Time-dependent Outward Current Research Articles

A Dual Role for the OsK5.2 Ion Channel in Stomatal Movements and K+ Loading into Xylem Sap.

The roles of potassium channels from the Shaker family in stomatal movements have been investigated by reverse genetics analyses in Arabidopsis (Arabidopsis thaliana), but corresponding information is lacking outside this model species. Rice (Oryza sativa) and other cereals possess stomata that are more complex than those of Arabidopsis. We examined the role of the outward Shaker K+ channel gene OsK5.2. Expression of the OsK5.2 gene (GUS reporter strategy) was observed in the whole stomatal complex (guard cells and subsidiary cells), root vasculature, and root cortex. In stomata, loss of OsK5.2 functional expression resulted in lack of time-dependent outward potassium currents in guard cells, higher rates of water loss through transpiration, and severe slowdown of stomatal closure. In line with the expression of OsK5.2 in the plant vasculature, mutant plants displayed a reduced K+ translocation from the root system toward the leaves via the xylem. The comparison between rice and Arabidopsis show that despite the strong conservation of Shaker family in plants, substantial differences can exist between the physiological roles of seemingly orthologous genes, as xylem loading depends on SKOR and stomatal closure on GORK in Arabidopsis, whereas both functions are executed by the single OsK5.2 Shaker in rice.

A Functional Role for the 'Fibroblast-Like Cells' in Gastrointestinal Smooth Muscles (J Physiol 2011;589[Pt 3]:697-710)

The physiologic role of fibroblast-like cells (FLCs) has not been well established. Recently Kurahashi et al1 suggested the involvement of FLCs in neurotransmission by Ca2+-activated K+ (SK3) channels. They demonstrated that a network of more extensively branching cells with platelet-derived growth factor receptor alpha (PDGFRα) existed between the circular and longitudinal muscles in the plane of the myenteric plexus. FLCs (PDGFRα+) were located in the same anatomical spaces and distributed in similar density as interstitial cells of Cajal (ICC), but PDGFRα+ cells were distinct from c-Kit+ ICC, as shown by double labeling. Other double labeling experiments with anti-SK3 antibody revealed that PDGFRα+ cells in both muscle layers and the region of the myenteric plexus expressed SK3. Some other experiments showed that PDGFRα+ cells labeled with enhanced green fluorescent protein (eGFP) produced robust expression of P2ry1 (P2Y1 receptors) and Kcnn3 (SK3 channels). Whole-cell patch clamp results suggested that PDGFRα+ cells express a high density of apamin-sensitive Ca2+-activated K+ channels, which is consistent with the expression of SK3 channels: small amplitude and time-dependent outward currents without voltage-dependent inward currents in step depolarization, and significant reduction of this currents by apamin 300 nM and charybdotoxin (block both BK and IK conductance Ca2+-activated K+ channels) caused a small but significant reduction in outward currents. Single channel patch recording (excised inside-out patch) to change Ca2+ at the intracellular surface produced the results consistent with previous reported data of Ca2+-activated K+ channels. Pharmacological data using purines (ATP, β-NAD and ADP) evoked significant outward currents in PDGFRα+ cells. Apamin and MRS2500 (P2Y1 antagonist) attenuated current amplitudes. The authors concluded that PDGFRα+ cells are a novel class of excitable cells with large current densities attributable to SK channels that are the molecular and ionic apparatus to mediate enteric inhibitory responses to purines in gastrointestinal (GI) muscles.

