Probing of NMDA channels with fast blockers

Using whole-cell patch-clamp techniques, we studied the interaction of open NMDA channels with tetraalkylammonium compounds: tetraethylammonium (TEA), tetrapropylammonium (TPA), tetrabutylammonium (TBA), and tetrapentylammoni um (TPentA). Analysis of the blocking kinetics, concentration, and agonist dependencies using a set of kinetic models allowed us to create the criteri a distinguishing the effects of these blockers on the channel closure, dese nsitization, and agonist dissociation. Thus, it was found that TPentA prohi bited, TBA partly prevented, and TPA and TEA did not prevent either the cha nnel closure or the agonist dissociation. TPentA and TBA prohibited, TPA sl ightly prevented, and TEA did not affect the channel desensitization. These data along with the voltage dependence of the stationary current inhibitio n led us to hypothesize that: (1) there are activation and desensitization gates in the NMDA channel; (2) these gates are distinct structures located in the external channel vestibule, the desensitization gate being located d eeper than the activation gate. The size of the blocker plays a key role in its interaction with the NMDA channel gating machinery: small blockers (TE A and TPA) bind in the depth of the channel pore and permit the closure of both gates, whereas larger blockers (TBA) allow the closure of the activati on gate but prohibit the closure of the desensitization gate; finally, the largest blockers (TPentA) prohibit the closure of both activation and desen sitization gates. The mean diameter of the NMDA channel pore in the region of the activation gate localization was estimated to be similar to 11 Angst rom.