and has a much shorter M3-M4 loop. To determine whether (or not) these segments are crucial for the function of a eukaryotic acetylcholine-glutamate Cys-loop chimeric receptor (a7-GluClbR), we deleted those segments of the chimera that are missing in GLIC. Ligand-binding assays performed on transfected living cells indicate that chimeras lacking most of the M3-M4 loop can readily bind 3H-a-bungarotoxin (a competitive antagonist) and nicotine (an agonist). These deletion chimeras were visualized on the cell surface by confocal microscopy using rhodaminylated a-bungarotoxin and specific antibodies. In addition, chimeras lacking the M3-M4 loop display AChinduced currents with unchanged EC50, Hill coefficient and ionic selectivity. In contrast, chimeras lacking the N-terminal helical segment do not bind 3H-a-bungarotoxin. However, these N-terminus-truncated receptors migrate as non-degraded proteins in SDS PAGE and are readily visualized on the surface of transfected cells with specific anti-HA tag antibodies. Electrophysiological experiments are currently performed to determine whether (or not) acetylcholine, nicotine or protons activate the N-terminus truncated chimeras. Supported by the Wolfson Family Foundation and the Israel Science Foundation. 1508-Pos Board B418Number of Extracellular-Transmembrane Interfaces Required for Activation of Homomeric Cys-Loop Receptors Natalia Andersen, Jeremias Corradi, Mariana Bartos, Steven M. Sine, Cecilia B. Bouzat. Each subunit in a homo-pentameric Cys-loop receptor contains a specialized transduction zone located at the extracellular-transmembrane interface that links the ligand binding domain to the ion conductive channel. To determine the contribution of each transduction zone to stability of the open channel, we constructed a subunit with both a disabled transduction zone and a reporter mutation that alters unitary conductance, and co-expressed mutant and normal subunits. The resulting receptors show single channel current amplitudes that are quantized according to the number of reporter mutations per receptor, allowing correlation of mean open time with the number of intact transduction zones. We find that each transduction zone contributes an equal increment to the stability of the open channel. However by combining subunits with either disabled agonist binding sites or transduction zones, we find that although each binding site is formed by a pair of subunits, detectable channel opening requires an intact transduction zone in both subunits. By manipulating the numbers and locations of transduction zones and binding sites, we find that a transduction zone in a subunit at an inactive binding site can still stabilize the open channel. The findings show that although the agonist binding sites and transduction zones contribute allosterically to open channel stability, their stoichiometry and positioning requirements are distinct. 1509-Pos Board B419Identification of the Binding Site for the Anthelmintic Drug Ivermectin in Cys-Loop Receptors Tali Gortler, Ruthi Tobi, Marina...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.