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.     

Estradiol inhibition of voltage-activated and gonadotropin-releasing hormone-induced currents in mouse gonadotrophs

We report the first study of voltage-activated and GnRH-induced plasma membrane currents and their modulation by estradiol (E2) in mouse gonadotrophs. In consideration of the pleiotropic effects of E2 on gonadotrophin secretion and the relationship between plasma membrane electrical excitability and secretion, our objective was to determine the role of E2 in modulating gonadotroph plasma membrane currents. We measured total voltage-activated and GnRH-induced currents using the perforated-patch configuration of the patch-clamp technique, which preserves signaling pathways, including GnRH-induced Ca2+ oscillations. We show that female mouse gonadotrophs are similar to those from other species in that the voltage-activated net current response exhibits an inward fast activating current that is inhibited by tetrodotoxin, which is characteristic of a Na+ current, and a larger magnitude outward current with a profile suggesting the presence of multiple K+ currents. Furthermore, in voltage-clamped mouse gonadotrophs, GnRH activates large amplitude current oscillations that are apamin sensitive and have a reversal potential of -90 mV, consistent with Ca2+-activated K+ currents. Significantly, E2 pretreatment for 2-5 d decreased the density of both the peak outward voltage-activated current and the peak GnRH-induced current. The specific linkage between the observed E2 effects on membrane currents and, ultimately, gonadotroph function remains to be established. However, because decreased K+ current density is associated with an increase in membrane electrical excitability, we postulate increased excitability is one of the modes of action of E2 in sensitizing the gonadotroph to GnRH, an event central to the regulation of cyclic gonadotrophin secretion.     

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.     

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.     

Glucocorticoid regulation of cardiac K+ currents and L-type Ca2+ current in neonatal mice.

Previous studies have reported that dexamethasone (Dex) prolongs cardiac action potential repolarization in mice and rats. However, the cellular mechanisms of this effect have not been addressed. Because action potential duration is influenced by a complex interplay of both inward and outward currents, this study evaluated the role of K+ currents and the L-type Ca2+ current in response to chronic in vivo Dex treatment. Accordingly, neonatal mice were randomly allocated to treatment with Dex (1 mg/kg per day) or placebo (saline) given subcutaneously for 5 days. At 14 to 15 days of age, the L-type Ca2+ current and K+ currents were recorded in ventricular myocytes using whole-cell patch-clamp techniques. The density of peak outward K+ currents was significantly decreased in the chronic Dex-treated group, but the current measured at the end of a 1-second depolarization pulse was similar in both groups. We further measured the magnitudes of the fast-inactivating (I(to)) and the slowly inactivating (I(slow)) currents that contribute to the peak outward K+ currents. I(to) was reduced from 17.5+/-3.0 pA/pF (control) to 10.6+/-2.5 pA/pF (Dex) at +50 mV (P<0.05), but I(slow) was not significantly different. These data suggest that downregulation of I(to) is responsible for the reduced peak outward current. Time courses of the onset and offset of in vivo Dex effects were also assessed. A period of 3 days of treatment was required to observe the Dex effect on peak outward K(+) currents, whereas a 7-day period after discontinuation of Dex was required to recover the baseline current density. Acute in vitro treatment with Dex (1 micromol/L) had no effect on K+ current densities. In addition, chronic Dex treatment significantly increased the density of the L-type Ca2+ current (I(Ca-L)) from -7.2+/-0.5 pA/pF of control to -8.9+/-0.6 pA/pF of Dex at +10 mV, P<0.05. In conclusion, chronic in vivo Dex treatment decreases I(to) and increases I(Ca-L) in neonatal mouse ventricular myocytes, both of which contribute to the prolongation of cardiac action potential repolarization induced by glucocorticoids.     

