Characterization of a G-protein-regulated outward K+ current in mesophyll cells of vicia faba L.

Whole-cell voltage-dependent currents in isolated mesophyll protoplasts of Vicia faba were investigated by patch-clamp techniques. With 104 mM K+ in the cytosol and 13 mM K+ in the external solution, depolarization of the plasma membrane from -47 mV to potentials between -15 and +85 mV activated a voltage- and time-dependent outward current (Iout). The average magnitude of Iout at +85 mV was 28.5 +/- 3.3 pA.pF-1. No inward voltage-dependent current was observed upon hyperpolarization of the plasma membrane from -55 mV to potentials as negative as -175 mV. Time-activated outward current was blocked by Ba2+ (1 mM BaCl2) and was not observed when K+ was eliminated from the external and internal solutions, indicating that this outward current was carried primarily by K+ ions. The voltage dependency of outward K+ current revealed a possible mechanism for K+ efflux from mesophyll cells. A GDP analogue guanosine 5'-[beta-thio]diphosphate (500 microM) significantly enhanced outward K+ current. The outward K+ current was inhibited by the GTP analogue guanosine 5'-[gamma-thio]triphosphate (500 microM) and by an increase in cytoplasmic free Ca2+ concentrations. Cholera toxin, which ADP-ribosylates guanine nucleotide-binding regulatory proteins, also inhibited outward K+ current. These findings illustrate the presence in mesophyll cells of outward-rectifying K+ channels that are regulated by GTP-binding proteins and calcium.     

Calcium currents in GH3 cultured pituitary cells under whole-cell voltage-clamp: inhibition by voltage-dependent potassium currents.

To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.     

Current fluctuations and oscillations in smooth muscle cells from hog carotid artery. Role of the sarcoplasmic reticulum

Electrical activity of enzymatically isolated, smooth muscle cells from hog carotid arteries was recorded under current clamp and voltage clamp. Under the experimental conditions, membrane potential usually was not stable, and spontaneous hyperpolarizing transients of approximately 100-msec duration were recorded. The amplitude of the transients was markedly voltage dependent and ranged from about 20 mV at a membrane potential of 0 mV to undetectable at membrane potentials negative to -60 mV. Under voltage clamp, transient outward currents displayed a similar voltage dependency. These fluctuations reflect a K+ current; they were abolished by 10 mM tetraethylammonium chloride, a K+ channel blocker, and the current fluctuations reversed direction in high extracellular K+ concentration. Modulators of intracellular Ca2+ concentration also affected electrical activity. Lowering intracellular Ca2+ concentration by addition of 10 mM EGTA to the pipette solution or suppressing sarcoplasmic reticulum function by superfusion with caffeine (10 mM), ryanodine (1 microM), or histamine (3-10 microM) blocked the rapid voltage and current spikes. However, caffeine and histamine induced a much slower hump of outward current before blocking the rapid spikes. This slower transient outward current could be elicited only once after external Ca2+ was removed and is consistent with an activation of K+ channels by Ca2+ released from internal stores. In contrast, removal of external Ca2+ alone failed to abolish the rapid spikes. These results suggest that 1) a Ca2+-dependent K+ conductance can markedly affect the electrical behavior of arterial smooth muscle cells and 2) internal Ca2+ stores, probably the sarcoplasmic reticulum, can support rapid and frequent releases of Ca2+. Exposure to a low concentration of histamine (3 microM) caused synchronization of the irregular, rapid fluctuations giving rise to slow, periodic oscillations of Ca2+-activated K+ conductance with a frequency of 0.1-0.3 Hz. These regular oscillations are reminiscent of periodic Ca2+-induced Ca2+ release, were inhibited by 10 mM caffeine, and point to a modulation of sarcoplasmic reticulum Ca2+ release by histamine.     

