Effects of dopamine on voltage-dependent potassium currents in identified rat lactotroph cells

The effects of dopamine (DA) on voltage-dependent potassium currents were investigated in rat lactotrophs maintained in primary culture. Lactotroph cells were identified using the reverse hemolytic plaque assay. Membrane currents and potentials of lactotroph cells were recorded using the patch-clamp recording technique in the 'whole-cell' configuration. In the presence of cobalt (2 mM), two types of voltage-dependent K+ currents were recorded, a voltage-activated delayed K+ current (IK) and a voltage-activated transient K+ current (IA). The current IK was activated at membrane potentials varying from -20 to +40 mV and did not inactivate during prolonged voltage steps (up to 25 s); it was blocked by tetraethylammonium (10 mM). The current IA was activated at membrane potentials higher than -45 mV and showed a voltage-dependent inactivation between -110 and -40 mV; it was slightly inhibited by 4-aminopyridine (5 mM). Under current-clamp conditions, the majority of the cells (60%) showed spontaneous Ca2(+)-dependent action potentials (APs) while silent cells (40%) were excitable by depolarizing current pulses. Bath application of 10 nM DA evoked a hyperpolarizing response, blocked spontaneous APs and decrease the amplitude of evoked APs. Only the hyperpolarizing response faded during the course of the whole cell recording experiments. Under voltage-clamp conditions, DA induced a reversible increase in both voltage-dependent outward K+ currents, without modifying their thresholds. Steady-state inactivation of IA was not affected by DA. These DA-induced responses were dose-dependent and they involved D2 receptor activation. They were mimicked by the specific D2 receptor agonist bromocriptine (10 nM) and blocked by the specific D2 receptor antagonist sulpiride (100 nM), the D1 antagonist SCH 23390 being ineffective. The ability of DA to increase voltage-dependent K+ currents cannot be observed without GTP in the recording pipette. It was pertussis-toxin-sensitive but was affected neither by bath application of 1 mM forskolin nor by the presence of 500 microM cyclic AMP with 500 microM 3-isobutyl-1-methylxanthine in the pipette solutions. We conclude that in lactotroph cells DA specifically increases two voltage-dependent K+ currents via a pertussis-toxin-sensitive guanine nucleotide regulatory protein and appears to be independent of intracellular cyclic AMP. This effect leads to a decrease in the excitability of the cell, explaining in part the inhibitory effect of DA on prolactin release.     

A role for a glibenclamide-sensitive, relatively ATP-insensitive K+ current in regulating membrane potential and current in rat aorta

OBJECTIVE: ATP-sensitive K+ channels have been classified based on their inhibition by cytoplasmic ATP. Recent evidence in vascular smooth muscle has suggested that these channels show weak sensitivity to intracellular ATP. However, it is not known whether these channels regulate the resting K+ conductance in vascular smooth muscles. Therefore, the aim of the present investigation was to characterize this current in rat aorta myocytes and to examine whether it contributes to setting the membrane potential. METHODS: The conventional and nystatin-permeablised whole cell patch clamp techniques were used to characterize the effect of glibenclamide on membrane potential and K+ current in enzymatically dispersed rat aorta myocytes. RESULTS: The mean resting potential measured in current clamp mode using the permeabilized patch approach was -54 +/- 5 mV (n = 8). Glibenclamide (10 microM) caused a reversible 24-mV depolarization in these cells. In symmetrical K+ (135 mM) solution an inward glibenclamide-sensitive (10 microM) current (-4.1 +/- 0.7 pA/pF; n = 5), hereafter termed Iglib, was observed at a membrane potential of -80 mV when cells held at -60 mV were ramped from -80 to +80 mV. In the absence of any nucleotide in the pipette solution, Iglib measured by the conventional whole-cell method was -23.69 +/- 4.65 pA/pF (n = 9). With 1 and 3 mM ATP in the pipette, the average current density was -25 +/- 6.3 pA/pF (n = 8), and -9.4 +/- 2.7 pA/pF (n = 9), respectively. In the absence of ATP, 1 mM GDP significantly (P < 0.01) increased Iglib (-44.8 +/- 8.4 pA/pF; n = 13). Inclusion of 1 mM ATP in the GDP-containing pipette solution had no significant effect on the current amplitude (-56.4 +/- 10.7 pA/pF; n = 7). Iglib fell to -11.0 +/- 2.9 pA/pF (n = 10) if 1 mM GDP and 3 mM ATP were present. In symmetrical K+, the Iglib observed in the presence of 1 mM ATP in the pipette was increased by more than two-fold in the presence of 10 microM levcromakalim. In PSS containing 5 mM K+, a significant glibenclamide-sensitive current was observed at -45 mV membrane potential when cells dialyzed with 1 mM ATP were ramped between -80 to 30 mV. CONCLUSION: These results demonstrate that Iglib channels in rat aorta myocytes differ from classical KATP channels, being relatively insensitive to intracellular ATP. Iglib therefore appears to have an important role in contributing to the maintenance of the resting potential in rat aortic smooth muscle.     

