Chicago - 4:00 PM - 5:00 PM
Stephen F Traynelis, Ph.D.
Professor of Pharmacology and Chemical Biology
Emory University School of Medicine
Abstract:
NMDA receptors mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the central nervous system (CNS). These receptors are ligand-gated ion channels that are tetrameric assemblies of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. There are four different genes encoding GluN2 (GRIN2A, GRIN2B, GRIN2C, GRIN2D), which are each expressed with a unique spatial profile throughout the CNS at different developmental stages. The different GluN2 subunits control the temporal signaling properties of the receptors, which likely allows different subunits to mediate different roles in development and circuit function. NMDA receptors require the binding of both glutamate and glycine in order to open the pore, which is triggered by protein rearrangements secondary to agonist-induced closure of a bi-lobed agonist binding domain that resembles a clamshell. We hypothesize that three key gating elements control opening of the ion-conducting pore following agonist binding. These elements include the 9 highly conserved residues (SYTANLAAF) that comprise the extracellular end of the M3 transmembrane helix, the short two turn helix that is parallel to the plane of the membrane and precedes the M1 transmembrane helix, and the short linker preceding the M4 transmembrane helix (Amin et al., 2018; Chen et al., 2017; Gibb et al., 2018, Yuan et al., 2014). A considerable amount of data implicates these regions in gating, including the observation that regions of the genes encoding these portions of the polypeptide chain are devoid of variation in the human population, yet are a locus for disease-causing mutations in patients with neurological disease (XiangWei et al., 2018). We have studied the functional consequences of these de novo mutations, which have diverse effects on the process of channel gating. Our results included the identification of a mutation in the pre-M1 helix that reduced single channel conductance (Ogden et al., 2017), which is rare for receptor mutations that lie outside of the pore-forming regions. These data suggest that the pre-M1 helix may influence the geometry and characteristics of the open pore. Consistent with this idea, we have identified several allosteric modulators that appear to act at the pre-M1 region and can also reduce single channel conductance, an effect not previously observed for exogenous modulators of NMDA receptor function. For example, the thienopyrimidone EU1622 series of positive allosteric modulators of NMDA receptor function (Perszyk et al., Soc Neuroscience Annual Meeting, 2014) have structural determinants in the pre-M1 region. Members of this class can reduce single channel conductance while still potentiating the maximally effective current. Chord conductance levels of NMDA receptors on cultured cortical neurons changed from 52, 44 pS in vehicle (0.2% DMSO) to 42, 35, and 28 pS in 50 M EU1622-14 (mean from 7 outside out patches recorded at -80 mV, SEM varied between 0.5-1.1 pS). Given that the pre-M1 region might influence the properties of the pore, we explored the mechanism underlying this effect, and discovered that one modulator that reduces conductance can also alter the relative permeability ratio for cations. We found that EU1622-14 reduced the relative permeability of Ca2+ to Na+ for recombinant GluN1/GluN2A and GluN1/GluN2B receptors expressed in HEK cells by more than 2-fold (p<0.05 for both receptors, One-way ANOVA, post-hoc t-test of drug vs vehicle, Bonferroni correction for multiple comparisons, F3,28 = 6.91). This represents the first example to our knowledge of an exogenous drug-like allosteric modulator that can interact with the NMDAR protein complex to alter the relative permeability of ions, which has important implications. The result is perhaps intuitively understandable, as any change in the diameter or electrostatic properties of the pore that alters unitary conductance would be unlikely to do so in a manner that equally affects the permeability of ions with different diameters and distinct hydration shells. Rather, it seems likely that ions with unique atomic radii will have a different relative permeability when the pore diameter or electrostatic environment changes. These findings highlight important biophysical considerations about how the NMDA receptor tetrameric complex controls pore diameter and related properties. In addition, the precedent that Ca2+ permeability can be controlled pharmacologically creates a new potential therapeutic target (i.e. regulation of ionic selectivity) with intriguing possibilities, such as future development of positive allosteric modulators of NMDA receptor function that do not elevate intracellular Ca2+ to potentially pathological levels.
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