Hydrogen bond-mediated self-assembly is a powerful strategy for generating large structures from smaller subunits. The synthesis of molecules containing two isophthalic acid units covalently attached to a rigid aromatic spacer is described. By normal pairing of carboxylic acids into hydrogen-bonded dimers, these molecules self-assemble in organic solvents to form either a series of linear aggregates or a cyclic hexamer. These molecules were linked to the core of a family of polyether dendrimers, which caused the hexamer to be formed preferentially. The stability of the hexamer depended on the generation number of the dendrimer. The largest of these hydrogen-bonded macromolecular assemblies is roughly disk-shaped with a 9-nanometer diameter and a 2-nanometer thickness. Its size and molecular mass (34,000 daltons) are comparable to that of small proteins.

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Neuroactive steroids are among the most efficacious modulators of the mammalian GABA-A receptor. Previous work has proposed that receptor potentiation is mediated by steroid interactions with a site defined by the residues ␣1Asn407/Tyr410 in the M4 transmembrane domain and residue ␣1Gln241 in the M1 domain. We examined the role of residues in the ␣1 subunit M1 domain in the modulation of the rat ␣1␤2␥2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the ␣1Q241W or ␣1Q241L mutations were insensitive to (3␣,5␣)-3-hydroxypregnan-20-one (3␣5␣P), albeit with different underlying mechanisms. The ␣1Q241S mutant was potentiated by 3␣5␣P, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the ␣1Q241L mutation had no effect on channel potentiation by (3␣,5␣)-3-hydroxymethylpregnan-20-one, but mutation of the neighboring residue, ␣1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5␣-pregnan-20-one) potentiated the wild-type receptor but not the ␣1Q241L mutant. The findings are consistent with a model in which the ␣1Ser240 and ␣1Gln241 residues shape the surface to which steroid molecules bind.Potentiating neurosteroids are among the most efficacious modulators of the mammalian GABA-A receptor having potential applications as anxiolytics, anticonvulsants, sedatives, and anesthetics. Recent work has given significant insights into the functional and structural mechanisms of steroid actions. Potentiating steroids [e.g., (3␣,5␣)-3-hydroxypregnan-20-one (3␣5␣P) and (3␣,5␤)-3-hydroxypregnan-20-one (3␣5␤P)] act on the GABA-A receptor by modifying the channel open and closed times, leading to an increase in the open probability of the channel, enhanced macroscopic peak current, and a slower current decay when exposure to agonist is terminated. The putative steroid binding site is located in the membrane-spanning regions of the ␣ subunit of the receptor, extending from the ␣1Gln241 residue in the M1 membrane-spanning region to the residues ␣1Asn407 and ␣1Tyr410 in the M4 domain (Hosie et al., 2006). Mutations that reduce the H-bonding ability of these residues reduce receptor potentiation by both 5␣-and 5␤-reduced steroids. It was proposed that a common interaction site mediates the effects of the two classes of steroids, the ␣1Gln241 residue acting as an H-bond acceptor to the 3␣-hydroxyl group of the steroid molecule and the ␣1Asn407/Tyr410 residues interacting with the ketone group in the side chain on the D ring of steroids (Hosie et al., 2006). Subsequent studies showed that mutations that disrupt channel potentiation by steroids also affect modulation by a tricyclic benz[e]indene neurosteroid analog (Li et al., 2006), enantiomers of natural steroids (Li et al., 2007a), and the marine cembranoid eupalmerin acetate (Li et al., 2008), suggesting that the site may function as a common interaction site for a number of GABA-A receptor modul...

In the K/B×N mouse model of arthritis, autoantibodies against glucose-6-phosphate isomerase cause joint-specific inflammation and destruction. We have shown using micro-positron emission tomography that these glucose-6-phosphate isomerase-specific autoantibodies rapidly localize to distal joints of mice. In this study we used micro-positron emission tomography to delineate the stages involved in the development of arthritis. Localization of Abs to the joints depended upon mast cells, neutrophils, and FcRs, but not on C5. Surprisingly, anti-type II collagen Abs alone did not accumulate in the distal joints, but could be induced to do so by coinjection of irrelevant preformed immune complexes. Control Abs localized to the joint in a similar manner. Thus, immune complexes are essential initiators of arthritis by sequential activation of neutrophils and mast cells to allow Abs access to the joints, where they must bind a target Ag to initiate inflammation. Our findings support a four-stage model for the development of arthritis and identify checkpoints where the disease is reversible.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(−) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action.