␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian CNS. Structures of the extracellular ligandbinding domain suggest that the extent of cleft closure in the ligand-binding domain controls the extent of activation of the receptor. Here we have developed a fluorescence resonance energy transfer-based probe that allows us to study the extent of cleft closure in the isolated ligand-binding domain in solution. These investigations show that the wild-type protein exhibits a graded cleft closure that correlates to the extent of activation, which is in qualitative agreement with the crystal structures. However, the changes in extent of cleft closure between the apo and agonist-bound states are smaller than that observed in the crystal structures. We have also used this method to study the L650T mutant and show that in solution the ␣-amino-5-methyl-3-hydroxy-4-isoxazole propionate-bound form of this mutant exists primarily in a conformation that is more closed than predicted based on the activity, indicating that the degree of cleft closure alone cannot be used as a measure of extent of activation of the receptor, and there are possibly other mechanisms in addition to cleft closure that mediate the subtleties in extent of activation by a given agonist.fluorescence ͉ glutamate ͉ ion channel L igand-gated ion channels are allosteric proteins that convert chemical signals into electrical signals by forming transmembrane ion channels upon ligand binding to an extracellular domain. Ionotropic glutamate receptors, a member of the ligand-gated ion channels, serve as an excellent paradigm for studying allostery in this family of proteins (1-6). The crystal structures of the isolated ligand-binding domain of the GluR2 subunit of the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor protein show a graded cleft closure with a direct correlation between the extent of cleft closure induced by a ligand and its extent of activation (5, 6), which is consistent with a multiple state-induced fit model. However, there is one exception to this correlation, the AMPA-bound form of the L650T mutation of the receptor crystallizes in two forms: one structure where the cleft is closed 11°and a second structure in which the cleft is closed 22°relative to the open apo form of the protein (7). These structures lie on either side of the cleft closure versus activation correlation observed for the wild-type protein, thus raising the question as to whether some ligands or modifications at the AMPA receptors can change the mechanism of activation from the multistate model to that defined by equilibrium of two distinct states.Here we have developed a fluorescence resonance energy transfer (FRET)-based assay that allows us to investigate the conformational changes in the ligand-binding domain in solution, thus allowing us to probe the conformational changes in this domain without the crystallographic constraints. In addition, we have u...

Purpose. To evaluate intraobserver and interobserver agreement in locating the scleral spur landmark (SSL) and anterior chamber angle measurements obtained using Fourier Domain Anterior Segment Optical Coherence Tomography (ASOCT) images. Methods. Two independent, masked observers (SR and AZC) identified SSLs on ASOCT images from 31 eyes with open and nonopen angles. A third independent reader, NPB, adjudicated SSL placement if identifications differed by more than 80 μm. Nine months later, SR reidentified SSLs. Intraobserver and interobserver agreement in SSL placement, trabecular-iris space area (TISA750), and angle opening distance (AOD750) were calculated. Results. In 84% of quadrants, SR's SSL placements during 2 sessions were within 80 μm in both the X- and Y-axes, and in 77% of quadrants, SR and AZC were within 80 μm in both axes. In adjudicated images, 90% of all quadrants were within 80 μm, 88% in nonopen-angle eyes, and 92% in open-angle eyes. The intraobserver and interobserver correlation coefficients (with and without adjudication) were above 0.9 for TISA750 and AOD750 for all quadrants. Conclusions. Reproducible identification of the SSL from images obtained with FD-ASOCT is possible. The ability to identify the SSL allows reproducible measurement of the anterior chamber angle using TISA750 and AOD750.

Purpose. To introduce a new anterior segment optical coherence tomography parameter, trabecular-iris circumference volume (TICV), which measures the integrated volume of the peripheral angle, and establish a reference range in normal, open angle eyes. Methods. One eye of each participant with open angles and a normal anterior segment was imaged using 3D mode by the CASIA SS-1000 (Tomey, Nagoya, Japan). Trabecular-iris space area (TISA) and TICV at 500 and 750 µm were calculated. Analysis of covariance was performed to examine the effect of age and its interaction with spherical equivalent. Results. The study included 100 participants with a mean age of 50 (±15) years (range 20–79). TICV showed a normal distribution with a mean (±SD) value of 4.75 µL (±2.30) for TICV500 and a mean (±SD) value of 8.90 µL (±3.88) for TICV750. Overall, TICV showed an age-related reduction (P = 0.035). In addition, angle volume increased with increased myopia for all age groups, except for those older than 65 years. Conclusions. This study introduces a new parameter to measure peripheral angle volume, TICV, with age-adjusted normal ranges for open angle eyes. Further investigation is warranted to determine the clinical utility of this new parameter.