Characterization of the Rapidly Activating Delayed Rectifier Potassium Current, I Kr, in HL-1 Mouse Atrial Myocytes

HL-1 is the adult murine cardiac cell line that can be passaged repeatedly in vitro without losing differentiated phenotype. The present study was designed to characterize the rapidly activating delayed rectifier potassium current, I (Kr), endogenously expressed in HL-1 cells using the whole-cell patch-clamp technique. In the presence of nisoldipine, depolarizing voltage steps applied from a holding potential of -50 mV evoked the time-dependent outward current, followed by slowly decaying outward tail current upon return to the holding potential. The amplitude of the current increased with depolarizations up to 0 mV but then progressively decreased with further depolarizations. The time-dependent outward current as well as the tail current were highly sensitive to block by E-4031 and dofetilide (IC(50) of 21.1 and 15.1 nM, respectively) and almost totally abolished by micromolar concentrations of each drug, suggesting that most of the outward current in HL-1 cells was attributable to I (Kr). The magnitude of I (Kr) available from HL-1 cells (18.1 +/- 1.5 pA pF(-1)) was sufficient for reliable measurements of various gating parameters. RT-PCR and Western blot analysis revealed the expression of alternatively spliced forms of mouse ether-a-go-go-related genes (mERG1), the full-length mERG1a and the N-terminally truncated mERG1b isoforms. Knockdown of mERG1 transcripts with small interfering RNA (siRNA) dramatically reduced I (Kr) amplitude, confirming the molecular link of mERG1 and I (Kr) in HL-1 cells. These findings demonstrate that HL-1 cells possess I (Kr) with properties comparable to those in native cardiac I (Kr) and provide an experimental model suitable for studies of I (Kr) channels.

Ventricular Fibrillation

Ventricular fibrillation (VF) is the leading cause of sudden cardiac death. This brief review addresses issues relevant to the dynamics of the rotors responsible for functional reentry and VF. It also makes an attempt to summarize present-day knowledge of the manner in which the dynamic interplay between inward and outward transmembrane currents and the heterogeneous cardiac structure establish a substrate for the initiation and maintenance of rotors and VF. The fragmentary nature of our current understanding of ionic VF mechanisms does not even allow an approach toward a "Theory of VF". Yet some hope is provided by recently obtained insight into the roles played in VF by some of the sarcolemmal ion channels that control the excitation-recovery process. For example, strong evidence supports the idea that the interplay between the rapid-inward sodium current and the inward-rectifier potassium current controls rotor formation, as well as rotor stability and frequency. Solid evidence also exists for an involvement of L-type calcium current in the control of rotor frequency and in determining VF-to-ventricular tachycardia conversion. Less clear, however, is whether or not time dependent outward currents through voltage-gated potassium channels affect the fibrillatory process. Hopefully, taking advantage of currently available approaches of structural, molecular and cellular biology, together with computational and imaging techniques, will afford us the opportunity to further advance knowledge on VF mechanisms.

Olanzapine prolongs cardiac repolarization by blocking the rapid component of the delayed rectifier potassium current

Prolongation of the QT interval has been observed during treatment with olanzapine, a thienobenzodiazepine antipsychotic agent. Our objectives were 1) to characterize the effects of olanzapine on cardiac repolarization and 2) to evaluate effects of olanzapine on the major time-dependent outward potassium current involved in cardiac repolarization, namely I(Kr) (I(Kr): rapid component of the delayed rectifier potassium current).Isolated, buffer-perfused guinea pig hearts (n = 40) were stimulated at different pacing cycle lengths (150-250 msec) and exposed to olanzapine at concentrations ranging from 1 to 100 microM. Olanzapine increased monophasic action potential duration measured at 90% repolarization (MAPD90) in a concentration-dependent manner by 6.7 +/- 0.7 msec at 3 microM but by 26.0 +/- 4.3 msec at 100 microM (250 msec cycle length). Increase in MAPD(90) was also reverse frequency dependent; 30 microM olanzapine increased MAPD90 by 28.0 +/- 6.2 msec at a pacing cycle length of 250 msec but by only 18.9 +/- 2.2 msec at a pacing cycle length of 150 msec. Experiments in HERG-transfected (HERG: human ether-a-gogo-related gene) HEK293 cells (n = 36) demonstrated concentration-dependent block of the rapid component (I(Kr)) of the delayed rectifier potassium current: tail current was decreased 50% at olanzapine 3.8 microM. Olanzapine possesses direct cardiac electrophysiological effects similar to those of class III anti-arrhythmic drugs. These effects were observed at concentrations that can be measured in patients under conditions of impaired drug elimination such as renal or hepatic insufficiency, during co-administration of other CYP1A2 substrates/inhibitors or after drug overdose. These results offer a new potential explanation for QT prolonging effects observed during olanzapine treatment in patients.