Outward potassium currents in freshly isolated smooth muscle cell of dog coronary arteries

Outward membrane currents were characterized in single coronary smooth muscle cells of adult beagle dogs. The cells averaged 96.4 x 7.1 microns and had a resting potential of -50.7 mV, an input resistance of 307.9 M omega, a capacitance of 32.3 pF, and a calculated membrane surface area of 4,037 microns2. The cells contracted in response to external application of acetylcholine or high K+. In voltage clamp by use of the suction pipette method, outward current began to appear at -50 mV and reached 15.2 nA at 50 mV with a current density of 376.5 microA/cm2. The current was reduced by external tetraethylammonium, Ba2+, and internal Cs+, and its reversal potential had a Nernst relation to external K+ concentration. Elevation of external Ca2+ (Ca2+o) from 0 to 0.3 mM increased total K+ current by up to 300%; elevation of internal Ca2+ (Ca2+i) to 5 x 10(-7) M by internal perfusion increased total outward current to a similar extent, suggesting a large difference in Ca2+ transmembrane sensitivity. Total whole-cell K+ current consisted of two components: an initial time-independent current (Ii) followed by a time-dependent current (It). Ii and It were through separate K+ channels based on differences in a) sensitivity to Ca2+09b) modulation by an inward Ca2+ current, c) current amplitudes and activation kinetics, and d) responses to pharmacological agents. It was the largest component, measuring 4.5 nA in 0 mM Ca2+o but increasing to 11.9 nA in 0.3 mM Ca2+o with a steep 2.5 power function. It activated with a biexponential time course; in Ca2+o-free solution, its time course was relatively insensitive to voltage changes but became voltage sensitive in the presence of Ca2+o. Further, such sensitivity was abolished or enhanced by Co2+ or Bay K 8644, respectively. We concluded that there are two types of Ca2+-sensitive K+ currents, Ii and It, in coronary smooth muscle cells. Via an inward Ca2+ channel Ca2+o strongly modulates It, both in amplitude and kinetics.     

Changes in left ventricular repolarization and ion channel currents following a transient rate increase superimposed on bradycardia in anesthetized dogs

INTRODUCTION: We previously demonstrated in dogs that a transient rate increase superimposed on bradycardia causes prolongation of ventricular refractoriness that persists for hours after resumption of bradycardia. In this study, we examined changes in membrane currents that are associated with this phenomenon. METHODS AND RESULTS: The whole cell, patch clamp technique was used to record transmembrane voltages and currents, respectively, in single mid-myocardial left ventricular myocytes from dogs with 1 week of complete AV block; dogs either underwent 1 hour of left ventricular pacing at 120 beats/min or did not undergo pacing. Pacing significantly heightened mean phase 1 and peak plateau amplitudes by approximately 6 and approximately 3 mV, respectively (P < 0.02), and prolonged action potential duration at 90% repolarization from 235+/-8 msec to 278+/-8 msec (1 Hz; P = 0.02). Rapid pacing-induced changes in transmembrane ionic currents included (1) a more pronounced cumulative inactivation of the 4-aminopyridine-sensitive transient outward K+ current, Ito, over the range of physiologic frequencies, resulting from a approximately 30% decrease in the population of quickly reactivating channels; (2) increases in peak density of L-type Ca2+ currents, I(Ca.L), by 15% to 35 % between +10 and +60 mV; and (3) increases in peak density of the Ca2+-activated chloride current, I(Cl.Ca), by 30% to 120% between +30 and +50 mV. CONCLUSION: Frequency-dependent reduction in Ito combined with enhanced I(Ca.L) causes an increase in net inward current that may be responsible for the observed changes in ventricular repolarization. This augmentation of net cation influx is partially antagonized by an increase in outward I(Ca.Cl).     

Calcium currents in rat myoballs and their inhibition by insulin

Two Ca2+ currents were found in myoballs prepared from primary culture of hindlimb muscles from rat embryos. One whole cell current, not described previously, was early, fast, and transient. It depended on the presence of Na+ in the bathing solution and was blocked by tetrodotoxin. Despite this behavior suggesting that it might be via a Na+ channel, its reversal potential exceeded 80 mV compared to 46 mV for the Na+ equilibrium potential. It was increased by increased Ca2+ concentration in the bathing solution and eliminated by Co2+ and by diltiazem. The other Ca2+ current resembled the slow inward Ca2+ current ICa(si), described in other cell types. Insulin decreased both Ca2+ currents; even at 64 pM insulin reduced ICa(si). When outward K+ currents were prevented, the myoball action potential was altered greatly, owing to the unopposed effect of ICa(si). Its duration was on the order of seconds. Insulin, in a concentration-dependent manner, beginning at 60 pM, reduced the duration of this action potential more effectively than nimodipine. This is the most sensitive response to insulin observed in skeletal muscle.     

