N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system.

] Institute of Physiology CAS Videnska 1083 142 20 Prague 4 Czech Republic [2] School of Pharmacy and Biomedical Sciences University of Central Lancashire Preston PR1 2HE UK [3] Institute of Medical Physiology School of Medicine University of Belgrade Visegradska 26 2 11000 Beograd Srbija

Charles University Prague Faculty of Science Albertov 6 128 43 Prague 2 Czech Republic

Institute of Chemical Technology Prague Technicka 5 166 28 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry CAS Flemingovo nam 2 166 10 Prague 2 Czech Republic

Institute of Physiology CAS Videnska 1083 142 20 Prague 4 Czech Republic

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