A picrotoxin-specific conformational change in the glycine receptor M2-M3 loop.

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg(271) Lys(276)) toward the N-terminal end of the homomeric alpha 1 GlyR M2 - M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound ( MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr(6') residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2 - M3 loop are mediated allosterically. This suggests that the M2 - M3 loop responds differently to the occupation of different binding sites.

Expansion tube operating conditions for studying nonequilibrium radiation relevant to Titan Aerocapture

The predictions of nonequilibrium radiation in the shock layer for a Titan aerocapture aeroshell vary significantly amongst Computational Fluid Dynamics (CFD) analyses and are limited by the physical models of the nonequilibrium flow processes. Of particular interest are nonequilibrium processes associated with the CN molecule which is a strong radiator. It is necessary to have experimental data for these radiating shock layers which will allow for validation of the CFD models. This paper describes the development of a test flow condition for subscale aeroshell models in a superorbital expansion tunnel. We discuss the need for a Titan gas condition that closely simulates the atmospheric composition and present experimental data of the free stream test flow conditions. Furthermore, we present finite-rate CFD calculations of the facility to estimate the remaining free stream conditions, which cannot be directly measured during experiments.