TOK homologue in Neurospora crassa: first cloning and functional characterization of an ion channel in a filamentous fungus.

In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the "filamentous" polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K(+) channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K(+) channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K(+) efflux. NcTOKA channel gating was sensitive to extracellular K(+) such that channel activation was dependent on the reversal potential for K(+). However, expression of NcTOKA was able to overcome the K(+) auxotrophy of a yeast mutant missing the K(+) uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K(+) influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K(+) currents at potentials negative of E(K). NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca(2+), verapamil, quinine, and TEA(+) but was insensitive to Cs(+), 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

Pharmacological enhancement of hemi-gap-junctional currents in Xenopus oocytes

Hemichannels formed by expressing connexin subunits in Xenopus oocytes provide a valuable tool for revealing the gating properties of intercellular gap junctions in electrically coupled cells. We used the two electrode voltage-clamp technique to demonstrate that activation of the time-dependent outward hemichannel currents brings into play a sodium current of similar time course and opposite polarity; the interaction between these opposing currents had not been explored previously. Using the endogenous connexin (Cx38) of Xenopus oocytes as a model system, we have shown that substituting choline for sodium in the bath solution eliminates the sodium current, thereby unmasking large hemichannel currents, and enabling pharmacological studies of agents that are known to modulate gap-junctional conductances. The cinchona alkaloid quinine also effectively blocked the inward current, and in addition, enhanced significantly the Cx38 hemichannel currents in a dose-dependent fashion; the Hill coefficient of 1.9 suggests that the binding of at least two molecules of quinine is required to produce the effect. Intracellular quinine had no effect on hemichannel currents, and experiments on the displacement of quinine suggest that binding is at an external site near or within the mouth of the hemichannel. Intracellular acidification suppressed the quinine-enhanced hemichannel currents, indicating that quinine does not block the proton binding site. We found that retinoic acid (RA) and carbenoxolone, agents that block gap-junctional channels in coupled neurons and other cell types, also suppressed Cx38 hemichannel currents with an IC 50 of approximately 2 and 34 μM for RA and carbenoxolone, respectively. Raising extracellular calcium to 3 mM suppressed both the hemichannel current and the inward sodium current. These results provide a foundation upon which to further characterize the gating of hemichannel currents mediated by connexins expressed in Xenopus oocytes.

Large Mg 2+-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae

Two genes in Saccharomyces cerevisiae, ALR1 and ALR2, encode proteins putatively involved in Mg 2+ uptake. The present study supports this role for ALR1 and provides the first electrophysiological characterisation of this protein. The patch-clamp technique was used to measure whole-cell ion currents in protoplasts prepared from the wild-type strain, the alr1 alr2 double mutant (CM66), and the double mutant over-expressing the ALR1 gene (CM66+ALR1). With 50 mM Mg 2+ in the bathing solution, the inward current in protoplasts of CM66+ALR1 averaged −264±48 pA at −150 mV. Inward currents measured in the wild-type and CM66 protoplasts were more than five-fold smaller. When Mg 2+ was the major cation in the pipette solution, time-dependent outward currents were also detected in CM66+ALR1 protoplasts suggesting ALR1 can facilitate Mg 2+ efflux as well as uptake. We conclude that the ALR1 gene encodes a transport protein. The large magnitude of the Mg 2+-dependent currents suggests that ALR1 could function as a cation channel.

