Slip dynamics at a patterned rubber/glass interface during stick-slip motions.

We report on an experimental study of heterogeneous slip instabilities generated during stick-slip motions at a contact interface between a smooth rubber substrate and a patterned glass lens. Using a sol-gel process, the glass lens is patterned with a lattice of parallel ridges (wavelength, 1.6 µm, amplitude 0.35 µm). Friction experiments using this patterned surface result in the systematic occurrence of stick-slip motions over three orders of magnitude in the imposed driving velocity while stable friction is achieved with a smooth surface. Using a contact imaging method, real-time displacement fields are measured at the surface of the rubber substrate. Stick-slip motions are found to involve the localized propagation of transverse interface shear cracks whose velocity is observed to be remarkably independent on the driving velocity.

A (beta)-strand in the (gamma)2 subunit lines the benzodiazepine binding site of the GABA A receptor: structural rearrangements detected during channel gating.

Benzodiazepines (BZDs) exert their effects in the CNS by binding to a modulatory site on GABA(A) receptors. Individual amino acids have been implicated in BZD recognition and modulation of the GABA(A) receptor, but the secondary structure of the amino acids contributing to the BZD binding site has not been elucidated. In this report we used the substituted cysteine accessibility method to understand the structural dynamics of a region of the GABA(A) receptor implicated in BZD binding, gamma(2)Y72-gamma(2)Y83. Each residue within this region was mutated to cysteine and expressed with wild-type alpha(1) and beta(2) subunits in Xenopus oocytes. Methanethiosulfonate (MTS) reagents were used to modify covalently the engineered cysteines, and the subsequent effects on BZD modulation of the receptor were monitored functionally by two-electrode voltage clamp. We identified an alternating pattern of accessibility to sulfhydryl modification, indicating that the region gamma(2)T73-gamma(2)T81 adopts a beta-strand conformation. By monitoring the ability of BZD ligands to impede the covalent modification of accessible cysteines, we also identified two residues within this region, gamma(2)A79 and gamma(2)T81, that line the BZD binding site. Sulfhydryl modification of gamma(2)A79C or gamma(2)T81C allosterically shifts the GABA EC(50) of the receptor, suggesting that certain MTS compounds may act as tethered agonists at the BZD binding site. Last, we present structural evidence that a portion of the BZD binding site undergoes a conformational change in response to GABA binding and channel gating (opening and desensitization). These data represent an important step in understanding allosteric communication in ligand-gated ion channels.

Identification of benzodiazepine binding site residues in the gamma2 subunit of the gamma-aminobutyric acid(A) receptor.

gamma-Aminobutyric acid(A) receptor gamma-subunits are important for benzodiazepine (BZD) binding and modulation of the gamma-aminobutyric acid-mediated Cl(-) current. Previously, by using gamma2/alpha1 chimeric subunits, we identified two domains of the gamma2-subunit, Lys-41-Trp-82 and Arg-114-Asp-161, that are, in conjunction, necessary and sufficient for high-affinity BZD binding. In this study, we generated additional gamma2/alpha1 chimeric subunits and gamma2 point mutants to identify specific residues within the gamma2 Lys-41-Trp-82 region that contribute to BZD binding. Mutant gamma2 and gamma2/alpha1 chimeric subunits were expressed with wild-type alpha1 and beta2 subunits in HEK 293 cells, and the binding of several BZDs was measured. We present evidence that the gamma2 region Met-57-Ile-62 is important for flunitrazepam binding and that, in particular, gamma2 Met-57 and gamma2 Tyr-58 are essential determinants for conferring high-affinity binding. Furthermore, we identify an additional residue, gamma2 Ala-79, that not only is important for high-affinity binding by flunitrazepam (a strong positive modulator) but also plays a crucial role in the binding of the imidazobenzodiazepines Ro15-1788 (a zero modulator) and Ro15-4513 (a weak negative modulator) in the BZD binding pocket. Results from site-directed mutagenesis of gamma2 Ala-79 suggest that this residue may be part of a microdomain within the BZD binding site that is important for binding imidazobenzodiazepines. This separation of drug-specific microdomains for competitive BZD ligands lends insight into the structural determinants governing the divergent effects of these compounds.