Sergey Missan

Tyrosine kinase and phosphatase regulation of slow delayed-rectifier K+ current in guinea-pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the regulation of the cardiac slowly activating delayed‐rectifier K+ current (IKs) that is important for action potential repolarization. Constitutive IKs recorded from guinea‐pig ventricular myocytes was suppressed by broad‐spectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC50, 4.1 ± 0.6 μm), tyrphostin A25 (IC50, 12.1 ± 2.1 μm) and genistein (IC50, 64 ± 4 μm), but was relatively insensitive to the inactive analogues tyrphostin A1, tyrphostin A63, daidzein and genistin. IKs was unaffected by AG1478 (10 μm), an inhibitor of epidermal growth factor receptor TK, and was strongly suppressed by the Src TK inhibitor PP2 (10 μm) but not by the inactive analogue PP3 (10 μm). The results of experiments with forskolin, H89 and bisindolylmaleimide I indicate that the suppression of IKs by TK inhibitors was not mediated via inhibition of (IKs‐stimulatory) protein kinases A and C. To evaluate whether the suppression was related to lowered tyrosine phosphorylation, myocytes were pretreated with TK inhibitors and then exposed to the phosphotyrosyl phosphatase inhibitor orthovanadate (1 mm). Orthovanadate almost completely reversed the suppression of IKs induced by broad‐spectrum TK inhibitors at concentrations around their IC50 values. We conclude that basal IKs is strongly dependent on tyrosine phosphorylation of Ks channel (or channel‐regulatory) protein.

Block of cardiac delayed‐rectifier and inward‐rectifier K+ currents by nisoldipine

The objective of this study was to determine the concentration‐dependent effects of nisoldipine, a dihydropyridine Ca2+ channel blocker, on K+ currents in guinea‐pig ventricular myocytes. Myocytes in the conventional whole‐cell configuration were bathed in normal Tyrodes solution or K+‐free Tyrodes solution for the measurement of the effects of 0.01–100 μM nisoldipine on rapidly activating delayed‐rectifier K+ current (IKr), slowly activating delayed‐rectifier K+ current (IKs), inwardly rectifying K+ current (IK1), and reference L‐type Ca2+ current (ICa,L). Nisoldipine inhibited IKr with an IC50 of 23 μM, and IKs with an IC50 of 40 μM. The drug also had weak inhibitory effects on inward‐ and outward‐directed IK1; the IC50 determined for outward‐directed current was 80 μM. Investigation of nisoldipine action on IKs showed that inhibition occurred in the absence of previous pulsing, and with little change in the time courses of activation and deactivation. However, the drug‐induced inhibition was significantly weaker at +30 mV than at +10 mV. We estimate that nisoldipine is about 30 times less selective for delayed‐rectifier K+ channels than for L‐type Ca2+ channels in fully polarised guinea‐pig ventricular myocytes, and several orders less selective in partially depolarised myocytes.

Selective phenylalkylamine block of IKr over other K+ currents in guinea‐pig ventricular myocytes

Previous studies on verapamil and D600 have established that the Ca2+‐channel blockers also inhibit delayed‐rectifier K+ currents in cardiac tissues and myocytes. However, estimated IC50 values range over two to three orders of concentration, and it is unclear whether this reflects a high selectivity by one or both of the phenylalkylamines for particular K+ channels. The purpose of the present study was to determine the concentration‐dependent actions of verapamil and D600 on three defined cardiac K+ currents. Guinea‐pig ventricular myocytes in the conventional whole‐cell configuration were bathed with normal Tyrodes or K+‐free solution, and pulsed from −80 mV for measurement of the effects of 0.01 μM to 3 mM verapamil and D600 on the inwardly‐rectifying K+ current (IKl) and the two delayed‐rectifier K+ currents, rapidly‐activating IKr and slowly‐activating IKs. The phenylalkylamines inhibited both inward‐ and outward‐directed IKl. The IC50 values for outward IKl were approximately 220 μM. Verapamil and D600 were approximately equipotent inhibitors of the delayed‐rectifier K+ currents. They inhibited IKr with IC50 near 3 μM, and IKs with IC50 280 μM. These results are discussed in relation to previous findings on K+ currents and to the clinical actions of the drugs.

CESE: Cell Electrophysiology Simulation Environment.

UNLABELLED Cell electrophysiology simulation environment (CESE) is an integrated environment for performing simulations with a variety of electrophysiological models that have Hodgkin-Huxley and Markovian formulations of ionic currents. CESE is written in Java 2 and is readily portable to a number of operating systems. CESE allows execution of single-cell models and modification and clamping of model parameters, as well as data visualisation and analysis using a consistent interface. Model creation for CESE is facilitated by an object-oriented approach and use of an extensive modelling framework. The Web-based model repository is available. AVAILABILITY CESE and the Web-based model repository are available at http://cese.sourceforge.net/.

Osmotic modulation of slowly activating IKs in guinea-pig ventricular myocytes

AIMS The aims of the study were to determine the effects of anisosmotic bathing solution on selected properties of I(Ks), the slowly activating delayed-rectifier K(+) current important for repolarization of the action potential in cardiac cells. METHODS AND RESULTS Guinea-pig ventricular myocytes were voltage-clamped using either the ruptured-patch or perforated-patch technique, and the amplitude, time course, and voltage dependence of I(Ks) were determined before [isosmotic (1T)] and during superfusion of hyposmotic (<1T) or hyperosmotic (>1T) bathing solution. Hyposmotic solution increased the amplitude of I(Ks), and hyperosmotic solution decreased it. Anisosmotic-induced changes in I(Ks) amplitude were complete in 2-5 min, well-maintained, reversible, and not accompanied by significant changes in I(Ks) time course and voltage dependence. There was little difference in the results obtained with the ruptured-patch technique and those obtained with the perforated-patch technique. The amplitude of I(Ks) was sensitive to small (±10%) changes in osmolarity, maximally increased by hyposmotic solution with T < 0.7, and strongly decreased by hyperosmotic solution with T > 1.5. Experimental data on a plot of relative (1T = 1.0) I(Ks) amplitude vs. the reciprocal of relative osmolarity are well-described by a Hill equation that has a lower asymptote of 0.0, an upper asymptote of 2.0, and a slope factor of 1.87 ± 0.07. CONCLUSION Modulation of I(Ks) amplitude by anisosmotic solution is independent of patch configuration, unaccompanied by changes in current gating, and well-described by a Hill dose-response relation that predicts relatively strong responses of I(Ks) to small perturbations in external osmolarity.