Large Mg2+-dependent currents are associated with the increased expression ofALR1inSaccharomyces cerevisiae

Two genes in Saccharomyces cerevisiae, ALR1 and ALR2, encode proteins putatively involved in Mg(2+) uptake. The present study supports this role for ALR1 and provides the first electrophysiological characterisation of this protein. The patch-clamp technique was used to measure whole-cell ion currents in protoplasts prepared from the wild-type strain, the alr1 alr2 double mutant (CM66), and the double mutant over-expressing the ALR1 gene (CM66+ALR1). With 50 mM Mg(2+) in the bathing solution, the inward current in protoplasts of CM66+ALR1 averaged -264+/-48 pA at -150 mV. Inward currents measured in the wild-type and CM66 protoplasts were more than five-fold smaller. When Mg(2+) was the major cation in the pipette solution, time-dependent outward currents were also detected in CM66+ALR1 protoplasts suggesting ALR1 can facilitate Mg(2+) efflux as well as uptake. We conclude that the ALR1 gene encodes a transport protein. The large magnitude of the Mg(2+)-dependent currents suggests that ALR1 could function as a cation channel.

Permeability Characteristics of Monovalent Cations in Atrial Myocytes of the Rat Heart

We investigated the permeability of Cs+ and Na+ through various ion channels in rat atrial myocytes using the whole-cell voltage-clamp technique. With isotonic CsCl (140 mM) on both sides of the membrane and nominally [Ca2+]o-free conditions, depolarising clamp pulses induced an increase of outward currents which showed a biphasic time course. Repolarisation to the holding potential induced inward tail currents. With isotonic NaCl, depolarisation also induced outward currents which showed a monotonic decay, but inward tail currents were not observed. Both in NaCl and CsCl, currents were hardly affected by TEA (10 mM), 4-AP (5 mM) and DIDS (100 microM). Nicardipine (1 M) almost completely blocked time-dependent outward currents in isotonic NaCl solution, leaving only time-independent currents which showed linear I-V relationship. In isotonic CsCl conditions, nicardipine blocked outward current considerably, but there still remained time-dependent outward currents and inward tail currents. Addition of E-4031 (2-20 M) which is known as a specific blocker of the rapidly activating delayed rectifier K+ current (IKr) completely blocked these time-dependent outward and inward currents, leaving only a time-independent current. Time-independent currents recorded in the presence of nicardipine and E-4031 were inhibited by GdCl3, which is known to block non-selective cation (NSC) currents. From these results, it was suggested that NSC current in atrial myocytes can be investigated in isotonic Cs+ or Na+ solution in the presence of Ca2+ channel and IKr blockers.

Role of Na-Ca exchange in the action potential changes caused by drive in cardiac myocytes exposed to different Ca2+loads

The role of Na-Ca exchange in the membrane potential changes caused by repetitive activity ("drive") was studied in guinea pig single ventricular myocytes exposed to different [Ca2+]o. The following results were obtained. (i) In 5.4 mM [Ca2+]o, the action potentials (APs) gradually shortened during drive, and the outward current during a train of depolarizing voltage clamp steps gradually increased. (ii) The APs shortened more and were followed by a decaying voltage tail during drive in the presence of 5 mM caffeine; the outward current became larger and there was an inward tail current on repolarization during a train of depolarizing steps. (iii) These effects outlasted drive so that immediately after a train of APs, currents were already bigger and, after a train of steps, APs were already shorter. (iv) In 0.54 mM [Ca2+]o, the above effects were much smaller. (v) In high [Ca2+]o APs were shorter and outward currents larger than in low [Ca2+]o. (vi) In 10.8 mM [Ca2+]o, both outward and inward currents during long steps were exaggerated by prior drive, even with steps (+80 and +120 mV) at which there was no apparent inward current identifiable as I(Ca). (vii) In 0.54 mM [Ca2+]o, the time-dependent outward current was small and prior drive slightly increased it. (viii) During long steps, caffeine markedly increased outward and inward tail currents, and these effects were greatly decreased by low [Ca2+]o. (ix) After drive in the presence of caffeine, Ni2+ decreased the outward and inward tail currents. It is concluded that in the presence of high [Ca2+]o drive activates outward and inward Na-Ca exchange currents. During drive, the outward current participates in the plateau shortening and the inward tail current in the voltage tail after the action potential.

