Bernd Schubert

Use- and voltage-dependent depression by ethmozine (moricizine) of the rapid inward sodium current in single rat ventricular muscle cells

Summary: Effects of ethmozine (moricizine) on the rapid inward sodium current (INa) were studied in freshly isolated single cells of rat ventricular myocardium. INa was measured by means of a patch clamp method for observing integral ionic currents. Ethmozine was applied extracellularly to a small cell membrane patch at concentrations of 10, 20, and 40 μM. At a stimulation frequency of 0.1 Hz the drug decreased the peak INa without producing a shift of the current–voltage curve, but shifted the V0.5 of the steady-state inactivation curve by −6 mV. At frequencies of 2–5 Hz the ethmozine-induced block exhibited a prominent use dependence, with trains of depolarizing clamp pulses 5–50 ms in duration eliciting maximal INa from holding potentials at which the steady-state inactivation variable h∞ was close to 1. The use-dependent inhibition of INa became more pronounced with an increase in both stimulation rate and pulse duration. In contrast to what has been observed in the node of Ranvier of the frog, the present results indicate that ethmozine binds to both inactivated and open Na+ channels, but that the contribution of the open channel block to the overall block at depolarizing clamp step durations of several hundred milliseconds is small in comparison with the contribution of the block of inactivated channels.

Cell-attached patch clamp measurement of macroscopic rapid inward sodium current in cultured heart cell reaggregates

A megaohm seal, cell-attached patch clamp technique utilizing plastic suction pipettes for measuring macroscopic rapid inward sodium current (INa) was applied to cell membrane patches 30 to 100 micron2 in area at the periphery of reaggregates of myocardial cells from newborn rats cultured for 3 to 7 days. The reaggregates were composed of about 20 to several thousand of electrically well-coupled cells, the latter forming spheroidal reaggregates with a diameter of maximally 300 microns. This system was shown to guarantee satisfactory voltage-clamp control during ionic current measurements. The time and voltage dependence of the INa recorded during periods of up to 90 min was similar to that observed in single cardiac cell preparations from adult rats. INa inactivation was described by two time constants (tau h1, and tau h2). For maximal INa tau h1 and tau h2 had values of 1.5 +/- 0.25 ms and 8.7 +/- 3.9 ms (n = 4), respectively. The time course for recovery of INa from inactivation at the reaggregate resting potential exhibited also two time constants (tau re1 = 9.8 +/- 3 ms, tau re2 = 193 +/- 50 ms, n = 5). Estimated current density was about 10 pA/micron2. The concentration of tetrodotoxin needed to reduce maximal INa by 75% was 85 times lower in the reaggregates than it was in freshly isolated heart muscle cells from adult rats. The present system offers a combination of features that should make it well-suited for the study of both short- and long-term effects on the sodium channels in neonatal heart cells: Easy handling of the object, great electric stability, high time and amplitude resolution during ionic current measurements, and viability for at least one week.

A patch clamp study of the action of ethacyzine on the rapid inward sodium current in single rat heart muscle cells

The effect of ethacyzine (a diethylamine analogue of ethmozine) on the rapid inward sodium current (INa) was studied in single rat ventricular muscle cells by a patch clamp technique. Extracellular application of 3 microM ethacyzine depressed the INa in a use-dependent fashion. Rest recovery from the use-dependent block by ethacyzine followed an exponential time course with a time constant of about 215 +/- 40 s (n = 4, mean +/- S.E.).