Inhibition of pacemaker current by the bradycardic agent ZD 7288 is lost use-dependently in sheep cardiac Purkinje fibres

Abstract

The inhibition of the pacemaker current (if) in sheep cardiac Purkinje fibers by ZD 7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium++ + chloride] is lost use-dependently. This disinhibition of if was investigated by using the two-microelectrode voltage-clamp technique. The pulse protocol consisted of a rest period (holding potential of about -50 mV, 1-10 micromol/l ZD 7288) followed by a train of test pulses (potential negative to -100 mV, stimulation frequency 0.05 Hz). At the beginning of the first test pulse there was an immediate reduction of if but inhibition was lost during continued stimulation. Activation of if is sigmoidal and the early delay in current activation was prolonged from 33 ms (no ZD 7288) to 424 ms (10 micromol/l ZD 7288). Therefore hardly any disinhibition occurred during short test pulses (0.5s). During longer test pulses (5 s, -120 mV, 10 micromol/l) disinhibition developed with a time constant of about 2 s. The inhibition of if by ZD 7288 was lost voltage-dependently. With 10 micro mol/l ZD 7288 the half-maximal disinhibition occurred at -92 mV and the slope factor of the disinhibition/voltage curve (Boltzmann relation) was 4.8 mV. The voltage-dependent disinhibition could be abolished largely by extracellular application of protease (0.5 mg/ml, 7 min). After prior disinhibition, reinhibition at the holding potential (about -50 mV) followed a bi-exponential time course indicating that inhibition may be produced by a fast (tau=0.7 min) and a slow component (tau=20-30 min). Increasing ZD 7288 concentration from 1 to 10 mu mol/l accelerated reinhibition, mainly by an increase of the amplitude (A) of the fast component. The ratio Afast/Aslow was 0.399 at 1 micromol/l and 2.65 at 10 micromol/l ZD 7288. The reinhibition of if was unchanged by shifting the holding potential from -50 mV to -20 mV. Trials to wash out the effects of 10 micromol/l ZD 7288 gave two results. The inhibition of if was slightly reversed after a wash-out of 1.5 h with drug-free solution. A second effect of the drug, the fast reinhibition, could be completely removed by wash-out. In summary if is inhibited by ZD 7288 at membrane potentials at which the virtual if gate is closed. Disinhibition occurs during long-lasting hyperpolarization but will hardly be operative in unclamped fibres under physiological conditions.

Blocker State Dependence and Trapping in Hyperpolarization-Activated Cation Channels

“…The most remarkable property of ZD7288 blockade of mHCN1 channels is its steep voltage dependence, corresponding to approximately four elementary charges moving through the entire transmembrane field. It has been previously demonstrated that the blocking effect of ZD7288 is relieved by hyperpolarization (BoSmith et al, 1993;Harris and Constanti, 1995;Gasparini and Di-Francesco, 1997) and that the voltage dependence of disinhibition was 4.8 mV/e-fold change (Berger et al, 1995). This slope value is equivalent to a z␦ of 5.3 (similar to z␦ Ϸ 4.2 measured here for mHCN1).…”

Effects of 17β-estradiol on action potentials and ionic currents in male rat ventricular myocytes

“…For GH 3 pituitary tumor cells it is known that membrane-bound estrogen receptors are removed by trypsin (Watson et al 1995). Furthermore, we have shown that drug-induced effects in sheep Purkinje fibres are manipulated severely by protease treatment (Berger et al 1995b).…”

The effects of over-expression of the FK506-binding protein FKBP12.6 on K+ currents in adult rabbit ventricular myocytes

“…At very positive potentials (>+40 mV), the rate of inactivation after FKBP12.6 expression was enhanced compared to the control values. I to is active during phase 1 of the cardiac action potential, and drug-induced reduction of I to is known to prolong action potential duration [2]. A more rapid inactivation (as observed after FKBP12.6 over-expression) might be expected to prolong the action potential duration, but this link has not been reported.…”