelopes of tail amplitudes demonstrated that this component was absent in solutions containing 30-100 microM La3+. The remaining time-dependent current, IK, activated with a sigmoidal time course that was well-characterized by three time constants. Nonlinear least-squares fits of a four-state Markovian chain model (closed - closed - closed - open) to IK activation were therefore compared to other models previously used to characterize IK gating: n2 and n4 Hodgkin-Huxley models and a Markovian chain model with only two closed states. In each case the four-state model was significantly better (P less than 0.05). The failure of the Hodgkin-Huxley models to adequately describe the macroscopic current indicates that identical and independent gating particles should not be assumed for this K channel. The voltage-dependent terms describing the rate constants for the four-state model were then derived using a global fitting approach for IK data obtained over a wide range of potentials (-80 to +70 mV). The fit was significantly improved by including a term representing the membrane dipole forces (P less than 0.01). The resulting rate constants predicted long single-channel openings (greater than 1 s) at voltages greater than 0 mV. In cell-attached patches, single delayed rectifier channels which had a mean chord conductance of 5.4 pS at +60 mV ([K]o = 4.5 mM) were recorded for brief periods. These channels exhibited behavior predicted by the four-state model: long openings and latency distributions with delayed peaks. These results suggest that the cardiac delayed rectifier undergoes at least two major transitions between closed states before opening upon depolarization.     

Alteration of the transmembrane K+ gradient during development of delayed rectifier in isolated rat pulmonary arterial cells

The properties of the tail current associated with the delayed rectifier K+ current (IK) in isolated rat pulmonary artery smooth muscle cells were examined using the whole cell patch clamp technique. The tail currents observed upon repolarization to -60 mV after brief (e.g., 20 ms) or small (i.e. to potentials negative of 0 mV) depolarizations were outwardly directed, as expected given the calculated K+ reversal potential of -83 mV. The tail currents seen upon repolarization after longer (e.g., 500 ms) and larger (e.g., to +60 mV) depolarizations tended to be inwardly directed. Depolarizations of intermediate strength and/or duration were followed by biphasic tail currents, which were inwardly directed immediately upon repolarization, but changed direction and became outwardly directed before deactivation was complete. When cells were depolarized to +60 mV for 500 ms both IK and the subsequent inward tail current at -60 mV were similarly blocked by phencyclidine. Both IK and the inward tail current were also blocked by 4-aminopyridine. Application of progressively more depolarized 30 s preconditioning potentials inactivated IK, and reduced the inward tail current amplitude with a similar potential dependency. These results indicated that the inward tail current was mediated by IK. The reversal potential of the tail current became progressively more positive with longer depolarizations to +60 mV, shifting from -76.1 +/- 2.2 mV (n = 10) after a 20-ms step to -57.7 +/- 3.5 mV (n = 9) after a 500-ms step. Similar effects occurred when extracellular K+ and Na+ were replaced by choline. When extracellular K+ was raised to 50 mM, the tail current was always inwardly directed at -60 mV, but showed little change in amplitude as the duration of depolarization was increased. These observations are best explained if the dependencies of tail current direction and kinetics upon the duration of the preceding depolarization result from an accumulation of K+ at the external face of the membrane, possibly in membrane invaginations. A mathematical model which simulates the reversal potential shift and the biphasic kinetics of the tail current on this basis is presented.     

Kinetic study of N-type calcium current modulation by delta-opioid receptor activation in the mammalian cell line NG108-15

The voltage-dependent inhibition of N-type Ca2+ channel current by the delta-opioid agonist [D-pen2, D-pen5]-enkephalin (DPDPE) was investigated in the mammalian cell line NG108-15 with 10 microM nifedipine to block L-type channels, with whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells DPDPE reversibly decreased omega-conotoxin GVIA-sensitive Ba2+ currents in a concentration-dependent way. Inhibition was maximal with 1 microM DPDPE (66% at 0 mV) and was characterized by a slowing of Ba2+ current activation at low test potentials. Both inhibition and kinetic slowing were attenuated at more positive potentials and could be relieved up to 90% by strong conditioning depolarizations. The kinetics of removal of inhibition (de-inhibition) and of its retrieval (re-inhibition) were also voltage dependent. Both de-inhibition and re-inhibition were single exponentials and, in the voltage range from -20 to +10 mV, had significantly different time constants at a given membrane potential, the time course of re-inhibition being faster than that of de-inhibition. The kinetics of de-inhibition at -20 mV and of reinhibition at -40 mV were also concentration dependent, both processes becoming slower at lower agonist concentrations. The rate of de-inhibition at +80/+120 mV was similar to that of Ca2+ channel activation at the same potentials measured during application of DPDPE (approximately 7 ms), both processes being much slower than channel activation in controls (<1 ms). Moreover, the amplitude but not the time course of tail currents changed as the depolarization to +80/+120 mV was made longer. The state-dependent properties of DPDPE Ca2+ channel inhibition could be simulated by a model that assumes that inhibition by DPDPE results from voltage- and concentration-dependent binding of an inhibitory molecule to the N-type channel.     

