Swanger SA, Chen W, Wells G, Burger PB, Tankovic A,
Bhattacharya S, Strong KL, Hu C, Kusumoto H, Zhang J, Adams DR, Millichap JJ, Petrovski
S, Traynelis SF, Yuan H. Mechanistic Insight into NMDA Receptor Dysregulation
by Rare Variants in the GluN2A and GluN2B Agonist Binding Domains. Am J Hum
Genet. 2016 Dec 1;99(6):1261-1280.
Epilepsy and intellectual disability are associated with
rare variants in the GluN2A and GluN2B (encoded by GRIN2A and GRIN2B) subunits
of the N-methyl-D-aspartate receptor (NMDAR), a ligand-gated ion channel with
essential roles in brain development and function. By assessing genetic
variation across GluN2 domains, we determined that the agonist binding domain,
transmembrane domain, and the linker regions between these domains were
particularly intolerant to functional variation. Notably, the agonist binding
domain of GluN2B exhibited significantly more variation intolerance than that
of GluN2A. To understand the ramifications of missense variation in the agonist
binding domain, we investigated the mechanisms by which 25 rare variants in the
GluN2A and GluN2B agonist binding domains dysregulated NMDAR activity. When
introduced into recombinant human NMDARs, these rare variants identified in
individuals with neurologic disease had complex, and sometimes opposing,
consequences on agonist binding, channel gating, receptor biogenesis, and
forward trafficking. Our approach combined quantitative assessments of these
effects to estimate the overall impact on synaptic and non-synaptic NMDAR
function. Interestingly, similar neurologic diseases were associated with both
gain- and loss-of-function variants in the same gene. Most rare variants in
GluN2A were associated with epilepsy, whereas GluN2B variants were associated
with intellectual disability with or without seizures. Finally, discerning the
mechanisms underlying NMDAR dysregulation by these rare variants allowed
investigations of pharmacologic strategies to correct NMDAR function.
_________________________________________________________________________
In a recent article, Swanger et al. approached these issues
by focusing on genetic variants found in GRIN2A and GRIN2B, both of which are
members of the GRIN family of ligand-gated ion channels that code for the NMDA
receptor subunits Glu-N2A and GluN2B, respectively. These variants were
missense mutations, meaning that a single nucleotide was changed that switched
one amino acid in the protein, and were considered to be pathogenic. The
majority of the variants were associated with epilepsy, especially the GRIN2A
variants, but some of the GRIN2B variants were associated with intellectual
disability.
NMDA receptors are well-characterized proteins. Extensive
structure-function experiments, in combination with crystal structures of the
ligand-binding domains and well-defined electrophysiological assays, have led
to a good understanding of how different parts of these proteins bind
glutamate, flux ions, and assemble into functional tetramers. Using this
available breadth of knowledge, the authors first looked at which functional
domains of the proteins were most vulnerable to missense mutations. Although
missense mutations can be disease causing, another insight from human genetics
is that they, and other genetic variants, are also common in healthy people.
One way to determine whether a particular variant is pathogenic is to compare
the frequency of the variant in healthy people to those with neurologic disease.
Amino acids or regions of a protein that show higher variability in control
populations are less likely to harbor disease-causing variants, whereas those
with lower variability are more likely. Comparing the distribution of missense
variants in GRIN2A and GRIN2B in the healthy control group versus that in the
neurologic disease group showed that the regions of the proteins that bind
glutamate, the transmembrane domains, and the links between the two had very
few missense mutations in the control group but were overrepresented in the
disease group, suggesting that changes to amino acids in these regions are
particularly likely to cause functional, pathogenic changes.
Next, the authors cloned human GRIN2A and GRIN2B genes
harboring 25 disease-associated variants within these vulnerable regions and
expressed them in oocytes or HEK cells to assess their functional impact. They
put them through a battery of tests designed to measure fundamental aspects of
ligand-gated ion channel biology, such as the EC50 for glutamate and glycine,
the time course of the measured current, the probability of channel opening,
and the number of receptors that make it to the cell surface.
Many of the variants were in the glutamate-binding region of
the protein, so the authors first measured whether the potency of glutamate was
altered. Of the 22 variants that had a measurable response to glutamate, 20
caused a significant shift in the glutamate EC50; 11 variants had a higher EC50
(up to 1000-fold), and 9 had a lower EC50 (up to 4.5-fold). In agreement with
the changes in EC50 values, the rate of decay of the receptor currents in
response to brief applications of glutamate (deactivation) were also affected,
as these two measurements are highly correlated. Surface expression was also
reduced by all the variants that reduced glutamate potency, and even by some of
the variants that increased potency.
Because the effects of the variants on individual receptor
properties were variable and often opposing, the authors next attempted to
quantify the magnitude of the overall impact on receptor function by estimating
the degree of the increase or decrease in charge transfer through the
receptors. According to this estimation, about a quarter of the variants
enhanced receptor function (up to 3.7-fold), whereas the majority decreased
receptor function (down to 5.1 × 10−5-fold). Gain-of-function and
loss-of-function variants were found in both GRIN2A and GRIN2B, further
demonstrating that the variants resist simple classification schemes. Despite
this complexity, the authors did show that the gain-of-function variants were
sensitive to the NMDA receptor antagonist memantine, whereas the
loss-of-function variants showed enhanced charge transfer by three different
positive allosteric modulators, suggesting that these or similar drugs may be
beneficial to patients with GRIN variants.
By performing a detailed and thorough analysis of a
relatively large number of variants in two NMDA receptor-encoding genes, this
work advances our knowledge of the nature and functional consequences of GRIN
variants and gives a glimpse of what attempts at personalized therapies based
on identified genetic variants might look like. Based on this work, one could
imagine that an epilepsy patient with an identified NMDA receptor mutation
could have this mutant receptor cloned and expressed, parameters measured, and
then screened against a library of compounds to identify those that normalize
the overall impact on receptor function and treat the neurologic symptoms.
However, for many other epilepsy-causing mutations in less well-characterized
genes, this sort of personalized therapy procedure may not yet be feasible. At
the same time, this work also serves as a warning that the task of finding
common mechanisms may be daunting. Even though these variants were associated
with similar neurologic symptoms and caused single amino-acid changes in the
same domain of a protein, their functional effects were very different. This
suggests that higher-level cellular, circuit, and developmental mechanisms,
such as differential expression in excitatory versus inhibitory neurons, are
crucial to dictating the disease phenotype, and that each variant may lead the
brain down a unique path to epilepsy. To better understand these complex
mechanisms, we will need additional animal models and complementary model
systems, potentially one for each variant, to effectively design precision
therapies and achieve a more complete understanding of the underlying
mechanisms.
http://epilepsycurrents.org/doi/full/10.5698/1535-7597.17.6.381
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