Electrophysiological characterization of cloned m1 muscarinic receptors expressed in A9 L cells.

The electrophysiological properties of A9 L cells stably transfected with m1 muscarinic receptor cDNA were examined by using the whole-cell patch-clamp technique. In current-clamp recordings, acetylcholine (AcCho) elicited a hyperpolarization of all transfected cells studied but had no effect on nontransfected A9 L cells. In voltage-clamp recordings, AcCho elicited an outward current at -50 mV accompanied by an increase in conductance. The onset of the current response was consistently delayed by several seconds with respect to the onset of the application of AcCho and could not be accounted for by diffusion. The AcCho-induced currents were reversibly inhibited by the muscarinic receptor antagonist atropine (1 microM) but were unaffected by the nicotinic receptor antagonist tubocurarine (50 microM). Ion-substitution experiments replacing K+ with N-methyl-D-glucamine and Cl- with methanesulfonate indicated that the current was carried mainly by K+, although a minor part appeared to be carried by Cl-. The AcCho-induced current could be blocked by the K+ channel blocking agents tetraethylammonium ion, 4-aminopyridine, apamin, and Ba2+ but not by Cs+. The AcCho-induced current was inhibited when 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was included in the patch pipette or when extracellular Cd2+ or Co2+ was applied, indicating a role for intracellular Ca2+ in the generation of the response. Thus, these results show that cloned m1 muscarinic receptors expressed in A9 L cells can activate a Ca2+-dependent K+ conductance, possibly via a second-messenger system.     

Determinants of action potential duration in neonatal rat ventricle cells.

STUDY OBJECTIVE--The aim was to study the currents that determine the action potential duration in ventricular cells from neonatal rats. DESIGN--Microelectrode measurements of action potentials from ventricle strips were compared with action potentials obtained from isolated myocytes with the whole cell patch clamp method in current clamp mode. Ionic currents were studied in myocytes in voltage clamp mode using recognised modulators of channel activity. EXPERIMENTAL MATERIAL--Neonatal rats (2 d old) were decapitated and myocytes were prepared from the apical third of collagenase treated hearts. MEASUREMENTS AND MAIN RESULTS--Modification of the action potential by 1.8-5.0 mM Ca, 2.0 mM Co, 8 mM 4-aminopyridine, 1.8 mM Sr, and 20 mM tetraethylammonium suggested the presence of the slowly inactivating Ca current ICa,L, an early outward current Ieo, and at least one other K current. Action potentials from myocytes and ventricular strips were comparable. Voltage clamp experiments were confirmatory and revealed currents with the following properties: (1) ICa,L: a Ca current with a current density of 21.7 microA.cm-2, activated between -30 and -20 mV, saturated at 1.8 Cao, inactivated faster at 5 than at 1.8 mM Cao, more permeable to Ba and Sr than to Ca, and with Sr as charge carrier blocked by Ca; (2) Ieo: the peak current had a linear I/V relation between 0 and 70 mV and was abolished by 4 mM 4-aminopyridine; (3) IK1: the current was an inward rectifier that showed a relaxation at potentials negative to -90 mV. CONCLUSIONS--Action potentials obtained from neonatal rat ventricle with microelectrodes are comparable with those measured in myocytes in current clamp mode. The action potential duration is mainly determined by ICa,L, Ieo, and IK1, and there is no evidence for the presence of a delayed rectifier.     

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.     

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.     

