No abstract

Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric ␣1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19 or 22 positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19C increased by ϳ20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and ␤-alanine were weak partial agonists at the ␣1R19C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or ␤-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists. Glycine receptor (GlyR)4 chloride channels mediate inhibitory neurotransmission in the central nervous system (1). They comprise an assembly of five subunits that are each composed of a large N-terminal extracellular ligand-binding domain and four transmembrane ␣-helices (M1-M4). Cryo-electron microscopy images of the homologous Torpedo electropax nicotinic acetylcholine receptor (nAChR) transmembrane region reveals that the pore-lining M2 domains are kinked radially inward to form a central constriction at the membrane midpoint (2). There is currently a great deal of interest in understanding how M2 domains move to open the channel. Although the original model proposed a drastic rotation of M2 domains about their long axes (3), more recent evidence suggests that there is little if any rotation during receptor activation (4 -6). The precise nature of the structural change remains a matter for debate.The central pore kink is likely to introduce a degree of structural discontinuity because the hydrogen bonds responsible for maintaining ␣-helix rigidity are most likely broken. The kink may therefore act as a swivel enabling the outer half of M2 to move asynchronously with the inner half, where the gate is most likely positioned. Indeed, several lines of evidence suggest gating is mediated by a backbone rearrangement at this midpoint (7-10). In addition, a rate equilibrium fr...

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the ␤19 position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (⌬F͞F) Ϸ10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for ⌬F agreed well with those for epibatidine-induced currents, but were shifted Ϸ100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the ␣␥-binding site than to the ␣␦ site and (ii) ACh binds with reverse-site selectivity, these data suggest that ⌬F monitors an event linked to binding specifically at the ␣␦-subunit interface. In experiments with flash-applied agonists, the earliest detectable ⌬F occurs within milliseconds, i.e., during activation. T he muscle nicotinic acetylcholine receptor (nAChR) is a well studied member of the Cys-loop family of neurotransmittergated ion channels. A 4.6-Å structure (1) shows five Ϸ160-Å-long rod-shaped subunits surrounding a central channel. From the extracellular side the subunits have a counterclockwise order of ␣␥␣␦␤ (2, 3). Each subunit has a large extracellular Nterminal or ligand-binding domain followed by four transmembrane regions, M1-M4 (4). The structure of the extracellular ligand-binding sites, at the ␣␥ and ␣␦ interfaces, resembles that of a homologous molluscan ACh-binding protein (3). Numerous biochemical and electrophysiological experiments indicate that M2 lines the channel (5).The nAChR exists in at least four distinct, interconvertible conformational states: resting, open, fast-onset-desensitized, and slow-onset-desensitized (6). The open and the fast-onsetdesensitized states presumably have moderate affinity for ACh, are metastable (on millisecond time scales), and are present in low concentrations at equilibrium. The supralinear doseresponse relation (Hill coefficient Ͼ1) suggests that the open state of the channel is much more likely to be associated with the presence of two bound agonist molecules than with a single bound agonist (7). In the prevailing kinetic scheme, receptors in the resting state bind two agonist molecules, isomerize to the open state, and in the continued presence of agonist, desensitize.After removal of agonist, the agonist-receptor complex dissociates and the channel closes within milliseconds; but desensitized receptors isomerize more slowly to the resting state (tens of milliseconds to hundreds of seconds). Thermodynamic considerations suggest that the resting state has low affinity for agonist, whereas the slow-onset-desensitized state is the most stable state in the presence of agonist because of its high affinity.Kinetic analyses of single-channel and macroscopic function suggests that in the resting state, the affinity of ACh for the two sites differs by a factor of...

