One RNA aptamer sequence, two structures: a collaborating pair that inhibits AMPA receptors.

RNA is ideally suited for in vitro evolution experiments, because a single RNA molecule possesses both genotypic (replicable sequence) and phenotypic (selectable shape) properties. Using systematic evolution of ligands by exponential enrichment (SELEX), we found a single 58-nt aptamer sequence that assumes two structures with different functions, both of which are required to inhibit the GluR2 AMPA receptor channel. Yet, the two structures, once formed during transcription, appear to be incapable of interconverting through unfolding and refolding, presumably due to their extraordinary structural stability. Thus, our results suggest more broadly that natural RNA molecules can evolve to acquire alternative structures and associated functions. Such divergence of RNA phenotype may precede gene duplication at the genome level.

Flip and flop: a molecular determinant for AMPA receptor channel opening.

Alternative splicing in the extracellular ligand binding domain of the AMPA receptors generates two variants, i.e., flip and flop. The flop variant of the GluR2 AMPA receptor is known to desensitize faster than the flip counterpart, whereas the GluR1 flip and flop variants exhibit the same rate of desensitization. However, whether the alternative splicing affects the channel opening kinetic properties of these receptors is unknown. Using a laser-pulse photolysis technique, we have characterized the channel opening kinetic mechanism for the flip and flop channels of GluR1 and GluR2, respectively. We find that the flop variant of GluR2 opens the channel, following the binding of glutamate, with the same rate as the flip channel, but closes its channel more rapidly. The difference in the kinetic properties between the two receptor isoforms can be described by a model we proposed previously in which the channel closing rate, a measure of the stability of the open channel state, controls an apparent tendency of the channel to desensitize, most likely, through the closed channel state. Specifically, the flop sequence of GluR2 promotes the channel to close more rapidly and consequently to desensitize with a faster rate than the flip sequence. For GluR1, the alternative splicing does not seem to affect the channel opening kinetics, since the flip and flop variants of GluR1 have the same channel opening rate, and the same channel closing rate. As expected and indeed observed, the flop variant desensitizes with the same rate as the flip variant does. On the basis of these results, we hypothesize that the flip/flop sequence cassette of AMPA receptors, in a sequence-dependent manner, regulates the rate of the channel closing process, in the microsecond time domain, through which it further regulates the channel desensitization in the millisecond time region.

RNA aptamers selected against the GluR2 glutamate receptor channel.

The excessive activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, a subtype of glutamate ion channels, has been implicated in various neurological diseases such as cerebral ischemeia and amyotrophic lateral sclerosis. Inhibitors of AMPA receptors are drug candidates for potential treatment of these diseases. Using the systematic evolution of ligands by exponential enrichment (SELEX), we have selected a group of RNA aptamers against the recombinant GluR2Qflip AMPA receptor transiently expressed in HEK-293 (human embryonic kidney) cells. One of the aptamers, AN58, is shown to competitively inhibit the receptor. The nanomolar affinity of AN58 rivals that of NBQX (6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione), one of the best competitive inhibitors. Like NBQX, AN58 has the highest affinity for GluR2, the selection target, among all AMPA receptor subunits. However, AN58 has a higher selectivity for the GluR4 AMPA receptor subunit and remains potent even at pH = 6.8 (i.e., a clinically relevant acidic pH), as compared with NBQX. Furthermore, this RNA molecule possesses stable physical properties. Therefore, AN58 serves as a unique lead compound for developing water-soluble inhibitors with a nanomolar affinity for GluR2 AMPA receptors.

Receptor occupancy and channel-opening kinetics: a study of GLUR1 L497Y AMPA receptor.