Insensitivity of cardiac delayed‐rectifier IKr to tyrosine phosphorylation inhibitors and stimulators

1 The rapidly activating delayed‐rectifying K+ current (IKr) in heart cells is an important determinant of repolarisation, and decreases in its density are implicated in acquired and inherited long QT syndromes. The objective of the present study on IKr in guinea‐pig ventricular myocytes was to evaluate whether the current is acutely regulated by tyrosine phosphorylation. 2 Myocytes configured for ruptured‐patch or perforated‐patch voltage‐clamp were depolarised with 200‐ms steps to 0 mV for measurement of IKr tail amplitude on repolarisations to −40 mV. 3 IKr in both ruptured‐patch and perforated‐patch myocytes was only moderately (14–20%) decreased by 100 μM concentrations of protein tyrosine kinase (PTK) inhibitors tyrphostin A23, tyrphostin A25, and genistein. However, similar‐sized decreases were induced by PTK‐inactive analogues tyrphostin A1 and daidzein, suggesting that they were unrelated to inhibition of PTK. 4 Ruptured‐patch and perforated‐patch myocytes were also treated with promoters of tyrosine phosphorylation, including phosphotyrosyl phosphatase (PTP) inhibitor orthovanadate, exogenous c‐Src PTK, and four receptor PTK activators (insulin, insulin‐like growth factor‐1, epidermal growth factor, and basic fibroblast growth factor). None of these treatments had a significant effect on the amplitude of IKr. 5 We conclude that Kr channels in guinea‐pig ventricular myocytes are unlikely to be regulated by PTK and PTP.

Cardiac Na+–Ca2+ exchanger current induced by tyrphostin tyrosine kinase inhibitors

Tyrosine kinase (TK) inhibitors genistein and tyrphostin A23 (A23) inhibited Ca2+ currents in guinea‐pig ventricular myocytes investigated under standard whole‐cell conditions (K+‐free Tyrodes superfusate; EGTA‐buffered (pCa–10.5) Cs+ dialysate). However, the inhibitors (100 μM) also induced membrane currents that reversed between −40 and 0 mV, and the objective of the present study was to characterize these currents. Genistein‐induced current behaved like Cl− current, and was unaffected by either the addition of divalent cations (0.5 mM Cd2+; 3 mM Ni2+) that block the Na+–Ca2+ exchanger (NCX), or the removal of external Na+ and Ca2+. A23‐induced current was independent of Cl− driving force, and strongly suppressed by addition of Cd2+ and Ni2+, and by removal of either external Na+ or Ca2+. These and other results suggested that A23 activated an NCX current driven by submembrane Na+ and Ca2+ concentrations higher than those in the bulk cytoplasm. Improved control of intracellular Na+ and Ca2+ concentrations was obtained by suppressing cation influx (10 μM verapamil) and raising dialysate Na+ to 7 mM and dialysate pCa to 7. Under these conditions, stimulation by A23 was described by the Hill equation with EC50 68±4 μM and coefficient 1.1, tyrphostin A25 was as effective as A23, and TK‐inactive tyrphostin A1 was ineffective. Phosphotyrosyl phosphatase inhibitor orthovanadate (1 mM) antagonized the action of 100 μM A23. The results suggest that activation of cardiac NCX by A23 is due to inhibition of genistein‐insensitive TK.

Inward-rectifier K+ Current in Guinea-pig Ventricular Myocytes Exposed to Hyperosmotic Solutions

Superfusion of heart cells with hyperosmotic solution causes cell shrinkage and inhibition of membrane ionic currents, including delayed-rectifer K+ currents. To determine whether osmotic shrinkage also inhibits inwardly-rectifying K+ current (IK1), guinea-pig ventricular myocytes in the perforated-patch or ruptured-patch configuration were superfused with a Tyrode’s solution whose osmolarity (T) relative to isosmotic (1T) solution was increased to 1.3–2.2T by addition of sucrose. Hyperosmotic superfusate caused a rapid shrinkage that was accompanied by a negative shift in the reversal potential of Ba2+-sensitive IK1, an increase in the amplitude of outward IK1, and a steepening of the slope of the inward IK1-voltage (V) relation. The magnitude of these effects increased with external osmolarity. To evaluate the underlying changes in chord conductance (GK1) and rectification, GK1-V data were fitted with Boltzmann functions to determine maximal GK1 (GK1max) and voltage at one-half GK1max (V0.5). Superfusion with hyperosmotic sucrose solutions led to significant increases in GK1max (e.g., 28 ± 2% with 1.8T), and significant negative shifts in V0.5 (e.g., −6.7 ± 0.6 mV with 1.8T). Data from myocytes investigated under hyperosmotic conditions that do not induce shrinkage indicate that GK1max and V0.5 were insensitive to hyperosmotic stress per se but sensitive to elevation of intracellular K+. We conclude that the effects of hyperosmotic sucrose solutions on IK1 are related to shrinkage-induced concentrating of intracellular K+.