C O M M U N I C A T I O N S
Acknowledgment. We thank Dr. Roger Nicoll, Hillel Adesnik,
and Dr. Ming An for helpful discussions, Anang Shelat for
calculating the polar surface area, and Alex Ward for preparing
oocytes. The GluR1 clone was a gift from Stephen F. Heinemann.
This work was supported by a generous grant from the Sandler
Foundation.
Figure 2. Glutamate-evoked current traces from a single oocyte following
the indicated (A-F) treatments. Currents are irreversibly knocked down
following irradiation with UV light (100 W, 30 s) in the presence of ANQX
(F). Control experiments (B-E) show that peak currents are unaffected
following treatment with UV light alone, UV light in the presence of
glutamate or DNQX, or ANQX in the absence of UV light (E).
Supporting Information Available: Molecular docking details,
GluR1 DNA and mRNA preparation protocols, electrophysiological
recording and photolysis conditions, bar graph showing the mean
currents ((SEM) from seven oocytes treated as described in Figure 2,
and bar graph showing mean currents ((SEM) from oocytes expressing
kainate and N-methyl-D-aspartate before and after treatment with
ANQX + UV light. This material is available free of charge via the
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Figure 3. Time course for photochemically knocking out AMPAR currents
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of the nitro group to the amine, followed by diazotization and
displacement of the arenediazonium ion with azide, afforded
6-azido-1,4-dihydroquinoxaline-2,3-dione. Finally, nitration with
fuming nitric acid provided ANQX in 44% overall yield.
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DNQX (0.5 µM), demonstrating that ANQX is a potent AMPAR
antagonist. As shown in Figure 2, glutamate-evoked currents were
irreVersibly inhibited upon photolysis in the presence of ANQX.
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(10) The apparent cross-linking efficiency of ANQX in these experiments was
likely limited by the size (∼1 mm diameter), shape (spherical), and opacity
of the oocytes. In preliminary experiments on cultured neurons expressing
endogenous AMPARs, irradiation in the presence of ANQX completely
inactivated AMPAR currents after only 2 s of photolysis and with
significantly less UV light (data not shown).
In summary, we have designed, synthesized, and evaluated a
photoreactive AMPAR antagonist that provides a means of directly
characterizing the role of AMPAR trafficking in synaptic plasticity.
The membrane impermeability of ANQX will allow for exceptional
spatial resolution, targeting only surface-exposed receptors, and the
photoreactivity of the azido group will allow for unparalleled
temporal resolution, knocking out receptors on the minute time
scale.11
(11) The measured octanol/buffer (pH 7.5) partition coefficient (-0.72) suggests
that ANQX is a fairly hydrophilic compound. In addition, the calculated
polar surface area (171.33 Å2) for ANQX predicts that ANQX is
membrane impermeable. Finally, the photochemical knock-out experiments
require only brief (<1 min) exposure to ANQXsa period too short to
permit penetration of this compound through the plasma membrane.
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