Direct inhibition of the pacemaker (If) current in rabbit sinoatrial node cells by genistein

Abstract

Genistein is a tyrosine kinase inhibitor which interferes with the activity of several ionic channels either by altering modulatory phosphorylating processes or by direct binding. In whole-cell conditions, genistein induces a partial inhibition of the pacemaker (I(f)) current recorded in cardiac sinoatrial and ventricular myocytes. We investigated the mechanism of action of genistein (50 microM) on the I(f) current in whole-cell, cell-attached, and inside-out configurations, and the measured fractional inhibitions were similar: 26.6, 27.2, and 33.6%, respectively. When ATP was removed from the whole-cell pipette solution no differences were revealed in the effect of the drug when compared to metabolically active cells. Genistein fully maintained its blocking ability even when herbimycin, a tyrosine kinase inhibitor, was added to the whole-cell ATP-free pipette solution. Genistein-induced block was independent of the gating state of the channel and did not display voltage or current dependence; this independence distinguishes genistein from all other f-channel blockers. When inside-out experiments were performed to test for a direct interaction with the channel, genistein, superfused on the intracellular side of the membrane, decreased the maximal I(f) conductance, and slightly shifted the current-activation curve to the left. Furthermore, the effect of genistein was independent of cAMP modulation. We conclude that, in addition to its tyrosine kinase-inhibitory properties, genistein also blocks I(f) by directly interacting with the channel, and thus cannot be considered a valuable pharmacological tool to investigate phosphorylation-dependent modulatory pathways of the I(f) current and of cardiac rhythm.

Figure 1

Genistein reduces the whole-cell I_f current. (a) I_f current was elicited every 6 s by a 2-s hyperpolarizing step to −75 mV from a holding potential of −35 mV. Sample traces recorded in control solution (1), during superfusion of genistein (50 μM, 2) and after washout (3) are shown. (b) Time course of I_f amplitude at −75 mV; the horizontal bar indicates the time of genistein superfusion (gen); the calculated inhibition was 24.2%. Continuous line represents the single exponential fitting of the onset (τ=17.3 s) and removal (τ=27.4 s) of the block.

Figure 2

Action of genistein on whole-cell I_f in the absence of intracellular ATP and PTK activity. (a) Time course of steady-state I_f amplitudes at −75 mV (holding potential, −35 mV); horizontal bars indicate duration of genistein superfusion (gen); genistein was applied twice at an interval of 10 min. Drug-induced I_f reductions were 26.9 and 28.8% during the first and second exposures, respectively. Sample I_f traces in control conditions (1,3) and during superfusion of genistein (50 μM; 2,4) are shown in the insets. (b) Current traces recorded at −95 mV in control condition (cont) was started (gen) 9 min after superfusion of genistein (50 μM) and after washout (ret). The pipette solution contained regular intracellular solution plus herbimycin (35 μM) and no ATP. (c) Graph bar showing the genistein-induced mean (±s.e.m.) I_f reductions obtained with a regular intracellular pipette solution containing ATP 2 mM, no ATP, and no ATP plus herbimycin 35 μM; blocks were 26.6±2.5% (n=11), 26.6±2.8% (n=5), and 22.9±3.2% (n=5), for the three conditions, respectively. All recordings started at least 6 min after establishing the whole-cell configuration. Blocks were not significantly different.

Figure 3

Genistein-induced block of closed f-channels. A train of activating/deactivating steps (−105 mV*3 s/+5 mV*0.75 s; 1/6 Hz) was delivered to the cell under study and stable I_f traces were recorded (left). Current stimulation was then stopped for 72 s by clamping the cell at the holding potential (−35 mV). Genistein (50 μM) superfusion started at the beginning of this silent period, and was maintained throughout the whole time of the experiment. Train stimulation was then resumed (right). When traces recorded just after the interruption (b) and at the end of the experiment (c) were compared to the trace recorded just before the interruption (a), the calculated ratios b/a and c/a were 0.63 and 0.64, respectively, while the c/b ratio yielded a value of 1.01.

Figure 4

Genistein blocks I_f in a voltage independent way. (a) The I_f current was elicited by hyperpolarizing pulses to the voltages indicated and genistein (50 μM) was applied only after steady-state activation was reached. Evaluation of steady-state blocks yielded values of 28.5, 29.4, and 27.8% for currents recorded at −85, −105, and −125 mV, respectively. (b) Mean±s.e.m.% block obtained at different voltages (−75, −85, −105, and −125 mV) are plotted against voltage; statistical analysis did not reveal any voltage dependence of the block.

Figure 5

Action of genistein on I_f in cell-attached and inside-out macropatches. (a) I_f recordings obtained in cell-attached conditions during hyperpolarizations to −105 mV in control solution (1), superfusion of genistein (50 μM; 2), and after washout (3); the steady-state block was 24.3%. (b) I_f traces recorded in inside-out conditions during hyperpolarizations to −125 mV; the recordings were taken at various times in control conditions (1, 4), during superfusions of cAMP (10 μM; 2) and genistein (50 μM; 3, 5); traces 4 and 5 were recorded 300 s after traces 1, 2, and 3, and correspond to the third exposure to the drug (second exposure not shown). The holding potential was set for all recordings to −35 mV and activating steps delivered at 0.1 Hz.

Figure 6

Action of genistein on inside-out I_f in the presence of cAMP. (a, b) I_f currents recorded during hyperpolarizations to −125 mV applied every 10 s from a holding potential of −35 mV; the traces correspond to various times in control conditions and during superfusion of genistein (50 μM) either alone or in combination with cAMP (0.2 μM), as shown in (c). (c) Time course of I_f amplitude at −125 mV; horizontal bars indicate superfusion of the different solutions.

Figure 7

Action of genistein on current– and conductance–voltage relations. (a, top) Hyperpolarizing ramps from −35 to −145 mV were applied at a rate of −110 mV min⁻¹ to an inside-out membrane patch. Note that time runs backward. (a, bottom) Steady-state I–V relations in control solution and during superfusion of genistein (50 μM) as indicated. (b) g_f(V) curves obtained as detailed in Methods in control and in the presence of genistein.

Figure 8

Concentration–response curve of the I_f current inhibition by genistein. I_f was recorded in inside-out conditions during hyperpolarizing steps to −125 mV applied every 10 s (holding potential −35 mV) in control solution and during superfusion of increasing concentrations of genistein (10, 30, and 100 μM). (b) Time course of I_f amplitude at −125 mV; horizontal bars indicate the period of drug superfusion at each concentration used. (c) Concentration–response curve. Fitting of data points with the Hill equation y=y_max*(1/(1+(IC₅₀/x)n_H)) yielded a maximal fractional inhibition (y_max) of 0.90, a half-block concentration (IC₅₀) of 60.9 μM and a Hill coefficient (n_H) of 1.2. The numbers of patches tested at the various concentrations are indicated near data points.