Inward current generated by Na-Ca exchange during the action potential in single atrial cells of the rabbit.

To investigate the underlying ionic mechanism of the late plateau phase of the action potential in rabbit atrium the whole-cell patch-clamp technique with intracellular perfusion was used. We recorded the inward current during repolarizations following a brief 2 ms depolarizing pulse to +40 mV from a holding potential of between -70 and -80 mV. The development of this current coincides with the onset of the late plateau phase of the action potential. Peak activation of the current occurs about 10 ms from the beginning of the depolarizing pulse, and it decays spontaneously with a slow timecourse. Its voltage dependency from -40 mV to +40 mV shows very steep activation (-40 to -20 mV) and shows almost the same maximum magnitude between -10 mV and +40 mV. This behaviour is quite different from that of the calcium current. The inward current and the late plateau phase of the action potential were both abolished by the application of 5 mM EGTA, 1 microM ryanodine and by reducing the Na+ gradient. The fully activated current-voltage relation of the inward current was plotted as the difference current before and after treatment with Ryanodine, Diltiazem, 20 mM Na+ inside or 30% Na+ outside and shows an exponential voltage dependence with the largest magnitude of the current occurring at negative potentials. The current-voltage (I-V) curve was well fitted by the Na-Ca exchange equation, i = A exp (-(1 - r)EF/RT). The results suggest that the inward current contributes to the generation of the late plateau phase of the rabbit atrial action potential, and is activated by intracellular calcium released from the sarcoplasmic reticulum. Sarcoplasmic reticulum calcium release appears to be triggered both by the membrane voltage and by the calcium current. It is concluded that the inward current is generated by Na-Ca exchange.     

Voltage-gated transient currents in bovine adrenal fasciculata cells. II. A-type K+ current

In whole cell patch clamp recordings on enzymatically dissociated adrenal zona fasciculata (AZF) cells, a rapidly inactivating A-type K+ current was observed in each of more than 150 cells. Activation of IA was steeply voltage dependent and could be described by a Boltzmann function raised to an integer power of 4, with a midpoint of -28.3 mV. Using the "limiting logarithmic potential sensitivity," the single channel gating charge was estimated to be 7.2 e. Voltage-dependent inactivation could also be described by a Boltzmann function with a midpoint of -58.7 mV and a slope factor of 5.92 mV. Gating kinetics of IA included both voltage-dependent and -independent transitions in pathways between closed, open, and inactivated states. IA activated with voltage-dependent sigmoidal kinetics that could be fit with an n4h formalism. The activation time constant, tau a, reached a voltage- independent minimum at potentials positive to 0 mV. IA currents inactivated with two time constants that were voltage independent at potentials ranging from -30 to +45 mV. At +20 mV, tau i(fast) and tau i(slow) were 13.16 +/- 0.64 and 62.26 +/- 5.35 ms (n = 34), respectively. In some cells, IA inactivation kinetics slowed dramatically after many minutes of whole cell recording. Once activated by depolarization, IA channels returned to the closed state along pathways with two voltage-dependent time constants which were 0.208 s, tau rec-f and 10.02 s, tau rec-s at -80 mV. Approximately 90% of IA current recovered with slow kinetics at potentials between -60 and -100 mV. IA was blocked by 4-aminopyridine (IC50 = 629 microM) through a mechanism that was strongly promoted by channel activation. Divalent and trivalent cations including Ni2+ and La3+ also blocked IA with IC50's of 467 and 26.4 microM, respectively. With respect to biophysical properties and pharmacology, IA in AZF cells resembles to some extent transient K+ currents in neurons and muscle, where they function to regulate action potential frequency and duration. The function of this prominent current in steroid hormone secretion by endocrine cells that may not generate action potentials is not yet clear.     

Whole-cell chloride conductances in cultured brushed human nasal epithelial cells.

Human airway epithelial cells were obtained by nasal brushing, thus avoiding the use of proteolytic enzymes for cell isolation. Whole-cell Cl- conductances were studied in these cells by means of the patch-clamp technique. During whole-cell recordings, cell swelling activated a Cl- conductance that was blocked by indanyloxyacetic acid (48 +/- 10% inhibition at 50 microM). The swelling-induced current outwardly rectified and showed inactivation at depolarizing voltages (> or = +60 mV) and activation at hyperpolarizing voltages (< or = -30 mV). The voltage sensitivity of current activation was approximately twice that of inactivation. Another Cl- current with different kinetics was observed when nonswollen airway cells were stimulated with ionomycin (2 microM) in the presence of 1 mM Ca2+. The Ca(2+)-induced current exhibited activation during depolarizing voltage steps (> or = +40 mV) and inactivation during hyperpolarizing voltage steps (< or = -40 mV). In contrast to the swelling-induced current, the activation of Ca(2+)-induced current was less sensitive to voltage compared with its inactivation. Tail current analysis suggested that Cl- channels having a linear current-voltage relation mediate the response to Ca2+. This study indicates that brushed human nasal epithelial cells possess Cl- conductances that are regulated by cell swelling and Ca2+ and that they represent a useful in vitro model for studying ion transport in epithelia.