Effects of K+ channel blockers on vascular tone in the perfused rat lung

To learn more of the role of K+ channel activity in the regulation of pulmonary vascular tone, we compared the pressor effects of the differential blockers of numerous K+ channels, tetraethylammonium chloride and 4-aminopyridine, and the inhibitor of ATP-sensitive K+ channels glibenclamide in meclofenamate-treated salt solution-perfused rat lungs. Tetraethylammonium (1 to 20 mM) and 4-aminopyridine (1 to 10 mM), but not glibenclamide (1 to 20 microM) caused vasoconstriction in the normoxic lung. The Ca++ channel blocker nifedipine (0.1 microM) and the alpha adrenoceptor antagonist phentolamine (10 microM) inhibited the 4-aminopyridine response by about 50% and reduced slightly the smaller tetraethylammonium response. 4-Aminopyridine and, to a lesser extent, tetraethylammonium, but not glibenclamide, also potentiated peak vasoconstriction to angiotensin II and airway hypoxia. Nifedipine, but not phentolamine, inhibited hypoxic vasoconstriction and prevented the potentiation by 4-aminopyridine. These results suggest that Ca(++)- and/or voltage-activated (not ATP-sensitive) K+ channels may be important in maintaining low pulmonary vascular tone.     

Protein kinase C activation enhances the delayed rectifier potassium current in guinea-pig heart cells

The possible involvement of protein kinase C in modulating membrane currents was investigated in isolated guinea-pig ventricular cells. In a Na(+)-and K(+)-free external solution, the delayed rectifier K+ current (IK) was increased by the activator of protein kinase C (PKC), 12-O-tetradecanoylphorbol-13-acetate (TPA). The amplitude of the IK tail elicited by a return from a depolarizing pulse for 3 s at + 50 mV to a holding potential of -30 mV was increased by 32 +/- 4% (mean +/- S.E., (n = 6) after the external application of 1 nM TPA, and by 60 +/- 17% (n = 5) after 10 nM. The increase in IK produced by 1 nM TPA was abolished by the inhibitor of PKC, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 microM). In addition, the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG, 125 microM) also increased IK (58 +/- 9%, n = 3). PKC purified from bovine brain remarkably increased IK (151 +/- 101%, n = 5) in the presence of 1 nM TPA when it was internally applied using the cell dialysis method. The concentration-response curve of IK for the intracellular concentration of Ca2+ was shifted to the left by 1 nM TPA, suggesting a Ca2(+)-dependent action of PKC and/or altered Ca2(+)-sensitivity of IK channels by phosphorylation. On the other hand, 1 nM TPA had no substantial influence on the Ca2+ current (decreased by 7 +/- 4%, n = 5) or the inward-rectifier K+ current (decreased by 5 +/- 5% in outward component, and 3 +/- 8% in inward component, n = 6). Therefore, the action of PKC was to specifically increase IK without affecting the other two currents.     

Single calcium channels in native sarcoplasmic reticulum membranes from skeletal muscle.

Electrical properties of native sarcoplasmic reticulum membranes from rabbit skeletal muscle were investigated using the patch-clamp technique. Bilayers were assembled at the tip of patch pipettes from monolayers formed at the air-water interface of sarcoplasmic reticulum membrane suspensions. The membranes were found to contain a spontaneously active cation channel of small conductance (5 pS in 200 mM CaCl2, symmetrical solutions) that was selective for Ca2+ and Ba2+. Between 50 and 200 mM CaCl2 (symmetrical) the increase in conductance as a function of [Ca2+] fit a hyperbola (K0.5, 83 mM, and gamma max, 7.9 pS) that extrapolated to a single-channel conductance of 0.5 pS at physiological Ca2+ levels. The channel opened in bursts followed by long silent periods of up to a minute. During a burst the channel fluctuated very rapidly with time constants in the millisecond range. The mean burst duration was voltage dependent, increasing from 1.8 s at a pipette voltage of +60 mV to 4.1 s at +80 mV. Over this range, burst frequency decreased with increasing voltage such that the fraction of time spent in the open state (fb) remained constant. Application of 1.6 mM caffeine resulted in activation of the channel that appeared as an increase in mean burst duration. In contrast, 50 microM dantrolene significantly decreased burst frequency, whereas 10 microM nitrendipine had no effect. The functional and pharmacological properties of this Ca2+ channel suggest that it may be important in mediating Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling.     

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 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.     

