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.

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 (GABA_A) 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 GABA_A 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 GABA_A receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABA_A 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. Neurosteroids are cholesterol metabolites produced by neurons and glial cells that participate in central nervous system (CNS) development, regulate neuronal excitability, and modulate complex behaviors such as mood. Exogenously administered neurosteroid analogues are effective sedative hypnotics and are being developed as antidepressants and anticonvulsants. Gamma amino-butyric acid Type A (GABA_A) receptors, the principal ionotropic inhibitory neurotransmitter receptors in the brain, are the primary functional target of neurosteroids. Understanding the molecular details of neurosteroid interactions with GABA_A receptors is critical to understanding their mechanism of action and developing specific and effective therapeutic agents. In the current study, we developed a suite of neurosteroid analogue affinity labeling reagents, which we used to identify three distinct binding sites on GABA_A receptors and to determine the orientation of neurosteroid binding in each site. Electrophysiological studies performed on receptors with mutations designed to disrupt the identified binding sites showed that two of the three sites contribute to neurosteroid modulation of GABA_A currents. The distinct patterns of neurosteroid affinity, binding orientation, and effect provide the potential for the development of isoform-specific agonists, partial agonists, and antagonists with targeted therapeutic effects.

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 α1Asn407/Tyr410 in the M4 transmembrane domain and residue α1Gln241 in the M1 domain. We examined the role of residues in the α1 subunit M1 domain in the modulation of the rat α1β2γ2L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the α1Q241W or α1Q241L mutations were insensitive to (3α,5α)-3-hydroxypregnan-20-one (3α5αP), albeit with different underlying mechanisms. The α1Q241S mutant was potentiated by 3α5αP, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the α1Q241L mutation had no effect on channel potentiation by (3α,5α)-3-hydroxymethyl-pregnan-20-one, but mutation of the neighboring residue, α1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5α-pregnan-20-one) potentiated the wild-type receptor but not the α1Q241L mutant. The findings are consistent with a model in which the α1Ser240 and α1Gln241 residues shape the surface to which steroid molecules bind.