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade extracellular matrix (ECM) components such as collagen and have important roles in multiple biological processes, including development and tissue remodeling, both in health and disease. The activity of MMPs is influenced by the expression of MMPs and tissue inhibitors of metalloproteinase (TIMPs). In the eye, MMP-mediated ECM turnover in the juxtacanalicular region of the trabecular meshwork (TM) reduces outflow resistance in the conventional outflow pathway and helps maintain intraocular pressure (IOP) homeostasis. An imbalance in the MMP/TIMP ratio may be involved in the elevated IOP often associated with glaucoma. The prostaglandin analog/prostamide (PGA) class of topical ocular hypotensive medications used in glaucoma treatment reduces IOP by increasing outflow through both conventional and unconventional (uveoscleral) outflow pathways. Evidence from in vivo and in vitro studies using animal models and anterior segment explant and cell cultures indicates that the mechanism of IOP lowering by PGAs involves increased MMP expression in the TM and ciliary body, leading to tissue remodeling that enhances conventional and unconventional outflow. PGA effects on MMP expression are dependent on the identity and concentration of the PGA. An intracameral sustained-release PGA implant (Bimatoprost SR) in development for glaucoma treatment can reduce IOP for many months after expected intraocular drug bioavailability. We hypothesize that the higher concentrations of bimatoprost achieved in ocular outflow tissues with the implant produce greater MMP upregulation and more extensive, sustained MMP-mediated target tissue remodeling, providing an extended duration of effect. IOP, intraocular pressure; MMP, matrix metalloproteinase; MT-MMP, membrane-type MMP; TIMP, tissue inhibitor of metalloproteinase.MMPS AND GLAUCOMA TREATMENT 209 210 WEINREB ET AL.

We report on the first, to our knowledge, successful detection of a fluorescent unnatural amino acid (fUAA), Lys(BODIPYFL), incorporated into a membrane protein (the muscle nicotinic acetylcholine receptor, nAChR) in a living cell. Xenopus oocytes were injected with a frameshift-suppressor tRNA, amino-acylated with Lys(BODIPYFL) and nAChR (alpha/beta19'GGGU/gamma/delta) mRNAs. We measured fluorescence from oocytes expressing nAChR beta19'Lys(BODIPYFL), using time-resolved total internal reflection fluorescence microscopy. Under conditions of relatively low receptor density (<0.1 receptors/microm(2)), we observed puncta with diffraction-limited profiles that were consistent with the point-spread function of our microscope. Furthermore, diffraction-limited puncta displayed step decreases in fluorescence intensity, consistent with single-molecule photobleaching. The puncta densities agreed with macroscopic ACh-induced current densities, showing that the fUAA was incorporated, and that receptors were functional. Dose-response relations for the nAChR beta19'Lys(BODIPYFL) receptors were similar to those for wild-type receptors. We also studied nAChR beta19'Lys(BODIPYFL) receptors labeled with alpha-bungarotoxin monoconjugated with Alexa488 (alphaBtxAlexa488). The nAChR has two alphaBtx binding sites, and puncta containing the Lys(BODIPYFL) labeled with alphaBtxAlexa488 yielded the expected three discrete photobleaching steps. We also performed positive control experiments with a nAChR containing enhanced green fluorescent protein in the gamma-subunit M3-M4 loop, which confirmed our nAChR beta19'Lys(BODIPYFL) measurements. Thus, we report on the cell-based single-molecule detection of nAChR beta19'Lys(BODIPYFL).

