Erika Suzuki

Modulation of AMPA receptor kinetics differentially influences synaptic plasticity in the hippocampus

Prior studies showed that positive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators facilitate long-term potentiation (LTP) and improve the formation of several types of memory in animals and humans. However, these modulators are highly diverse in their effects on receptor kinetics and synaptic transmission and thus may differ also in their efficacy to promote changes in synaptic strength. The present study examined three of these modulators for their effects on synaptic plasticity in field CA1 of hippocampal slices, two of them being the benzamide drugs 1-(quinoxalin-6-ylcarbonyl)piperidine (CX516) and 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546) which prominently enhance synaptic transmission yet differ in their relative impact on amplitude versus duration of the synaptic response. The third drug was cyclothiazide which potently blocks AMPA receptor desensitization. Effects on plasticity were assessed by measuring (i) the likelihood of obtaining stable potentiation when using theta-burst stimulation with three instead of four pulses per burst, (ii) the maximum amount of potentiation under optimal stimulation conditions, and (iii) the effect on long-term depression (LTD). Both benzamides facilitated the formation of stable potentiation induced with three-pulse burst stimulation which is normally ineffective. CX546 in addition increased maximally inducible potentiation after four-pulse burst stimulation from about 50% to 100%. Burst response analysis revealed that CX546 greatly prolonged the duration of depolarization by slowing the decay of the response which thus presumably leads to a more continuous N-methyl-D-aspartate (NMDA) receptor activation. Cyclothiazide was ineffective in increasing maximal potentiation in either field or whole-cell recordings. CX546, but not CX516, also enhanced nearly two-fold the NMDA receptor-dependent long-term depression induced by heterosynaptic 2 Hz stimulation. Tests with recombinant NMDA receptors (NR1/NR2A) showed that CX516 and CX546 have no direct effects on currents mediated by these receptors. These results suggest that (1) modulation of AMPA receptors which increases either response amplitude or duration can facilitate LTP formation, (2) modulators that effectively slow response deactivation augment the maximum magnitude of LTP and LTD, and (3) receptor desensitization may have a minor impact on synaptic plasticity in the hippocampus. Taken together, our data indicate that AMPA receptor modulators differ substantially in their ability to enhance synaptic potentiation or depression, depending on their particular influence on receptor kinetics, and hence that they may also be differentially effective in influencing higher-order processes such as memory encoding.

The fast kinetics of AMPA GluR3 receptors is selectively modulated by the TARPs γ4 and γ8

Transmembrane AMPA receptor-associated regulatory proteins (TARPs) modulate the kinetics of AMPA receptors and increase their surface expression. This study compared the effects of the TARPs gamma2, gamma3, gamma4 and gamma8 on the AMPA receptor subunits GluR1-3. We found that the deactivation and desensitization time constants of GluR3 receptors were greatly increased by gamma4 and gamma8 (approximately 3 fold) whereas gamma2 and gamma3 caused minimal changes. This stands in contrast to the effects on GluR1 and GluR2 receptors which were similar across all four TARPs. Other response parameters like the current density, the EC(50) for glutamate-induced peak currents, and kainate-induced currents were in general more effectively modulated by gamma2 and gamma3 in all subunits, including GluR3. All TARPs were effective in increasing the unitary conductance. The differential TARP effects on deactivation time constants of the GluR3 subunit are likely to have a significant impact on synaptic responses across different neurons in the brain.

Stability of a Recombinant Adenoviral Vector: Optimization of Conditions for Storage, Transport and Delivery

Recombinant viral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, little is known about the viability and stability of such recombinant viruses during storage, transport and delivery under various conditions. We describe here an analysis of the stability of an adenoviral vector in crude solutions of cell lysates during freezing and thawing and during storage at various temperatures in the presence and in the absence of glycerol. For example, the titer of adenoviruses in crude lysates of infected cells was reduced only ten‐fold or three‐fold after two hundred rounds of freezing and thawing or after incubation at 28°C for 14 days, respectively. Our observations indicate that recombinant adenoviral vector was more stable than expected both during freezing and thawing and during storage at low temperatures. Our results confirm the importance of appropriate conditions for the delivery and transport of recombinant adenoviral vectors.

Physiological significance of high- and low-affinity agonist binding to neuronal and recombinant AMPA receptors

Radioligand binding studies have shown that AMPA receptors exist in two variants that differ about twenty-fold in their binding affinities, with brain receptors being mainly of the low-affinity type and recombinantly expressed receptors having almost exclusively high affinity. However, the physiological correlate of high- and low-affinity binding is not yet known. In this study we examined if physiological experiments similarly reveal evidence for two distinct receptor variants. We therefore measured equilibrium desensitization by glutamate and determined IC(50) values for neuronal receptors and for the homomeric receptors GluR1-4 expressed in HEK293 cells. Contrary to the prediction that these IC(50) values exhibit large differences commensurate with those of high- and low-affinity binding, values for homomeric receptors (1-18 microM) were on an average not different from those of neuronal receptors (3-10 microM). Moreover, simulations with kinetic receptor models suggest that the IC(50) values for neuronal and recombinant receptors correspond to the binding affinity of the low-affinity receptor variant. These findings indicate that the high-affinity binding measured in heterologous expression systems represents an immature receptor variant that does not contribute to the currents recorded from these cells, and that the functional low-affinity receptors are present in such small number that they are effectively masked in binding assays by the high-affinity receptors. Thus, in order to compare experimentally determined saturation binding profiles with those predicted by kinetic receptor models and with dose-response curves from physiological studies, it will be imperative to develop methods for isolating first the low-affinity receptors.