AMPA glutamate ion channels are tetrameric receptors in which activation to form the open channel depends on the binding of possibly multiple glutamate molecules. However, it is unclear whether AMPA receptors bound with a different number of glutamate molecules (i.e. one being the minimal and four being the maximal number of glutamate molecules) open the channels with different kinetic constants. Using a laser pulse photolysis technique that provides microsecond time resolution, we investigated the channel-opening kinetic mechanism of a nondesensitizing AMPA receptor, i.e. GluR1Q(flip) L497Y or a leucine-to-tyrosine substitution mutant, in the entire range of glutamate concentrations to ensure receptor saturation. We found that the minimal number of glutamate molecules required to bind to the receptor and to open the channel is two (or n = 2), and that the entire channel-opening kinetics can be adequately described by just one channel-opening rate constant, k(op), which correlates to n = 2. This result suggests that higher receptor occupancy (n = 3 and 4) does not give rise to different k(op) values or, at least, not appreciably if the k(op) values are different. Furthermore, compared with the wild-type receptor (Li, G., and Niu, L. (2004) J. Biol. Chem. 279, 3990-3997), the channel-opening and channel-closing rate constants of the mutant are 1.5- and 13-fold smaller, respectively. Thus, the major effect of this mutation is to decrease the channel-closing rate constant by stabilizing the open channel conformation.

GluR3 flip and flop: differences in channel opening kinetics.

Ample evidence from earlier studies of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, GluR3 included, suggests that alternative splicing not only enriches AMPA receptor diversity but also, more importantly, creates receptor variants that are functionally different. However, it is not known whether alternative splicing affects the receptor channel opening that occurs in the microsecond time domain. Using a laser-pulse photolysis technique combined with whole-cell recording, we characterized the channel opening rate process for two alternatively spliced variants of GluR3, i.e., GluR3flip and GluR3flop. We show that the alternative splicing that generates flip and flop variants of GluR3 receptors regulates the channel opening process by controlling the rate of channel closing but not the rate of channel opening or the glutamate binding affinity. Specifically, the flop variant closes its channel almost 4-fold faster than the flip variant. We therefore propose that the function of the flip-flop sequence module in the channel opening process of AMPA receptors is to stabilize the open channel conformation, presumably by its pivotal structural location. Furthermore, a comparison of the flip isoform among all AMPA receptor subunits, based on the magnitude of the channel opening rate constant, suggests that GluR3 is kinetically more similar to GluR2 and GluR4 than to GluR1.

Enhancing protein expression in single HEK 293 cells.

Recombinant proteins are routinely expressed in heterologous expression systems such as human embryonic kidney 293 (HEK 293) cells. The efficiency of the expression is critical when the expressed protein must be characterized at the single-cell level. Here we describe a simple method by which the protein expression efficiency in single HEK 293 cells is enhanced by coexpressing simian virus 40 large T antigen (TAg), a powerful oncoprotein. Using the GluR2 ionotropic glutamate receptor as an example, we found that the receptor expression in single HEK 293S cells increased approximately seven-fold. The ratio of the plasmid amount of TAg to that of the receptor was optimized at 1:10, while the receptor function was unaffected in the presence of TAg. We further used fluorescence imaging from a population of cells as an independent detection method and found a similar increase in expression of green fluorescent protein (GFP) by TAg coexpression. This method is thus applicable for enhancing the expression of both membrane and soluble proteins at the single-cell level. More importantly, the function of a protein can be studied directly in intact cells, a feature particularly useful for studying membrane proteins.

Channel-opening kinetics of GluR2Q(flip) AMPA receptor: a laser-pulse photolysis study.

AMPA receptors mediate fast excitatory neurotransmission in the central nervous system. GluR2 is an AMPA receptor subunit that controls some key heteromeric AMPA receptor properties, such as calcium permeability. The kinetic properties of GluR2, relevant to the time scale of its channel opening, however, are poorly understood. Here, to measure the channel-opening kinetics, we use a laser-pulse photolysis technique, which permits glutamate to be liberated photolytically from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (caged glutamate) with a time constant of approximately 30 micros. We show that GluR2Q(flip), an unedited and Ca(2+) permeable isoform, is by far the fastest ligand-gated channel with the channel-opening and -closing rate constants being (8.0 +/- 0.49) x 10(4) and (2.6 +/- 0.20) x 10(3) s(-1), respectively. Therefore, the shortest rise time (20-80% of the receptor current response) or the fastest observed time by which the GluR2Q(flip) channel can open is predicted to be 17 micros. The minimal kinetic mechanism for the channel opening is further consistent with the binding of two glutamate molecules with the channel-opening probability of 0.96. These results suggest that GluR2 is a temporally, highly efficient receptor to transduce the binding of chemical signals (i.e., glutamate) into an electrical impulse.