Excitation-secretion coupling: ionic currents in glomerulosa cells: effects of adrenocorticotropin and K+ channel blockers

The ionic conductance of cultured rat glomerulosa cells has been studied using the whole cell variant of the patch-clamp technique. We have identified and partially characterized three currents: a transient outward current, a slow outward current, and a slow inward current. The transient outward current activated rapidly and then inactivated slowly on maintained depolarization. Activation was initiated at -30 mV, and zero current was seen at -60 to -50 mV. The slow outward current did not inactivate with time and was initiated around 0 mV; its zero current voltage was difficult to evaluate. The two outward currents were present in different proportions, which explains the different time course of the total outward current from one cell to another. A slow inward current was also found which activated near -30 mV and reached its reversal potential between 80 and 100 mV. This current was blocked by Co2+, increased with [Ca2+]o, and was insensitive to Na+-free external medium. ACTH, a potent stimulant of steroid output, was found to block the transient outward current, but was ineffective on the slow outward current and the slow inward current. Tetraethylammonium and 4-aminopyridine, K+ channel inhibitors, also blocked the transient outward current.     

Ionic currents in single smooth muscle cells of the canine renal artery.

Membrane currents from single smooth muscle cells enzymatically isolated from canine renal artery were recorded using the patch-clamp technique in the whole-cell and cell-attached configurations. These cells exhibited a mean resting potential, input resistance, membrane time constant, and cell capacitance of -51.8 +/- 2.1 mV, 5.2 +/- 0.98 G omega, 116.2 +/- 16.4 msec, and 29.1 +/- 2.0 pF, respectively. Inward current, when elicited from a holding potential of -80 mV, activated near -50 mV, reached a maximum near 0 mV and was sensitive to the dihydropyridine agonist Bay K 8644 and dihydropyridine antagonist nisoldipine. Two components of macroscopic outward current were identified from voltage-step and ramp depolarizations. The predominant charge carrier of the net outward current was identified as K+ by tail-current experiments (reversal potential, -61.0 +/- 0.8 mV in 10.8 mM [K+]o 0 mM [K+]i). The first component was a small, low-noise, voltage- and time-dependent current that activated between -40 and -30 mV (IK(dr)), and the second component was a larger, noisier, voltage- and time-dependent current that activated at potentials positive to +10 mV (IK(Ca)). Both IK(dr) and IK(Ca) displayed little inactivation during long (4-second) voltage steps. IK(Ca) and IK(dr) could be pharmacologically separated by using various Ca2+ and K+ channel blockers. IK(Ca) was substantially inhibited by external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), tetraethylammonium (Ki at +60 mV, 307 microM), and charybdotoxin (100 nM) but was insensitive to 4-aminopyridine (0.1-10 mM). IK(dr) was inhibited by 4-aminopyridine (Ki at +10 mV, 723 microM) and tetraethylammonium (Ki at +10 mV, 908 microM) but was insensitive to external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), and charybdotoxin (100 nM). Two types of single K+ channels were identified in cell-attached patches. The most abundant K+ channel that was recorded exhibited voltage-dependent activation, was blocked by external tetraethylammonium (250 microM), and had a large single-channel conductance (232 +/- 12 pS with 150 mM K+ in the patch pipette, 130 +/- 17 pS with 5.4 mM K+ in the patch pipette). The second channel was also voltage dependent, was blocked by 4-aminopyridine (5 mM), and exhibited a smaller single-channel conductance (104 +/- 8 pS with 150 mM K+ in the patch pipette, 57 +/- 6 pS with 5.4 mM K+ in the patch pipette). These results suggest that depolarization of canine renal artery cells opens dihydropyridine-sensitive Ca2+ channels and at least two K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+ -activated K+ conductance in internally perfused snail neurons is enhanced by protein phosphorylation.

Depolarizing voltage steps induce inward and outward currents in voltage-clamped, internally perfused neurons from the snail Helix roseneri. Addition of the catalytic subunit of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) to the internal perfusing medium results in an increase in the net outward current, with no apparent effect on the inward current. Catalytic subunit inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) is without effect, indicating that the increase in net outward current results from protein phosphorylation rather than an unspecific effect of protein perfusion. Decreasing the external Ca2+ concentration from 10 to 1 mM eliminates the effect of catalytic subunit, suggesting that Ca2+ plays an important role in this response. This suggestion is supported by the fact that the stimulation by catalytic subunit can be mimicked by increasing the Ca2+ concentration in the internal perfusion medium and can be prevented by intracellular perfusion with 10 mM EGTA. The results are consistent with the hypothesis that cyclic AMP-dependent protein phosphorylation regulates the Ca2+-activated K+ conductance in these cells.     

