Differential Effects on Current Kinetics by Point Mutations in the lurcher Motif of NR1/NR2A Receptors

Abstract

The extracellular proton site has emerged as the key site in regulating N-methyl-D-aspartate (NMDA) receptor function. However, the molecular determinants for the proton-sensitive gating of NMDA receptors are still not clearly delineated. The highly conserved lurcher motif plays an important role in determining the proton sensitivity of NMDA receptors. Mutations of several residues in the lurcher motif of either NR1 or NR2A significantly reduce proton sensitivity of recombinant NR1/NR2A receptors. It remains uncertain how these residues play a role in proton inhibition of NMDA receptors. Mutations of these residues could directly reduce the proton affinity. Alternatively, they could alter the proton IC 50 indirectly by increasing channel open probability. In the present study, we recorded the macroscopic NMDA currents in HEK 293 cells with a piezo-based rapid solution exchange system. We show that zinc slows the deactivation of NR1a(A653T)/NR2A receptors and NR1a/NR2A(A651T) receptors. However, NR1a(T648C)/NR2A, NR1a/NR2A(T646C), NR1a(A649C)/ NR2A, NR1a/NR2A(A647C), and NR1a(A653T)/NR2A exhibit significantly slower rise time and deactivation time constants under nominally zinc-free conditions. Our data suggest that the channel open probability for these mutant receptors may be significantly increased. The reduction in proton sensitivity by these mutations could be accounted for, at least partially, by the increased channel open probability. In contrast, NR1a/NR2A(A651T) exhibits normal macroscopic currents, suggesting that the reduction of proton sensitivity by this mutation cannot be attributed to any significant change of open probability. Further experiments are needed to determine the exact role of this residue in proton-sensitive gating of NMDA receptors.

The NR1 M3 Domain Mediates Allosteric Coupling in theN-Methyl-D-aspartate Receptor

“…Taverna et al, 2000). Consistent with our own results, the lurcher mutation increases glutamate potency and alters activation kinetics when introduced into NR1, but not NR2 (Kohda et al, 2000;Hu and Zheng, 2005), and it exhibits dominance in heteromeric GluR2 and GluR6 receptors (Schwarz et al, 2001). Furthermore, current potentiation and/or constitutive activity have been observed after modification of several substituted cysteines within the SYTANLAAF region (Beck et al, 1999;Jones et al, 2002;Sobolevsky et al, 2002bSobolevsky et al, , 2003, and the decreased sensitivity to competitive antagonists, pore blockers, and allosteric modulators displayed by our A7 mutants bears a striking resemblance to MTSEA-modified A7C receptors .…”

“…Recent studies have also shown that M3 is the only transmembrane domain contributing to the deepest portion of the pore, supporting a prominent role in gating (Sobolevsky et al, 2007). In vivo, the lurcher mouse phenotype, a neurodegenerative condition caused by excitotoxic cell death, has been attributed to a constitutive mutation within the GluR␦2 SYTANLAAF region (A8T) (Zuo et al, 1997), and introduction of the lurcher mutation into other glutamate receptors results in numerous gain-of-function phenotypes, including increased agonist potency, slower deactivation rates, and inhibition of desensitization (Kohda et al, 2000;Klein and Howe, 2004;Hu and Zheng, 2005;Schmid et al, 2007).…”

Probing the activation sequence of NMDA receptors with lurcher mutations

“…Similarly, when introduced in AMPA- or kainate-type iGluRs, the lurcher substitution (A8T) produces substantial constitutive activity and slows desensitization and deactivation kinetics of agonist-activated whole-cell currents (Schwarz et al, 2001). In contrast, in NMDA receptors, lurcher mutations cause only mild kinetic changes whether present on GluN1 (N1) or GluN2 (N2) subunits (Zuo et al, 1997; Kohda et al, 2000; Hu and Zheng, 2005a,b). Instead, A7Y mutations (lurcher-like) and other bulky side chains introduced at A7 sites render receptors constitutively open (Jones et al, 2002; Yuan et al, 2005; Blanke and VanDongen, 2008).…”

Molecular Determinants of Glycine-Independent Desensitization of NR1/NR2A Receptors

“…It also is interesting that five of these six mutants share the following common characteristics: 1) slower deactivation time constants (Hu and Zheng, 2004), 2) reduced proton sensitivity (Low et al, 2003), and 3) reduced glycine-independent desensitization. Although it is possible that these common characteristics could be just coincidental, we propose that it is an indication for a close link between these processes at a molecular level.…”

“…at ASPET Journals on May 9, 2018 jpet.aspetjournals.org Downloaded from tants in the lurcher motif of NR1 and NR2A and identified two residues that are critical for proton-sensitive gating of NMDA receptors [i.e., NR1a(T648C) and NR1a(A649C) and corresponding residues in NR2A; Low et al, 2003;Hu and Zheng, 2004]. If proton-sensitive gating and desensitization are closely associated, these two residues should also play a critical role in glycine-independent desensitization.…”