We have examined whether F-actin integrity is involved in activation of a volume-regulated Cl- current (VRChlC) in B-lymphocytes. VRChlC activation was initiated in response to establishing a whole cell recording in the presence of a hyposmotic gradient. Parallel confocal microscopy experiments using Rhodamine-Phalloidin (R-P) as a specific marker of F-actin showed that the submembrane actin ring is reversibly disrupted in response to an hyposmotic gradient. Disruptions of cortical F-actin integrity by 50 microM cytochalasin B (CB) does not trigger activation of VRChlC under isosmotic conditions or potentiate the rate of activation when the osmolarity of the extracellular solution was decreased by 75%. However, incubation with CB increased the rate of VRChlC activation in response to a 90% hyposmotic gradient. Phalloidin, a stabilizer of F-actin, decreases the rate of VRChlC activation in response to a 90% gradient, but has no effect in response to a 75% gradient. These observations suggest that disassembly of cortical F-actin is not critical for VRChlC activation in B-lymphocytes. The integrity of cortical F-actin, however, can exert a modulatory effect on the rate of VRChlC activation in the presence of a hyposmotic gradient.
However, due to the limited resolution of the crystal structures they do not provide a comprehensive atomistic picture of specific protein-ligand interactions that are important in inhibitor binding. In this study, molecular docking along with molecular dynamics (MD) simulations and binding energy calculations were carried out to assess the stability of crystallographically determined binding modes and explore potential alternative binding modes of inhibitors GYKI, CP, and PMP. Our MD simulation results provide structural insights into interactions of these inhibitors with AMPA receptors and highlight the features of the AMPA receptor non-competitive inhibitor binding pocket that are important in accommodating structurally different inhibitors. This information will aid in structure-based design of new non-competitive inhibitors that target AMPA receptors. 533-PosLife in the Fast Lane: Binding to Glutamate Receptors Ionotropic glutamate receptors (iGluRs) mediate neurotransmission at the majority of excitatory synapses in the brain. Little is known, however, about how the neurotransmitter glutamate reaches the recessed binding pocket in iGluR ligand-binding domains (LBDs). Here we report the process of glutamate binding to a prototypical iGluR, GluA2, in atomistic detail using both enhanced sampling and equilibrium molecular dynamics simulations. Charged residues on the LBD surface are found to form pathways that facilitate glutamate binding by effectively reducing a three-dimensional diffusion process to a spatiallyconstrained two-dimensional one. Free energy calculations identify residues that metastably interact with glutamate and help guide it into the binding pocket. These simulations also reveal that glutamate can bind in an inverted conformation and also reorient while in its pocket. Electrophysiological recordings demonstrate that eliminating these transient binding sites slows activation and deactivation, consistent with slower glutamate binding and unbinding. These results suggest that binding pathways have evolved to optimize rapid responses of GluA-type iGluRs at synapses.
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