C-terminal truncation affects kinetic properties of GluR1 receptors.

GluR1flop receptors in which the C-terminal 52 amino acids had been recombinantly removed were characterized with whole-cell recording and binding assays. Compared to wildtype GluR1, truncated receptors showed faster desensitization and deactivation and they recovered more slowly from desensitization. The EC50 for glutamate was increased 2-fold. In binding tests, K(D)s for [3H]fluorowillardiine were 1.5 times larger for truncated receptors. According to receptor simulations, most differences can be explained if the C-terminal domain is assumed to stabilize the ligand-bound closed and open states. The effects on response waveforms are different from those caused by phosphorylation, suggesting that the C-terminus influences receptor function in multiple ways. Truncated forms of GluR1 identical or similar to the one examined here may also be generated by calcium-activated proteases during intense synaptic activity. The lowered affinity and faster inactivation of these receptors suggests that their presence does not represent a risk for neuronal viability.

Spontaneous mutations in the human gene for p53 in recombinant adenovirus during multiple passages in human embryonic kidney 293 cells

Infectious recombinant adenovirus (rAd) is usually produced in human embryonic kidney 293 cells that harbor the E1 gene and rAd has been shown to be an efficient tool for gene transfer both in vivo and in vitro. It also has considerable potential in human gene therapy. However, rates of spontaneous mutations in genes introduced into host cells after multiple passages remain to be clarified. We have characterized the spontaneous mutation of genomes derived from human adenovirus type 5 (Ad5) and of human p53-rAd during multiple passages by two different methods, namely, a plaque assay and a molecular cloning assay, with subsequent direct nucleotide sequencing. Using the plaque assay, we found no mutations in the E1A and p53 genes derived from infectious Ad5 and p53-rAd, respectively. By contrast, we found spontaneous mutations in the E1A gene of Ad5, with a mutation rate of 9.28 x 10(-8) per base pair per plaque, in the molecular cloning assay. The rate of mutation of the p53 gene of p53-rAd, as determined by the molecular cloning assay, ranged from 1.50 x 10(-7) to 3.25 x 10(-7) per base pair per passage. The mutations in the p53 gene of p53-rAd were localized mainly in the transcriptional activation domain, the SH3 domain, and the regulation domain and they were rarely found in the DNA-binding domain, which is a major site for mutations in human cancers. Our results indicate that multiple passages can generate a heterogeneous population of p53-rAd and that the molecular cloning assay is an efficient technique with which to search for mutations in the genome of p53-rAd that cannot be detected by a plaque assay.

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank

Viral vectors are required as gene‐delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds.

Factors affecting guanine nucleotide binding to rat AMPA receptors

Glutamate receptors are competitively inhibited by guanine nucleotides. Insight into the physiological function of this inhibition would be greatly advanced if nucleotide binding could be eliminated through mutations without altering other aspects of receptor function, or if compounds were discovered that selectively prevent nucleotide binding. It was previously reported that a lysine in the chick kainate binding protein (cKBP) is specifically involved in guanine nucleotide binding. In the present study we mutated the equivalent lysine in the rat AMPA receptor subunit GluR1 flip to alanine (K445A) and assessed changes in nucleotide affinity from the displacement of [(3)H]fluorowillardiine. As in the cKBP, the affinity for nucleotides was greatly reduced while the binding affinity for agonists remained unchanged. The reduction in affinity was largest for GTP (factor of 5.8) and GDP (4.4) and minor for GMP and guanosine. This suggests that K445 is involved in stabilizing the second phosphate of the nucleotide. Given that bulkier analogs like GDP-fucose are also accommodated at this site, it seems likely that nucleotides bind in such a way that their phosphates project out of the cleft. In excised-patch recordings using short pulses of glutamate, the K445A mutation increased the EC(50) for the peak response 1.8-fold and accelerated desensitization and deactivation. This indicates that the effects of this mutation are not as specific as previously suggested. Efforts to selectively eliminate inhibition by nucleotides may therefore depend on mapping out further the docking site. In a first attempt using point mutations we ruled out several amino acids around the cleft as being involved in nucleotide binding. Also, the AMPA receptor modulator PPNDS which competitively inhibits nucleotide binding to purinergic receptors did not affect nucleotide inhibition, suggesting that there are major differences in the topography between purinergic and glutamate receptors. Thus new approaches, including crystallography, may be called for to identify residues uniquely involved in nucleotide binding.