An inward rectifier and a voltage-dependent K+ current in single, cultured pericytes from bovine heart

OBJECTIVE: The purpose of this study was to describe passive electrical properties and major membrane currents in coronary pericytes. METHODS: 78 single, cultured bovine pericytes were studied with the patch-clamp technique in the whole-cell mode. RESULTS: The membrane potential of the cells was -48.9+/-9.6 mV (mean+/-S.D.) with 5 mM and -23.2+/-2.2 mV with 60 mM extracellular K+. The membrane capacitance was 150.2+/-123.2 pF. The current-voltage relation of the pericytes was dominated by an inward current at hyperpolarized potentials and an outward current at depolarized potentials. Increasing extracellular K+ from 5 to 60 mM led to an increase of the inward current and to a shift of this current to more depolarized potentials. The inward current was very sensitive to extracellular barium (50 microM). The maximum slope conductance of the cells at hyperpolarized potentials was 2.9+/-2.8 nS. Inward rectification of whole-cell currents was steep (slope factor = 6.8 mV). With elevated external K+ the outward current reversed near the potassium equilibrium potential. Onset of the outward current was sigmoid and inactivation of this current was monoexponential, slow (time constant = 12.8 s) and incomplete. Voltage-dependence of outward current steady-state activation was steep (slope factor = 4.6 mV). The outward current was very sensitive to 4-aminopyridine (dissociation constant = 0.1 mM). The maximum slope conductance at depolarized potentials was 16.6+/-15.6 nS. CONCLUSION: We report for the first time, patch-clamp recordings from coronary pericytes. An inward rectifier and a voltage-dependent K+ current were identified and characterized. Regulation of these currents may influence coronary blood flow.     

Unblock of the slow inward current induces the arrhythmogenic transient inward current in isolated guinea-pig myocytes.

This study investigated whether abrupt changes in extracellular Ca2+ concentration or washout of the Ca2+ antagonists Mn2+ or verapamil, could induce transient inward current (ITI) in enzymatically disaggregated guinea-pig myocytes. Single electrode voltage-clamp techniques were used. ITI was elicited upon repolarization to various voltage steps from an activating step to +20 mV. The holding potential was -80 mV. Slow inward current (ICa) was induced by steps to -10 mV. Continuous exposure to either 2.5 or 6.0 mM Ca2+ did not induce ITI; however, following exposure of cells to 0.5 mM Ca2+ for 20 min which decreased ICa, return to 2.5 or 6.0 mM Ca2+ induced ITI. ITI could be observed for 10 to 20 min following sudden elevations of Ca2+. Similar effects also were seen when Ca2+ was increased from 2.5 to 6.0 mM. Exposure to 2.0 mM Mn2+ or 2.0 microM verapamil blocked ICa. Washout of either blocker induced ITI, particularly in 6.0 mM Ca2+. Peak ITI occurred upon repolarization at c. -70 mV; a reversal potential could not be demonstrated. Thus, abrupt changes in Ca2+ influx, produced either by sudden changes in external Ca2+ or by washout of Ca2+ antagonists, induced ITI with characteristics similar to those described for ITI induced by toxic concentrations of cardiac glycosides.     

Hyperpolarization of the membrane potential caused by somatostatin in dissociated human pituitary adenoma cells that secrete growth hormone.

