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J. Biol. Chem., Vol. 282, Issue 3, 1863-1872, January 19, 2007

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Disruption of Interdomain Interactions in the Glutamate Binding Pocket Affects Differentially Agonist Affinity and Efficacy of N-methyl-D-aspartate Receptor Activation^*

Wolfgang Maier¹, Rudolf Schemm², Christof Grewer^¶, and Bodo Laube³

From the Max-Planck-Institute for Brain Research, Frankfurt 60528, Germany, the Max-Planck-Institute for Biophysics, Frankfurt 60438, Germany, and the ^¶University of Miami School of Medicine, Department of Physiology and Biophysics, Miami, Florida 33136

In ionotropic glutamate receptors, agonist binding occurs in a conserved clam shell-like domain composed of the two lobes D1 and D2. Docking of glutamate into the binding cleft promotes rotation in the hinge region of the two lobes, resulting in closure of the binding pocket, which is thought to represent a prerequisite for channel gating. Here, we disrupted D1D2 interlobe interactions in the NR2A subunit of N-methyl-D-aspartate (NMDA) receptors through systematic mutation of individual residues and studied the influence on the activation kinetics of currents from NR1/NR2 NMDA receptors heterologously expressed in HEK cells. We show that the mutations affect differentially glutamate binding and channel gating, depending on their location within the binding domain, mainly by altering k_off and k_cl, respectively. Whereas impaired stability of glutamate in its binding site is the only effect of mutations on one side of the ligand binding pocket, close to the hinge region, alterations in gating are the predominant consequence of mutations on the opposite side, at the entrance of the binding pocket. A mutation increasing D1D2 interaction at the entrance of the pocket resulted in an NMDA receptor with an increased open probability as demonstrated by single channel and whole cell kinetic analysis. Thus, the results indicate that agonist-induced binding domain closure is itself a complex process, certain aspects of which are coupled either to binding or to gating. Specifically, we propose that late steps of domain closure, in kinetic terms, represent part of channel gating.

Received for publication, August 24, 2006 , and in revised form, November 9, 2006.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant LA 1086/4-1 (to B. L.) and by the Hertie-Stiftung (to B. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

¹ Present address: Friedrich Miescher Institute for Biomedical Research, Basel 4058, Switzerland.

² Present address: MPI for Biophysical Chemistry, Göttingen 37077, Germany.

³ To whom correspondence should be addressed: AG Cellular Neurophysiology, Dept. of Biology, Technical University Darmstadt, Schnittspahnstr. 3, 64287 Darmstadt, Germany. Tel.: 49-69-96769-295; Fax: 49-69-96769-441; E-mail: laube{at}bio.tu-darmstadt.de.

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