Abstract

Inhibitors of AMPA-type glutamate ion channels are useful as biochemical probes for structure-function studies and as drug candidates for a number of neurological disorders and diseases. Here, we describe the identification of an RNA inhibitor or aptamer by an in vitro evolution approach and a characterization of its mechanism of inhibition on the sites of interaction by equilibrium binding and on the receptor channel opening rate by a laser-pulse photolysis technique. Our results show that the aptamer is a noncompetitive inhibitor that selectively inhibits the GluA2Q(flip) AMPA receptor subunit without any effect on other AMPA receptor subunits or kainate or NMDA receptors. On the GluA2 subunit, this aptamer preferentially inhibits the flip variant. Furthermore, the aptamer preferentially inhibits the closed-channel state of GluA2Q(flip) with a K(I) = 1.5 μM or by ∼15-fold over the open-channel state. The potency and selectivity of this aptamer rival those of small molecule inhibitors. Together, these properties make this aptamer a promising candidate for the development of water-soluble, highly potent, and GluA2 subunit-selective drugs.

Highlights

  • Which the drug molecule binds is linked to the pathogenesis of a disease

  • We found that AF44/AF42 did not affect the rest of amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, i.e. GluA1, -3, and -4

  • We have described a de novo design, isolation, and mechanistic characterization of a novel aptamer pair (i.e. AF44/AF42) that acts as a potent, noncompetitive inhibitor

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Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Receptor Expression—The cDNAs encoding various subunits of glutamate ion channels were used for transient receptor expression in HEK-293S cells [35]. The cell culture, transfection, and preparation of membrane lipids containing functional, intact GluA2Qflip receptors for aptamer selection were carried out according to the protocols reported previously [35]. Aptamer Selection—The preparation of the RNA library with ϳ1015 random sequences and the running of in vitro selection of aptamers against GluA2Qflip in membrane lipids were described previously [35] (see supplemental Fig. S1). To suppress enrichment of nonspecific RNAs bound to any unwanted “targets,” such as lipids, we ran three negative selections at rounds 4, 8, and 13 in which plain HEK-293 cell membrane fragments lacking only GluA2Qflip receptors were used to absorb nonspecific RNAs. For identifying consensus sequences, the DNA pools from rounds 12 and 14 (i.e. 14 was the final round) were cloned separately into the pGEM-T easy vector (Invitrogen) and sequenced. Assuming a one-site binding model, the Kd of an aptamer was estimated by Equation 1 [36], Y ϭ

Kd ϩ
KI KI ϩ I ϩ kcl
RESULTS
DISCUSSION

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