Membrane electrical properties and the response to somatostatin were examined in dissociated human pituitary adenoma cells that secrete growth hormone (GH). Under current clamp condition with a patch electrode, the resting potential was -52.4 +/- 8.0 mV, and spontaneous action potentials were observed in 58% of the cells. Under voltage clamp condition an outward K+ current, a tetrodotoxin-sensitive Na+ current, and a Ca2+ current were observed. Cobalt ions suppressed the Ca2+ current. The threshold of Ca2+ current activation was about -60 mV. Somatostatin elicited a membrane hyperpolarization associated with increased membrane permeability in these cells. The reversal potential of somatostatin-induced hyperpolarization was -78.4 +/- 4.3 mV in 6 mM K+ medium and -97.2 +/- 6.4 mV in 3 mM K+ medium. These reversal potential values and a shift with the external K+ concentration indicated that membrane hyperpolarization was caused by increased permeability to K+. The hyperpolarized membrane potential induced by somatostatin was -63.6 +/- 5.9 mV in the standard medium. This level was subthreshold for Ca2+ and Na+ currents and was sufficient to inhibit spontaneous action potentials. Hormone secretion was significantly suppressed by somatostatin and cobalt ions. Therefore, we suggest that Ca2+ entering the cell through voltage-dependent channels are playing an important role for GH secretion and that somatostatin suppresses GH secretion by blocking Ca2+ currents. Finally, we discuss other possibilities for the inhibitory effect of somatostatin on GH secretion.     

Effects of quinidine on plateau currents of guinea-pig ventricular myocytes

Effects of quinidine on membrane currents forming the plateau of action potentials were studied using an isolated single ventricular cell from guinea-pig hearts. Quinidine (5 mg/l) produced a fall and shortening of the early part of the plateau, and delayed its later part and final repolarization, without changes in resting membrane potential. Application of quinidine caused a reversible depression of the peak Ca2+ current by about 30% of the control. Delayed outward K+ current, iK, also decreased to less than 20% of the control. Thus, an outward tail current upon repolarization to -40 mV from depolarizing voltage steps of the plateau ranges became inward. Current values at the end of 200 ms pulses in response to voltage steps to -60-0 mV were always positive and were not changed by the drug. The inward current elicited at potentials negative to resting potential level, also, decreased by 13% to 23% of the control in the presence of the drug, but the effect was not reversible upon wash-out of the drug. These results suggest that quinidine causes a non-specific depression of inward rectifier K+ current, iK1, with minor degree but has little effect on the window sodium current. Therefore, changes in the action potential repolarization produced by quinidine can be explained by its effects on both calcium current and delayed outward K+ current.     

Acetylcholine increases voltage-activated Ca2+ current in freshly dissociated smooth muscle cells.

The regulation of voltage-activated Ca2+ current by acetylcholine was studied in single freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus by using the tight-seal whole-cell recording technique. Ca2+ currents were elicited by positive-going command pulses from a holding level near -80 mV in the presence of internal Cs+ to block outward K+ currents. Ca2+ current was greatest in magnitude at command potentials near 10 mV. At such command potentials, acetylcholine increased the magnitude of the inward current and slowed its decay. The effects of acetylcholine were seen in the absence of external Na+ or with low Cl- (aspartate replacement) in the bathing solution and could be mimicked by muscarine. The peak of the current-voltage relationship for the Ca2+ current was not discernibly shifted along the voltage axis by acetylcholine. These results demonstrate that activation of muscarinic receptors not only suppresses a K+ current (M-current), as we have previously demonstrated [Sims, S. M., Singer, J. J. & Walsh, J. V., Jr. (1985) J. Physiol. (London) 367, 503-529], but also increases the magnitude and slows the decay of Ca2+ current.     

Inositol trisphosphate promotes Na-Ca exchange current by releasing calcium from sarcoplasmic reticulum in cardiac myocytes

An early inward tail current evoked by membrane depolarization (from -80 to -40 mV) sufficient to activate sodium but not calcium current was studied in single voltage-clamped ventricular myocytes isolated from guinea pig hearts. Like forward-mode Na-Ca exchange, this early inward tail current required [Na+]o and [Ca2+]i and is thought to follow earlier reverse-mode Na-Ca exchange that triggers Ca2+ release from sarcoplasmic reticulum. The dependence of the early inward tail current on [Ca2+]i was supported by the ability of small (+10 mV) and large (+80 mV) voltage jumps from -40 mV to decrease and increase, respectively, the size of early inward tail currents evoked by subsequent voltage steps from -80 to -40 mV. As expected, tetrodotoxin selectively inhibited the early inward tail current but not the late inward tail current that followed voltage jumps to +40 mV test potentials. Although tetrodotoxin also blocked the fast Na+ current, replacement of extracellular Na+ by Li+ sustained the fast Na+ current. However, Li+, which does not support Na-Ca exchange, reversibly suppressed both the early and late inward tail currents. Inhibitors (ryanodine and caffeine) and promoters (intracellularly dialyzed inositol 1,4,5-trisphosphate) of sarcoplasmic reticulum Ca2+ release decreased and increased, respectively, the magnitude of the early inward tail current. The results substantiate the hypothesis that Ca2+ release from the sarcoplasmic reticulum participates in early Na-Ca exchange current and demonstrate that inositol 1,4,5-trisphosphate, by releasing Ca2+ from the sarcoplasmic reticulum, can promote Na-Ca exchange across the plasma membrane.     