Effect of actin microfilament on potassium current in guinea pig gastric myocytes

AIM: To investigate the effect of actin microfilament on potassium current and hyposmotic membrane stretch-induced increase of potassium current in gastric antral circular myocytes of guinea pig. METHODS: Whole-cell patch clamp technique was used to record potassium current in isolated gastric myocyes. RESULTS: When the membrane potential was clamped at -60mV, an actin microfilament disruptor, cytochanlasin-B (Cyt-B, 20μmol/L in pipette) increased calcium-activated potassium current (IK（Ca）) and delayed rectifier potassium current (IK（V）)to 138.4&#177;14.3% and 142.1&#177;13.1%respectively at +60mV. In the same condition, an actin microfilament stabilizer phalloidin (20μmol/L in pipette)inhibited IK（Ca） and IK（V） to 74.2&#177;7.1% and 75.4&#177;9.9% respectively. At the holding potential of -60mV, hyposmotic membrane stretch increased IK（Ca） and IK（V） by 50.6&#177;9.7% and 24.9&#177;3.3% at +60mV respectively. In the presence of cytochalasin-B and phalloidin (20μmol/L, in the pipette)condition, hyposmotic membrane stretch also increased IK（Ca） by 44.5&#177;7.9% and 55.7&#177;9.8% at +60mV respectively. In the same condition, cytochalasin-B and phalloidin also increased IK（V） by 23.0&#177;5.5% and 30.3&#177;4.5% respectively. However, Cyt-B and phalloidin did not affect the amplitude of hyposmotic membrane stretch-induced increase of IK（Ca） and IK（V）. CONCLUSION: Actin microfilaments regulate the activities of potassium channels, but they are not involved in the process of hyposmotic membrane stretch-induced increase of potassium currents in gastric antral circular myocytes of guinea pig.     

Amiodarone inhibits cardiac ATP-sensitive potassium channels

INTRODUCTION: ATP-sensitive K+ channels (K(ATP)) are expressed abundantly in cardiovascular tissues. Blocking this channel in experimental models of ischemia can reduce arrhythmias. We investigated the acute effects of amiodarone on the activity of cardiac sarcolemmal K(ATP) channels and their sensitivity to ATP. METHODS AND RESULTS: Single K(ATP) channel activity was recorded using inside-out patches from rat ventricular myocytes (symmetric 140 mM K+ solutions and a pipette potential of +40 mV). Amiodarone inhibited K(ATP) channel activity in a concentration-dependent manner. After 60 seconds of exposure to amiodarone, the fraction of mean patch current relative to baseline current was 1.0 +/- 0.05 (n = 4), 0.8 +/- 0.07 (n = 4), 0.6 +/- 0.07 (n = 5), and 0.2 +/- 0.05 (n = 7) with 0, 0.1, 1.0, or 10 microM amiodarone, respectively (IC50 = 2.3 microM). ATP sensitivity was greater in the presence of amiodarone (EC50 = 13 +/- 0.2 microM in the presence of 10 microM amiodarone vs 43 +/- 0.1 microM in controls, n = 5; P < 0.05). Kinetic analysis showed that open and short closed intervals (bursting activity) were unchanged by 1 microM amiodarone, whereas interburst closed intervals were prolonged. Amiodarone also inhibited whole cell K(ATP) channel current (activated by 100 microM bimakalim). After a 10-minute application of amiodarone (10 microM), relative current was 0.71 +/- 0.03 vs 0.92 +/- 0.09 in control (P < 0.03). CONCLUSION: Amiodarone rapidly inhibited K(ATP) channel activity by both promoting channel closure and increasing ATP sensitivity. These actions may contribute to the antiarrhythmic properties of amiodarone.     

Characteristics of two basolateral potassium channel populations in human colonic crypts.

The basolateral membrane of human colonic crypt cells contains Ca2+ and cAMP activated, Ba2+ blockable, low conductance (23 pS) K+ channels, which probably play an important part in intestinal Cl- secretion. This study has defined more clearly the basolateral K+ conductive properties of human colonic crypts using patch clamp recording techniques. High conductance (138 pS) K+ channels were seen in 25% of patches (one or two channels per patch), and significantly inhibited by the addition of 5 mM Ba2+, 1 mM quinidine or 20 mM tetraethylammonium chloride (TEA) to the cytosolic side of excised inside-out patches, whereas 1 mM diphenylamine-2-carboxylic acid (DPC) had no effect. In contrast, clusters of the 23 pS K+ channel (two to six channels per patch) were present in > 75% of patches, and channel activity was inhibited by quinidine and DPC, but not by TEA. Activity of the 138 pS K+ channel in inside-out patches was abolished almost completely by removal of bath Ca2+, but in contrast with its effect on the 23 pS K+ channel, addition of 0.1 mM carbachol had no effect on the 138 pS K+ channel in cell attached patches. It is concluded that human colonic crypt cells possess two discrete basolateral K+ channel populations, which can be distinguished by their responses to K+ channel blockers, and their different sensitivities to changes in intracellular Ca2+ concentration.     

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.     

Ca2+-activated synexin forms highly selective, voltage-gated Ca2+ channels in phosphatidylserine bilayer membranes.