Heterogeneous distribution of the two components of delayed rectifier K+ current: a potential mechanism of the proarrhythmic effects of methanesulfonanilideclass III agents.

To elucidate the regional difference of the K+ current blocking effects of methanesulfonanilide class III agents. Regional differences in action potential duration (APD) and E-4031-sensitive component (IKr) as well as -insensitive component (IKs) of the delayed rectifier K+ current (IK) were investigated in enzymatically isolated myocytes from apical and basal regions of the rabbit left ventricle using the whole-cell clamp technique. At 1 Hz stimulation, APD was significantly longer in the apex than in the base (223.1 +/- 10.6 vs. 182.7 +/- 14.5 ms, p < 0.05); application of 1 microM E-4031 caused more significant APD prolongation in the apex than in the base (32.5 +/- 6.4% vs. 21.0 +/- 8.8%, p < 0.05), resulting in an augmentation of regional dispersion of APD. In response to a 3-s depolarization pulse to +40 mV from a holding potential of -50 mV, both IK tail and IKs tail densities were significantly smaller in apical than in basal myocytes (IK: 1.56 +/- 0.13 vs. 2.09 +/- 0.21 pA/pF, p < 0.05; IKs: 0.40 +/- 0.15 vs. 1.43 +/- 0.23, p < 0.01), whereas IKr tail density was significantly greater in the apex than in the base (1.15 +/- 0.13 vs. 0.66 +/- 0.11 pA/pF, p < 0.01). The ratio of IKs/IKr for the tail current in the apex was significantly smaller than that in the base (0.51 +/- 0.21 vs. 3.09 +/- 0.89; p < 0.05). No statistical difference was observed in the voltage dependence as well as activation and deactivation kinetics of IKr and IKs between the apex and base. Isoproterenol (1 microM) increased the time-dependent outward current of IKs by 111 +/- 8% during the 3-s depolarizing step at +40 mV and its tail current by 120 +/- 9% on repolarization to the holding potential of -50 mV, whereas it did not affect IKr. The regional differences in IK, in particular differences in its two components may underlie the regional disparity in APD, and that methanesulfonanilide class III antiarrhythmic agents such as E-4031 may cause a greater spatial inhomogeneity of ventricular repolarization, leading to re-entrant arrhythmias.

Block of the rapid component of the delayed rectifier potassium current by the prokinetic agent cisapride underlies drug-related lengthening of the QT interval.