Voltage-gated currents of tilapia prolactin cells

The first recordings of neuron-like electrical activity from endocrine cells were made from fish pituitary cells. However, patch-clamping studies have predominantly utilized mammalian preparations. This study used whole-cell patch-clamping to characterize voltage-gated ionic currents of anterior pituitary cells of Oreochromis mossambicus in primary culture. Due to their importance for control of hormone secretion we emphasize analysis of calcium currents (I(Ca)), including using peptide toxins diagnostic for mammalian neuronal Ca(2+) channel types. These appear not to have been previously tested on fish endocrine cells. In balanced salines, inward currents consisted of a rapid TTX-sensitive sodium current and a smaller, slower I(Ca); there followed outward potassium currents dominated by delayed, sustained TEA-sensitive K(+) current. About half of cells tested from a holding potential (V(h)) of -90 mV showed early transient K(+) current; most cells showed a small Ca(2+)-mediated outward current. I-V plots of isolated I(Ca) with 15 mM [Ca(2+)](o) showed peak currents (up to 20 pA/pF from V(h) -90 mV) at approximately +10 mV, with approximately 60% I(Ca) for V(h) -50 mV and approximately 30% remaining at V(h) -30 mV. Plots of normalized conductance vs. voltage at several V(h)s were nearly superimposable. Well-sustained I(Ca) with predominantly Ca(2+)-dependent inactivation and inhibition of approximately 30% of total I(Ca) by nifedipine or nimodipine suggests participation of L-type channels. Each of the peptide toxins (omega-conotoxin GVIA, omega-agatoxin IVA, SNX482) alone blocked 36-54% of I(Ca). Inhibition by any of these toxins was additive to inhibition by nifedipine. Combinations of the toxins failed to produce additive effects. I(Ca) of up to 30% of total remained with any combination of inhibitors, but 0.1mM cadmium blocked all I(Ca) rapidly and reversibly. We did not find differences among cells of differing size and hormone content. Thus, I(Ca) is carried by high voltage-activated Ca(2+) channels of at least three types, but the molecular types may differ from those characterized from mammalian neurons.     

Automatic activity in depolarized guinea pig ventricular myocardium. Characteristics and mechanisms.

Membrane potential was changed uniformly in segments, 0.7-1.0 mm long, of guinea pig papillary muscles excised from the right ventricle by using extracellular polarizing current pulses applied across two electrically insulated cf preparations superfused with Tyrode's solution at maximum diastolic membrane potentials ranging from-35.2+/-7.5 (threshold) to +4.0+/-9.2 mV. The average maximum dV/dt of RAD ranged from 17.1 to 18.0 V/sec within a membrane potential range of -40 to +20 mV. Raising extracellular Ca2+ concentration [Ca2+]0 from 1.8 to 6.8 mM, or application of isoproterenol (10(-6)g/ml) enhanced the rate of RAD, but lowering [Ca2+]0 to 0.4 mM or exposure to MnCl2 (6 mM) abolished RAD. RAD were enhanced by lowering extracellular K+ concentration [K+]0 from 5.4 to 1.5 mM. RAD were suppressed in 40% of fibers by raising [K+]0 to 15.4 mM, and in all fibers by raising [K+]0 to 40.4 mM. This suppression was due to increased [K+]0 and not to K-induced depolarization because it persisted when membrane potential was held by means of a conditioning hyperpolarizing puled gradually after maximum repolarization. These observations suggest that the development of RAD in depolarized myocardium is associated with a time-dependent decrease in outward current (probably K current) and with increase in the background inward current, presumably flowing through the slow cha-nel carrying Ca or Na ions, or both.     