Synexin, a cytosolic protein that mediates Ca2+-dependent membrane fusion, was incorporated into acidic phospholipid bilayers, formed at the tip of a patch pipet. The pipet was filled with a high-Ca2+ solution (50 mM) and immersed in a chamber containing a low-Ca2+ solution (1 mM). Brief exposures of the bilayer to synexin increased the capacitance of the bilayer by a factor of 10 and decreased the membrane resistance by a factor of 20. Reduction of Ca2+ in the chamber to 1 microM caused an abrupt increase in the current required to hold the pipet potential at 0 mV. Under certain conditions channel events could be detected, often occurring in bursts. Consistently, open-time histograms were found to be voltage-dependent and to exhibit one time constant in the time range examined here. The slope conductance for the synexin channel was estimated as 10.2 +/- 2.1 pS for the large Ca2+ gradient with low chamber Ca2+. However, for symmetrical, low-Cl- solutions containing 25 mM Ca2+ the conductance was 26.5 +/- 5.2 pS. Ion-replacement studies showed the synexin channel to much prefer Ca2+ over Ba2+ or Mg2+. Cd2+, a potent blocker of other voltage-gated Ca2+ channels at 100 microM, blocked synexin channels only at very high concentrations (greater than or equal to 10 mM). Similarly, nifedipine, an inhibitor of the nonactivating Ca2+ channel, was effective only at extremely high concentrations (greater than 300 microM). The high selectivity for Ca2+ and the lack of response of the channel to various drugs known to block Ca2+ channels thus distinguish the synexin channel from other types of Ca2+ channels hitherto reported.     

Decreased inward rectifier current in adult rabbit ventricular myocytes maintained in primary culture: a single-channel study.

OBJECTIVE: Regulation of ion channel function in heart has been shown to be affected by changes in the cellular environment. Recently it was shown that rabbit ventricular myocytes kept in primary culture, show a strong reduction in inward rectifier current (IK1). The aim of the present study was to elucidate the mechanism underlying this decrease in IK1, using single-channel measurements. In addition, we studied the effects of primary culture on the ATP-regulated K+ (K.ATP) channel, also a member of the inwardly rectifying K+ channel family. METHODS: Adult rabbit ventricular myocytes were cultured for up to 3 days in Ham's F-10 medium complemented with 1% rabbit serum and 5% glutamine. IK1 and K.ATP channel activity was studied in the inside-out patch configuration of the patch-clamp technique with equimolar K+ concentrations (140 mM K+) on the intra- and extracellular side. Single channel characteristics were determined at various times during culture and compared to those present in freshly isolated myocytes. RESULTS: IK1 channels in freshly isolated myocytes (day 0) had a single-channel conductance of 56.1 +/- 2.5 pS (mean +/- SEM) and an open probability of 0.64 +/- 0.05 (mean +/- SEM). Neither the single-channel conductance nor the open probability (Po) underwent significant changes during culture. The mean number of channels per patch, however, was drastically reduced from 1.2 +/- 0.3 (mean +/- SEM) at day 0 to 0.17 +/- 0.06 at day three. K.ATP channel density and open probability, on the other hand, were both increased with an optimum at day two. Po increased from 0.27 +/- 0.06 at day 0 to 0.63 +/- 0.06 at day three. The mean number of channels per patch was 2.29 +/- 0.57 and 3.25 +/- 0.48 at days 0 and 3 respectively. The unitary current amplitude at -50 mV remained unchanged, suggesting no change in the K.ATP single-channel conductance. CONCLUSIONS: The decrease in IK1 in rabbit ventricular myocytes as has been observed during primary culture is the result of a reduction in the number of active channels and not of altered kinetic or conductive channel properties. The increase in K.ATP channel activity under the same conditions suggests that gene expression of both channel types is differently regulated.     

Local Ca2+ entry through L-type Ca2+ channels activates Ca2+-dependent K+ channels in rabbit coronary myocytes.

Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.     