Lengthening of the QT interval and torsades de pointes resulting in cardiac arrests and deaths have been noticed during treatment with cisapride, a newly developed gastrointestinal prokinetic agent. The rapid (I[Kr]) and slow (I[Ks]) components of the delayed rectifier current (I[K]) are candidate ionic currents to explain cisapride-related toxicity because of their role in repolarization of cardiac ventricular myocytes. Our objectives were to (1) characterize effects of cisapride on two major time-dependent outward potassium currents involved in the repolarization of cardiac ventricular myocytes, I(Kr) and I(Ks), and (2) determine action potential-prolonging effects of cisapride on isolated hearts. A first set of experiments was performed in isolated guinea pig ventricular myocytes with the whole-cell configuration of the patch-clamp technique. Cells were held at -40 mV while time-dependent outward currents were elicited by depolarizing pulses lasting either 250 ms (I[K250]) or 5000 ms (I[K5000]). Effects of cisapride on the I(Kr) component were assessed by measurement of time-dependent activating currents elicited by short pulses (250 ms; I[K250]) to low depolarizing potentials (-20, -10, and 0 mV). Time-dependent activating currents elicited by long pulses (5000 ms; I[K5000]) to positive potentials (>+30 mV) were recorded to assess effects of the drug on the I(Ks) component. A second set of experiments was conducted in isolated guinea pig hearts buffer-perfused in the Langendorff mode to assess effects of the drug on monophasic action potential duration measured at 90% repolarization (MAPD90). Hearts were exposed to cisapride 100 nmol/L at decremental pacing cycle lengths of 250, 225, 200, 175, and 150 ms to determine reverse frequency-dependent effects of the drug. Overall, 112 myocytes were exposed to seven concentrations of cisapride (10 nmol/L to 10 micromol/L). Cisapride inhibited I(Kr), the major time-dependent outward current elicited by short pulses (I[K250]) to low depolarizing potentials, in a concentration-dependent manner with an IC50 of 15 nmol/L (therapeutic levels, 50 to 200 nmol/L). Conversely, block of I(Ks) by the drug was less potent (estimated IC50 >10 micromol/L). In isolated hearts (n=9 experiments), cisapride 100 nmol/L increased MAPD90 by 23+/-3 (P<.05) at a basic cycle length of 250 ms but by only 7+/-1 ms (P<.05) at a basic cycle length of 150 ms. Block of I(Kr) gives an explanation to lengthening of cardiac repolarization observed in isolated guinea pig hearts. Potent block of I(Kr) is also likely to underlie prolongation of the QT interval observed in patients receiving clinically recommended doses of cisapride as well as severe cardiac toxicity (torsades de pointes) observed in patients with increased plasma concentrations of the drug.

Mechanisms of solute efflux from seed coats: whole-cell K+currents in transfer cell protoplasts derived from coats of developing seeds ofVicia fabaL.

In developing seed of Vicia faba L., solutes imported through the phloem of the coats move symplastically from the sieve elements to a specialized set of cells (the thin-walled parenchyma transfer cells) for release to the seed apoplast. Potassium (K + ) is the predominant cation released from the seed coats. To elucidate the mechanisms of K + efflux from seed coat to seed apoplast, whole-cell currents across the plasma membranes of protoplasts of thin-walled parenchyma transfer cells were measured using the whole-cell patch-clamp technique. Membrane depolarization elicited a time-dependent and an instantaneous outward current. The reversal potential (E R ) of the time-dependent outward current was close to the potassium equilibrium potential (E K ) and it shifted in the same direction as E K upon changing the external K + concentration, indicating that this current was largely carried by an efflux of K + . The activation of the time-dependent outward K + current could be well fitted by two exponential components plus a constant. The instantaneous outward current could also be carried by K + efflux as suggested by ion substitution experiments. These K + outward rectifier currents elicited by membrane depolarization are probably too small to represent the mechanism for the normal K + efflux from seed coat cells. Membrane hyperpolarization more negative than -80 mV activated a time-dependent inward current. K + influx was responsible for the inward current as the current reversed at membrane voltage close to E K and shifted in the same direction as E K when external [K + ] was varied. Activation of this K + inward rectifier current was well fitted with two exponential components plus a constant. A regulating function for this current is suggested.

ON THE ANTIARRHYTHMIC ACTIONS OF MAGNESIUM IN SINGLE GUINEA‐PIG VENTRICULAR MYOCYTES

1. The hypotheses that magnesium quickly abolishes arrhythmias by acting as a calcium antagonist or by increasing outward potassium currents were tested in guinea-pig isolated ventricular myocytes by recording membrane potentials and currents by means of a single microelectrode discontinuous voltage clamp method. 2. High [Mg2+]o (4-16 mmol/L) slightly increased the amplitude and duration of the action potential (AP) in some myocytes, but overall the changes were not significant. 3. High [Mg2+]o did not decrease the slow inward current (ICa) and had little effect on voltage- and time-dependent outward potassium currents whether or not ICa was allowed to flow. 4. Zero [Mg2+]o decreased the duration, but not amplitude, of the AP. Zero [Mg2+]o had little effect on ICa and on outward currents except for a small increase in outward current in the region of the negative slope of the inward rectifier current-voltage relationship. 5. In our myocytes, in contrast to [Mg2+]o, high [Ca2+]o significantly increased the amplitude and decreased the duration of the AP; at the same time, high [Ca2+]o increases ICa and the outward potassium current. 6. High [Mg2+]o decreased the amplitude of the oscillatory potentials (Vos)induced by various Ca(2+)-overloading procedures (high [Ca2+]o, noradrenaline, strophanthidin and barium). 7. It is concluded that the mechanisms by which high [Mg2+]o quickly suppresses cardiac arrhythmias are related to an extracellular action of Mg2+ and do not include a block of ICa or an increase in outward current. Mg2+ can be antiarrhythmic by decreasing Vos amplitude and possibly by screening the fixed negative charges at the external surface of the sarcolemma.