Ionic currents in single cells from human cystic artery.

The patch-clamp technique was used to study the electrophysiological properties of single smooth muscle cells obtained from the human cystic artery. These cells contracted on exposure to high K+ and had a mean resting potential of -36 +/- 7 mV. Under current clamp, regenerative responses could not be elicited when depolarizing pulses were applied. Voltage-clamp measurements demonstrated that a large fraction of the outward current was inhibited by tetraethylammonium (5-10 mM) or Ca2+ channel blockers and that it was enhanced by increasing [Ca2+]o, suggesting that it is a Ca(2+)-activated K+ current. In addition, spontaneous transient outward currents that were sensitive to extracellular Ca2+ were observed in some cells. In cell-attached patch-clamp recordings, Ca(2+)-activated K+ channels that had a conductance of 117 pS were consistently identified. At negative potentials (approximately -60 mV), these single-channel events deactivated completely and very quickly, suggesting that they do not control the resting membrane potential in healthy cystic artery cells. Ca2+ currents that were recorded using Ba2+ (10 mM) as the charge carrier were enhanced by the dihydropyridine agonist, Bay K 8644, and blocked by nifedipine (0.1 microM). Only one type of Ca2+ current, the L-type, could be identified in these cells. These results demonstrate that the major ionic currents in the human cystic artery are similar to other mammalian arteries and indicate that this tissue will be a useful model for studying the metabolic and pharmacological modulation of ionic currents in human vascular smooth muscle.     

A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca2+ blockage, and ionic selectivity.

Ca2+ ions control the cGMP-gated channel of rod photoreceptor cells from the external and internal face. We studied ion selectivity and blockage by Ca2+ of wild-type and mutant channels in a heterologous expression system. External Ca2+ blocks the inward current at micromolar concentrations in a highly voltage-dependent manner. The blockage at negative membrane voltages shows a steep concentration dependence with a Hill coefficient of approximately 2. The blockage from the internal face requires approximately 1000-fold higher Ca2+ concentrations. Neutralization of a glutamate residue (E363) in the putative pore region between transmembrane segments H4 and H5 induces outward rectification and changes relative ion conductances but leaves relative ion permeabilities nearly unaffected. The current blockage at -80 mV requires approximately 2000-fold higher external Ca2+ concentrations and the voltage dependence is almost abolished. These results demonstrate that E363 represents a binding site for monovalent and divalent cations and resides in the pore lumen.     

Potassium current suppression by quinidine reveals additional calcium currents in neuroblastoma cells.

Quinine and quinidine have been evaluated with regard to their effects on the electrical activity of neuroblastoma cells. Under voltage-clamp conditions, we have found that quinine and quinidine block both the voltage-dependent and Ca2+-dependent K+ conductances. Blockage of the voltage-dependent K+ channel is manifest as an increase in the amplitude and in the duration of the action potential. Blockage of the Ca2+-dependent K+ channel in Na+-free (replaced by Tris) solutions containing 6.8 mM Ca2+ and tetraethylammonium ion or 4-aminopyridine (to block the voltage-dependent K+ current) is seen as a further prolongation of the Ca2+ action potential and diminution of the after-hyperpolarization. A critical role of the Ca2+-dependent K+ conductance in modulation of the rate and duration of trains of Ca2+ action potentials is shown by the use of low concentrations (5-40 microM) of quinine or quinidine, which diminish the Ca2+-dependent K+ conductance in a graded manner. After complete blockade of K+ currents, the peak Ca2+ currents are enhanced at all voltages, especially at values more positive than -30 mV, where a steady-state inward current appears as well. In this same voltage range, the decay of the Ca2+ current exhibits two time constants--that of the transient inward current, which is about 20 msec, and a much slower (approximately 2000 msec) component. It is suggested that neuroblastoma cells have two types of calcium channels--one which generates the Ca2+ action potential and a second, distinguished by activation at more depolarized levels and by a slow rate of inactivation, which underlies the calcium entry necessary to activate the Ca2+-dependent K+ conductance.     