Purpose: To compare the dose–response profiles of bimatoprost sustained-release implant (Bimatoprost SR) and topical bimatoprost in lowering intraocular pressure (IOP) in normotensive beagle dogs. Methods: In 1 study, topical bimatoprost 0.001%, 0.01%, or 0.1% was administered twice daily in the study eye for 5 days. IOP was measured at baseline and up to hour 6 each day. Other studies evaluated the IOP response to a single administration of Bimatoprost SR at dose strengths ranging from 8 to 120 μg. IOP was measured before implant administration and during 3 months of follow-up; IOP in response to topical bimatoprost 0.03% was measured prestudy as an internal control. Results: Mean percentage decrease in IOP from baseline at hour 6 (peak effect) across study days was 15.7%, 36.1%, and 24.8% (2.8, 7.0, and 4.0 mmHg) in animals treated with topical bimatoprost 0.001%, 0.01%, and 0.1%, respectively. After Bimatoprost SR administration, mean percentage decrease in IOP from baseline across 3 months consistently increased with increasing dose strength and was 38.7% (7.2 mmHg) with Bimatoprost SR 120 μg. Mean percentage IOP decrease with topical bimatoprost 0.03% was 27.6% (5.9 mmHg). Conclusions: Topical bimatoprost demonstrated a U-shaped dose–response curve; increasing the bimatoprost concentration to 0.1% resulted in reduced IOP-lowering efficacy. In contrast, the dose–response curve for Bimatoprost SR showed consistently greater IOP lowering as the dose strength increased, with the dose strength producing maximum IOP lowering not yet determined. At 60- and 120-μg dose strengths, Bimatoprost SR produced greater IOP reductions than were achieved with topical dosing.

The central nervous system convulsant picrotoxin (PTX) inhibits GABA A and glutamate-gated Cl ؊ channels in a use-facilitated fashion, whereas PTX inhibition of glycine and GABA C receptors displays little or no use-facilitated block. We have identified a residue in the extracellular aspect of the second transmembrane domain that converted picrotoxin inhibition of glycine ␣1 receptors from non-use-facilitated to usefacilitated. In wild type ␣1 receptors, PTX inhibited glycine-gated Cl ؊ current in a competitive manner and had equivalent effects on peak and steady-state currents, confirming a lack of use-facilitated block. Mutation of the second transmembrane domain 15-serine to glutamine (␣1(S15Q) receptors) converted the mechanism of PTX blockade from competitive to non-competitive. However, more notable was the fact that in ␣1(S15Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in ␣1(S15N) receptors. The reciprocal mutation in the ␤2 subunit of ␣1␤2 GABA A receptors (␣1␤2(N15S) receptors) decreased the magnitude of use-facilitated PTX inhibition. Our results implicate a specific amino acid at the extracellular aspect of the ion channel in determining use-facilitated characteristics of picrotoxin blockade. Moreover, the data are consistent with the suggestion that picrotoxin may interact with two domains in ligand-gated anion channels.Glycine receptors belong to a superfamily of ligand-gated chloride channels that include GABA A 1 receptors, GABA C receptors, and glutamate-gated chloride channels (1). In native tissue, glycine receptors exist as either ␣ homomers or ␣␤ heteromers (1). They comprise five subunits (usually three ␣ subunits and two ␤ subunits) arranged asymmetrically around the ion pore. Each subunit is made up of a large extracellular N-terminal region, four transmembrane domains (TM), and a large cytoplasmic domain; TMII forms the channel lumen (2). Glycine receptors are targets of therapeutics such as anesthetics as well as toxins like the central nervous system convulsant picrotoxin (1).Picrotoxin inhibits all known anionic ligand-gated Cl Ϫ channels (3-5). The mechanism of action and the exact location of picrotoxin binding are still unknown (6 -12). However, several studies have indicated that TMII is the probable site for picrotoxin action (6, 13-22) (Fig. 1). For example, the TMII of the glycine ␤ subunit was found to be responsible for conferring resistance to picrotoxin in heteromeric glycine ␣ n ␤ receptors (n ϭ 1-3) (6). Subsequent work has defined the existence of a phenylalanine residue at the 6Ј position of the TMII glycine ␤ subunit in conferring insensitivity to picrotoxin (16). In addition, other TMII residues (2Ј and 19Ј) have also been implicated directly or indirectly in the mechanism by which picrotoxin inhibits these channels (13, 15,16). The mutations at positions 2Ј and 19Ј have been shown to affect the type of the inhibition (competitive versus non-competitive) by picrotoxin ...

Although photon noise at low luminance reduces the visual benefits of small pupils, the benefits of 2- to 3-mm artificial pupils are sufficient to enable >80% of distance-corrected presbyopes to read proficiently at near, even at the lowest text luminances found in interior environments.