Ionic transport in the plasma membrane of carrot protoplasts from embryogenic cell-suspension cultures

Ionic transport properties of protoplasts obtained from embryogenic carrot suspension cells were studied by the patch-clamp technique. In the whole-cell configuration, carrot protoplasts presented macroscopic time-dependent outward currents, showing kinetics of activation which did not depend appreciably on the amplitude of the stimulus. Time- and voltage-dependent whole-cell inward rectifying currents as well as instantaneous non-selective currents were also observed. Both time-dependent inward and outward currents are carried by potassium ions. In a cell-attached configuration, two types of single-channel signals, displaying conductances of 10 and 17 pS, were observed; the instantaneous 10 pS channel was also present in outside-out excised patches.

Different Actions Between Pindolol and Propranolol on the Ionic Currents in Spontaneously Beating Rabbit Sino-Atrial Node Cells

Electrophysiological effects of beta-adrenoceptor blocking drugs (pindolol and propranolol) on the action potentials (APs) and the membrane currents in spontaneously beating rabbit sino-atrial (SA) node cells were examined by the two-microelectrode voltage-clamp technique. Cumulative administrations of both blocking drugs (10(-7)-10(-6) M) prolonged the AP duration (APD) and caused a negative chronotropic effect in a concentration-dependent manner. Propranolol exhibited stronger responses than pindolol. At 10(-6) M, propranolol depolarized the maximum diastolic potential (MDP) and pindolol decreased the maximum rate of depolarization significantly, but the drugs had little or no effect on the AP amplitude (APA). In voltage-clamp experiments, both blockers (10(-7)-10(-6) M) inhibited the slow inward and the time-dependent outward currents in a concentration-dependent manner. Holding potential was -40 mV. The hyperpolarization-activated inward current was not affected by pindolol, but it was concentration-dependently inhibited by propranolol. The inactivation curve of Isi was not modified by either pindolol or propranolol. The activation curve of IK was unaffected by pindolol, but was shifted by propranolol 10(-6) M by approximately 11 mV in the depolarizing direction. These results indicate that both beta-adrenoceptor blocking drugs (pindolol and propranolol) inhibit the slow inward and the time-dependent outward currents, but produce different actions on the hyperpolarization-activated inward current and the activation kinetic of IK due to their pharmacological properties, thereby resulting in difference in decrease in the spontaneous activity of rabbit SA node cells.

A new hypoglycemic agent, A-4166, inhibits ATP-sensitive potassium channels in rat pancreatic beta-cells.

Effects of a new hypoglycemic drug, N-[trans-4-isopropylcyclohexy-carbonyl]-D-phenylalanine (A-4166), on membrane current were investigated using the patch-clamp technique in single pancreatic beta-cells isolated from rats. A-4166, at a concentration of 10 microM, depolarized membrane potential of beta-cells and evoked action potentials in the presence of 2.8 mM glucose. The single ATP-sensitive K+ channel (K-ATP channel) current recorded in cell-attached membrane patches was reversibly inhibited by A-4166 (> 0.1 microM) without a change in the single-channel conductance of the K-ATP channel. Both A-4166 and tolbutamide inhibited the whole cell K-ATP channel current with half-maximum inhibition (IC50) of 0.23 and 12.8 microM, respectively (Hill coefficient = 1). In inside-out membrane patches, the IC50 with A-4166 occurred at 4.5 nM, in contrast to 0.7 microM for tolbutamide. A-4166 did not affect L- and T-type Ca2+ channels or the time-dependent outward current. We conclude that A-4166 specifically blocks the K-ATP channel and that the blockade is more potent than that of tolbutamide. The action of A-4166 underlies the mechanism by which the drug stimulates insulin secretion from beta-cells.

