Voltage-dependent interaction of open-channel blocking molecules with gating of NMDA receptors in rat cortical neurons

1. The mechanisms by which four adamantane derivatives (IEM-1857, -1592, -1460 and -1754) block the open NMDA-activated channel were studied at membrane voltages (V-m) from -170 to +30 mV. The rate constants of channel block (k(+)) and of channel unblock (k(-)) were measured from the fully resolvable flicker of single-channel currents induced by each compound. 2. The k(+) of each compound exhibited a similar exponential dependence on voltage over the V-m range studied. 3. The k(-) of IEM-1857 and IEM-1592 over the V-m range studied, and of IEM-1754 and IEM-1460 from -30 to -90 mV, exhibited similar exponential dependencies on voltage. However, the k(-) of IEM-1754 and IEM-1460 at V-m values more hyperpolarized than -90 mV mere much more steeply voltage dependent, suggesting that at these V-m values the two drugs can occupy a deeper binding site. 4. Each of the drugs induced a concentration-dependent prolongation of the mean burst length at -90 mV, suggesting that while blocking they can interfere with channel closure. 5. The prolongation of mean burst length induced by the largest drug (IEM-1857) increased with hyperpolarization. The increase was consistent at each V-m with the predictions of the sequential scheme of block, suggesting that channel closure is prevented when IEM-1857 is bound. The prolongation of burst length induced by the smallest drug (IEM-1754) was less than predicted by the sequential scheme and the deviation increased with hyperpolarization. 6. The IEM-1857 concentration-dependence of number of blockages per unit open time had a slope equal to k(+) at -150 mV. The IEM-1754 concentration-dependence of number of blockages per unit open time revealed a slope about two times less than k(+) for this compound at -150 mV. 7. The mean patch current was not significantly altered by 3 mu M IEM-1857 at V-m values from -90 to -150 mV, as expected of a drug that prevents channel closure when blocking. Mean patch current significantly decreased with hyperpolarization beyond -90 mV in the presence of 1 mu M IEM-1754. 8. The data suggest that there are two blocking sites at different depths within the NMDA-activated channel. Channel closure is prevented when any of the IEM drugs occupy the shallow blocking site. Channel closure is permitted during occupation of a deeper blocking site that can be reached only by the smaller IEM drugs at hyperpolarized voltages.