Mechanisms for the positive inotropic effect of alpha 1-adrenoceptor stimulation in rat cardiac myocytes.

alpha 1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+ sensitivity and action potential prolongation, which can increase net Ca2+ entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2-0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+ entry and differentiate between the two mechanisms. At 22-23 degrees C the specific alpha 1-adrenoceptor agonist methoxamine (100 microM) prolonged action potential duration at 50% repolarization from 55 +/- 2 to 81 +/- 5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3 +/- 0.6 to 7.8 +/- 1 microns (n = 18). Reduction of the transient outward current and other K+ currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52 +/- 6 to 98 +/- 12 msec and increased unloaded cell shortening from 2.9 +/- 0.4 to 6.6 +/- 0.6 microns (n = 4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70-500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3 +/- 0.7 to 4.97 +/- 0.8 microns at pulse potentials positive to -40 mV. Similar alpha 1-adrenoceptor responses were observed at 35 degrees C during action potential and voltage-clamp experiments, which could be blocked by 10 microM prazosin. In myocytes loaded with the Ca2+ indicator indo-1, alpha 1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+ transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+ transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to alpha 1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+ current reduction. In addition, modulation of K+ channels could occur independent of changes in contractility and/or [Ca2+]i.     

Mechanism of calcium current modulation underlying presynaptic facilitation and behavioral sensitization in Aplysia.

Behavioral sensitization of the gill-withdrawal reflex of Aplysia is caused by presynaptic facilitation at the synapses of the mechanoreceptor sensory neurons of the reflex onto the motor neurons and interneurons. The presynaptic facilitation has been shown to be simulated by serotonin (the putative presynaptic facilitatory transmitter) and by cyclic AMP and to be accompanied by an increase in the Ca2+ current of sensory neuron cell bodies exposed to tetraethylammonium. This increase in the Ca2+ current could result from either a direct action on the Ca2+ channel or an action on an opposing K+ current. Here we report voltage clamp experiments which indicate that the increase in Ca2+ current associated with presynaptic facilitation results from a decrease in a K+ current. Stimulation of the connective (the pathway that mediates sensitization) or application of serotonin causes a decrease in a voltage-sensitive, steady-state outward current measured under voltage clamp as well as an increase in the transient net inward and a decrease in the transient outward currents elicited by brief depolarizing command steps. The reversal potential of the steady-state synaptic current is sensitive to extracellular K+ concentration, and both the steady-state synaptic current and the changes in the transient currents are blocked by K+ current blocking agents and by washout of K+. These results suggest that serotonin and the natural transmitter released by connective stimulation act to decrease a voltage-sensitive K+ current. The decrease in K+ current prolongs the action potential, and this in turn increases the duration of the inward Ca2+ current and thereby enhances transmitter release.     

GABAB-receptor-activated K+ current in voltage-clamped CA3 pyramidal cells in hippocampal cultures.

GABAB receptors are a subclass of receptors for gamma-amino-n-butyric acid (GABA) that are also activated by the antispastic drug beta-p-chlorophenyl-GABA (baclofen). One effect of baclofen is to inhibit excitatory transmission from CA3 to CA1 hippocampal pyramidal cells. To identify the ionic mechanism of GABAB-receptor-mediated depression, we have studied the effect of baclofen and GABA on ionic currents in voltage-clamped CA3 pyramidal cell somata in rat hippocampal slice cultures. Baclofen (10 microM) induced an inwardly rectifying outward current that reversed at -74 +/- 4.3 mV (mean +/- SD). This appeared to be a K+ current since (i) its reversal potential showed the expected shift when extracellular K+ concentration was changed and (ii) it was blocked by external Ba2+ or internal Cs+. The action of baclofen was closely imitated by GABA after the GABAA-mediated Cl- current had been abolished with pitrazepin (10 microM); under these conditions, GABA (100 microM) also produced an inwardly rectifying, Ba2+-sensitive current with a reversal potential identical to that of the baclofen-induced current. When outward currents were blocked with internal Cs+, the residual inward voltage-dependent Ca2+ current was not changed by baclofen. It is concluded that the primary effect of GABAB-receptor activation in these neurones is to increase K+ permeability rather than to reduce Ca2+ permeability.