Ouabain actions on the spontaneous activity and ionic currents in rabbit sino-atrial node cells

1. 1. The effects of ouabain on the action potentials and the membrane currents in spontaneously beating rabbit sino-atrial (SA) node cells were examined using the two-microelectrode technique. 2. 2. Cumulative administrations of ouabain (10 −8 to 10 −6M) caused a negative chronotropic effect in a concentration-dependent manner. The effect was not modified by atropine (10 −7 M). At 10 −6 M, ouabain prolonged the duration of action potentials, but other parameters were unaffected to any significant extent. Ouabain elicited an arrhythmia, and increasing concentrations increased the incidence of arrhythmia (75% at 3 × 10 −7M). 3. 3. Pretreatment with clonidine (10 −6 M), a selective agonist of presynaptic α 2-adrenoceptors, completely blocked the development of arrhythmia induced by ouabain (3 × 10 −7M). Prazosin (10 −6 M), an α 1 antagonist, had similar effects, and yohimbine (10 −6 to 10 −5 M), an α 2 antagonist, did not affect the arrhythmias. 4. 4. Ouabain (10 −8 to 10 −6 M) inhibited the slow inward and the time-dependent outward currents, but enhanced the hyperpolarization-activated inward current, in a concentration-dependent manner. The time course of inactivation phase for I si was composed of two (fast and slow) components. Ouabain decreased the fast component and increased the slow component. The voltage of half-maximum activation for the outward current was not affected. Ouabain elicited a transient inward current on the repolarizing step, and also on the depolarizing step. 5. 5. These results indicate that ouabain inhibits the slow inward and time-dependent outward currents, but elicits arrhythmias due to the induction of cellular calcium overload, which are modulated by α-adrenoceptors.

Characterization of an E4031-sensitive potassium current in quiescent AT-1 cells.

A cardiac culture cell line (AT-1) recently has been generated from transgenic mice. Initial studies have yielded opposing results as to the nature of the major repolarizing current(s) in these cells. The present study describes the ion selectivity, voltage dependence, and E4031 sensitivity of the major time-dependent outward current present in AT-1 cells. In addition, we have determined whether an outward current with the characteristics we observed could be capable of modulating action potential duration in a frequency-dependent manner (for stimulation cycle lengths between 250 and 1000 msec). Action potentials and membrane currents were recorded from nonconfluent cultures of quiescent AT-1 cells using the "perforated patch" technique. AT-1 cells showed a round appearance 1 or 2 days after plating. An E4031-insensitive transient outward current seemed to be absent in these cells. The main time-dependent outward current was a rapidly activating and rectifying potassium current with properties similar to those of IKr. Most of the potassium current was sensitive to the benzenesulfonamide E4031 (5 microM). The same concentration of E4031 led to a 38% increase in action potential duration. Action potential parameters were independent of the stimulation cycle length within the range of 250 to 1000 msec, thus suggesting that the membrane currents involved in the action potential of AT-1 cells are completely reset within a diastolic interval of approximately 150 msec. AT-1 cells present a unique electrophysiologic phenotype, which is clearly different from that reported for freshly dissociated adult atrial or ventricular myocytes from other species. AT-1 cells may be a good model to study IKr, since there seems to be minimal contamination by other outward conductances (such as IKs). In addition, the feasibility of culturing AT-1 cells provides us with a system where electrophysiologic experiments on IKr currents could be combined with biochemical or molecular biological studies requiring significant periods of incubation in a cell culture system.