Abstractα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system. Structures of the isolated ligand binding domain of this receptor have provided significant insight into the large scale conformational changes, which when propagated to the channel segments leads to receptor activation. However, in order to establish the role of specific molecular interactions in controlling such fine details as the magnitude of the functional response, we have used a multiscale approach, where changes at specific moieties of the agonists have been studied by vibrational spectroscopy, while large scale conformational changes have been studied using fluorescence resonance energy transfer (FRET) investigations. By exploiting the wide range of activations by the agonists, glutamate, kainate, and AMPA, for the wild type,Y450F, and L650T mutants and by using the multiscale investigation, we show that the strength of the interactions at the α-amine group of the agonist with the protein in all but one case tracks the extent of activation. Since the α-amine group forms bridging interactions at the cusp of the ligand binding cleft this appears to be a critical interaction through which the agonist controls the extent of activation of the receptor.α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, a member of the ionotropic glutamate receptor family, are the main mediators of fast excitatory synaptic transmission in the mammalian central nervous system. Signal transmission is initiated by glutamate binding to an extracellular ligand binding domain, which leads to the formation of cation specific transmembrane channels (1-6). Large scale expression of the isolated extracellular ligand binding domain (S1S2) has led the way for detailed structural studies of this domain. X-ray (7-10), nuclear magnetic resonance (NMR) (4,(11)(12)(13), and vibrational spectroscopic (14-16) investigations using this soluble protein have provided significant insight into the relationship between structure and function in this subtype.The structures of S1S2 show a graded cleft closure conformational change upon binding agonists with varying efficacy in the bilobed ligand binding domain (9,10). The extent of cleft closure induced by a given agonist in most cases exhibits a direct correlation to the extent of activation of the receptor, suggesting that this is one of the possible modes of coupling between the ligand binding domain and opening of the ion channel. Vibrational spectroscopic investigations using the S1S2 protein provide a more detailed view of the specific interactions between the agonist and the extracellular ligand binding domain and their role in the functioning of the receptor. The frequency shifts in the asymmetric carboxylate vibrational mode, which is sensitive to the strength of the non-covalent interactions at this moiety, indicate that partial *Address correspondence to: Vasanthi Jayaraman, Department of Integrative Biolo...

Purpose To evaluate the change in trabecular-iris circumference volume (TICV) after laser peripheral iridotomy (LPI) in primary angle closure (PAC) spectrum eyes Patients and Methods Forty-two chronic PAC spectrum eyes from 24 patients were enrolled. Eyes with anterior chamber abnormalities affecting angle measurement were excluded. Intraocular pressure, slit lamp exam, and gonioscopy were recorded at each visit. Anterior segment optical coherence tomography (ASOCT) with 3D mode angle analysis scans were taken with the CASIA SS-1000 (Tomey Corp., Nagoya, Japan) before and after LPI. Forty-two pre-LPI ASOCT scans and 34 post-LPI ASOCT scans were analyzed using the Anterior Chamber Analysis and Interpretation (ACAI, Houston, TX) software. A mixed-effect model analysis was used to compare the trabecular-iris space area (TISA) changes among 4 quadrants, as well as to identify potential factors affecting TICV. Results There was a significant increase in all average angle parameters after LPI (TISA500, TISA750, TICV500, and TICV750). The magnitude of change in TISA500 in the superior angle was significantly less than the other angles. The changes in TICV500 and TICV750 were not associated with any demographic or ocular characteristics. Conclusion TICV is a useful parameter to quantitatively measure the effectiveness of LPI in the treatment of eyes with PAC spectrum disease.