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.     

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.     

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.     

Effects of external pH on ionic currents in smooth muscle cells from the basilar artery of the guinea pig.

pHo is an important determinant of vascular tone in cerebral blood vessels. We investigated the effects of changes in pHo on isolated smooth muscle cells from the basilar artery of the guinea pig. Single cells contracted rapidly in response to an elevation in pHo (constant CO2), and contraction was blocked by nifedipine, suggesting a role for dihydropyridine-sensitive Ca2+ channels. In whole-cell patch-clamp experiments, changes in pHo (pHo 5.7-8.1, pHi 7.2 with 10 mM HEPES) strongly affected the amplitude of the peak Ca2+ channel current (10 mM Ba2+, +15 mV, holding potential of -55 mV), with an apparent pK of 6.9. The current-voltage curves were minimally shifted, indicating no important effect of surface charge. To separate the slowly inactivating L-type Ca2+ channel current from the more rapidly inactivating B-type current, the decaying portions of inward currents from cells studied with repetitive 1-second pulses (+15 mV, holding potential of -55 mV) were fit to a two-component model. Titration curves for the L-type and B-type currents indicated maximum increases by factors of 3.65 and 1.28 at alkaline pHo and gave apparent pK values of 7.71 and 6.47 (Hill coefficient unity). The time constant of inactivation for the B-type current at +15 mV was little affected by pHo, whereas that for the L-type current increased somewhat with increasing pHo. Additional experiments showed no significant effect of pHo on holding current or on voltage-activated outward currents (pCai 7 with 11 mM EGTA). Our results provide additional evidence for participation of Ca2+ channels in regulating basal tone in cerebral smooth muscle and indicate that pHo regulates current through slowly inactivating, dihydropyridine-sensitive L-type Ca2+ channels.     

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)     

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.     

Effects of palmatine on potassium and calcium currents in isolated rat hepatocytes

AIM: To study the effects of palmatine, a known inhibitoron delayed rectifier potassium current and L-type calciumcurrent (ICa,L) in guinea pig ventricular myocytes, on thepotassium and calcium currents in isolated rat hepatocytes.METHODS: Tight-seal wh ole-cell patch-clamp techniqueswere performed to investigate the effects of palmatine onthe delayed outward potassium currents (IK), inward rectifierpotassium current (IK1) and Ca2+ release-activated Ca2+current (ICRAC) in enzymatically isolated rat hepatocytes.RESULTS: Palmatine 0.3-100 μM reduced IK in a concentationdependent manner with EC50of 41.62±10.11 μM and nH,0.48±0.07 (n=8). The effect of the drug was poorly reversibleafter washout. When the bath solution was changed totetraethylammonium (TEA) 8 mM, IK was inhibited.Palmatine 10 μM and 100 μM shifted the I-V curves of IKdownward, and the block of IK was voltage-independent.Palmatine 0.3-100 μM also inhibited ICRAC in a concentration-dependent manner. The fitting parameters were as follows:ECs0=51.19±15.18 μM, and nH=0.46+0.07 (n=8). The peakvalue of ICRAC in the I-V relationship was decreased bypalmatine 10 μM and 100 μM. But the reverse potential ofIcRAcoCcurred at Voltage=0 mV in all cells. Palmatine 0.3-100 μM failed to have any significant effect on either inwardor outward components of IK1 at any membrane potentialexamined.CONCLUSION: The inhibitory effects on IK and ICRAC couldbe one of the mechanisms that palmatine exerts protectiveeffect on hepatocytes.     

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.     

Differential inhibition of potassium currents in rat ventricular myocytes by capsaicin.

