Quantitative analysis of tetrapentylammonium-induced blockade of open N-methyl-D-aspartate channels.

The blockade of open N-methyl-d-aspartate (NMDA) channels by tetrapentylammonium (TPentA) in acutely isolated rat hippocampal neurons was studied using whole-cell patch-clamp techniques. TPentA prevented the closure of the NMDA channel following what is known as the foot-in-the-door mechanism. Hooked tail currents appearing after termination of the agonist (aspartate) and TPentA coapplication were analyzed quantitatively according to the corresponding sequential kinetic model. Studies of the hooked tail current amplitude and the degree of the stationary current inhibition dependence on the blocker concentration led to a new method for estimation of fast foot-in-the-door blocker binding/unbinding rate constants. The application of this method to the NMDA channel blockade by TPentA allowed finding the values of its binding (1.48 microM(-1)s(-1)) and unbinding (14 s(-1)) rate constants. An analysis of the dependence of the electric charge carried during the hooked tail current on the blocker concentration led to a new method for estimation of the maximum NMDA channel open probability, P(0). The value of P(0) found in experiments with TPentA was 0.04.