Characterisation of the Na, K pump current in atrial cells from patients with and without chronic atrial fibrillation

OBJECTIVE: To assess the contribution of the Na, K pump current (I(p)) to the action potential duration (APD) and effective refractory period (ERP) in human atrial cells, and to investigate whether I(p) contributes to the changes in APD and ERP associated with chronic atrial fibrillation (AF). METHODS: Action potentials and ion currents were recorded by whole-cell patch clamp in atrial myocytes isolated from consenting patients undergoing cardiac surgery, who were in sinus rhythm (SR) or AF (>3 months). RESULTS: In cells from patients in SR, the I(p) blocker, ouabain (10 microM) significantly depolarised the membrane potential, V(m), from -80+/-2 (mean+/-S.E.) to -73+/-2 mV, and lengthened both the APD (174+/-17 vs. 197+/-23 ms at 90% repolarisation) and ERP (198+/-22 vs. 266+/-14 ms; P<0.05 for each, Student's t-test, n=7 cells, 5 patients). With an elevated pipette [Na(+)] of 30 mM, I(p) was measured by increasing extracellular [K(+)] ([K(+)](o)) from 0 to 5.4 mM. This produced an outward shift in holding current at -40 mV, abolished by 10 microM ouabain. K(+)- and ouabain-sensitive current densities were similar, at 0.99+/-0.13 and 1.12+/-0.11 pA/pF, respectively (P>0.05; n=9 cells), confirming the K(+)-induced current as I(p). I(p) increased linearly with increasing V(m) between -120 and +60 mV (n=25 cells). Stepwise increments in [K(+)](o) (between 0 and 10 mM) increased I(p) in a concentration-dependent manner (maximum response, E(max)=1.19+/-0.09 pA/pF; EC(50)=1.71+/-0.15 mM; n=27 cells, 9 patients). In cells from patients in AF, the sensitivity of I(p) to both V(m) and [K(+)](o) (E(max)=1.02+/-0.05 pA/pF, EC(50)=1.54+/-0.11 mM; n=44 cells, 9 patients) was not significantly different from that in cells from patients in SR. Within the group of patients in AF, long-term digoxin therapy (n=5 patients) was associated with a small, but significant, reduction in E(max) (0.92+/-0.07 pA/pF) and EC(50) (1.35+/-0.15 mM) compared with non-treatment (E(max)=1.13+/-0.08 pA/pF, EC(50)=1.76+/-0.14 mM; P<0.05 for each, n=4 patients). In cells from non-digoxin-treated patients in AF, the voltage- and [K(+)](o)-sensitivity (E(max) and EC(50)) were similar to those in cells from patients in SR. CONCLUSIONS: The Na, K pump current contributes to the human atrial cell V(m), action potential shape and ERP. However, the similarity in I(p) sensitivity to both [K(+)](o) and V(m) between atrial cells from patients with and without chronic AF indicates that I(p) is not involved in AF-induced electrophysiological remodelling in patients.     

Membrane potassium currents in human radial artery and their regulation by nitric oxide donor

OBJECTIVE: The human radial artery has demonstrated superior long-term results as a graft in coronary bypass surgery, but undesirable post-surgical spasm limits its clinical application. Few have examined its excitatory properties, especially the underlying ion channel mechanisms. In this study, we investigated the kinetic and pharmacological properties of the smooth muscle membrane potassium currents of this important artery. METHODS AND RESULTS: Using whole cell patch-clamp techniques, we found the K(+) current to be voltage-dependent and outwardly rectifying. Voltage-dependent inactivation was observed, being half-maximal at +28.0 mV but incomplete even at +40 mV. The K(+) currents were predominantly sensitive to the K(Ca) blocker tetraethylammonium (TEA; 63.9+/-12.1% inhibition, p<0.05), less sensitive to the Kv blocker 4-aminopyridine (4-AP; 32.8+/-4.4% inhibition, p<0.05), and the K(ATP) blocker glibenclamide (28.7+/-8.5% inhibition), at -20 mV testing potential. Resting membrane potential was -52.0+/-6.8 mV (n=5), and suppression of K(+) currents by TEA and iberiotoxin (IbTx) caused membrane depolarization. Western blot analysis with channel-specific antibodies confirmed the presence of K(Ca) and Kv channel proteins. TEA evoked 20.7+/-9.9% of the contractile response to 60 mM KCl, whereas IbTx caused about 10% of the above response at 10(-7) M. The nitric oxide donor SNAP augmented membrane K(+) currents in a concentration-dependent fashion; the augmentation was completely suppressed by TEA, but was relatively insensitive to the guanylate cyclase inhibitor ODQ. CONCLUSIONS: The radial artery manifests mainly Ca(2+)-dependent K(+) currents at rest; this current is augmented by nitric oxide through a cGMP- and protein kinase G-independent action. The relatively depolarized membrane potential, as well as its muscular structure, predisposes the radial artery to spasm. Agents that activate the Ca(2+)-dependent K(+) current could be of therapeutic value in preventing post-surgical vasospasm.