OBJECTIVE: Capsaicin is a pungent irritant present in peppers of the Capsicum family. Its major target of action is believed to be sensory neurones. Capsaicin has also been shown to prolong cardiac action potential in atrial muscle, perhaps by local release of calcitonin gene related peptide which in turn enhances inward calcium currents. However, capsaicin has been shown to inhibit K+ current in neurones. Since such an action could contribute to action potential prolonging activity of capsaicin in heart, the aim of the study was to examine the effects of capsaicin on cardiac K+ currents. METHODS: Ionic currents and action potentials were examined in isolated adult rat ventricular myocytes using the whole cell variant of the patch clamp technique at 25 degrees C. RESULTS: Capsaicin (10 microM) increased the action potential duration (APD50) from 45 ms to 166 ms. This effect was associated with an inhibition of three distinct K+ currents. The decreasing rank order of potency was: transient outward K+ current (ITO, IC50 = 6.4 microM), a voltage dependent non-inactivating outward current (IK, IC = 11.5 microM), and the inward rectifier K+ current (IK1, IC50 = 46.9 microM). Capsaicin induced block of ITO was characterised by a decrease in the peak current amplitude and an increase in the rate of inactivation. The inactivation of ITO in the absence of capsaicin was well described by a single exponential [tau = 77 (SEM 2) ms at +40 mV, n = 10]. However, in the presence of 10 microM capsaicin inactivation was best described by the sum of two exponentials [tau FAST = 4.4(0.5) ms; tau SLOW = 92.4(3.0) ms, n = 10] with the fast component contributing 46(2)% of the total decay. A small but consistent hyperpolarising shift (approximately 3 mV) in the steady state voltage dependence of inactivation of ITO was induced by 10 microM capsaicin. Capsaicin had no effect on the rate of ITO recovery from inactivation (tau = 49 ms and 48 ms for control and drug respectively). The capsaicin analogue, resiniferatoxin, which as an irritant is up to 10(4)-fold more potent than capsaicin, had no effect on any of the K+ currents when present at concentrations of up to 10 microM. In contrast another capsaicin analogue, zingerone (30 microM) blocked ITO by 52(12)% and IK by 35%. CONCLUSIONS: Capsaicin produces a prolongation of the rat ventricular action potential, an effect which is associated with inhibition of potassium currents.     

Gonadotropin-releasing hormone induces oscillatory membrane currents in rat gonadotropes

Electrophysiological studies were performed to characterize membrane currents of rat gonadotropes under basal conditions and after exposure to secretagogues. Gonadotropes were identified in primary cultures of rat anterior pituitaries by a reverse hemolytic plaque assay. Giga-seal patch clamp recording with the cell-attached configuration was used to monitor membrane currents in these cells. Spontaneous spikes in basal current were seen. These were blocked by methoxyverapamil and probably reflect Ca2+-dependent action potentials. Brief GnRH stimulation induced slow oscillatory changes in membrane current that evolved into a series of large amplitude inward pulses after about 8 min. Treatment with TRH had no effect, and depolarization with K+ led to delayed inward currents without any oscillatory behavior. Under conditions of Ca2+ channel blockade, GnRH stimulation did not induce pulses of inward current, but did lead to oscillatory activation of a small conductance ion channel apparently selective for K+. Taken together these results suggest that GnRH induces oscillations in intracellular Ca2+ and that these oscillations are controlled by biochemical processes.     

Ionic currents and action potentials in rabbit,rat, and guinea pig ventricular myocytes

Summary Distinct differences exist in action potentials and ionic currents between rabbit, rat, and guinea pig ventricular myocytes. Data obtained at room temperature indicate that about half of the rabbit myocytes show prominent phase 1 repolarization and transient outward current. Action potentials in guinea pig ventricular myocytes resemble those from rabbit myocytes not exhibiting phase 1 repolarization; and guinea pig myocytes do not develop transient outward current. Rat ventricular action potentials are significantly shorter than those from rabbit and guinea pig ventricular myocytes. Unlike rabbit and guinea pig myocytes, rat ventricular myocytes also exhibit a prominent phase 1 and lack a well defined plateau phase during repolarization. All rat ventricular myocytes exhibit a transient outward current which can be best fitted by a double exponential relation. There are no significant differences between the amplitude, voltage dependence and inactivation kinetics of the inward calcium currents observed in rabbit, rat and guinea pig. The steady-state current-voltage relations between –120 mV and –20 mV, which mostly represent the inward rectifier potassium current are similar in rabbit and guinea pig. The amplitude of this current is significantly less in rat ventricular myocytes. The outward currents activated upon depolarization to between –10 and +50 mV are different in the three species. Only a negligible, or absent, delayed rectifier outward current has been observed in rabbit and rat; however, a relatively large delayed rectifier current has been found in guinea pig. These large interspecies variations in outward membrane currents help explain the differences in action potential configurations observed in rabbit, rat, and guinea pig.     

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.