The broad-spectrum anthelmintic drug ivermectin (IVM) activates and stabilizes an open-channel conformation of invertebrate chloride-selective glutamate receptors (GluClRs), thereby causing a continuous inflow of chloride ions and sustained membrane hyperpolarization. These effects suppress nervous impulses and vital physiological processes in parasitic nematodes. The GluClRs are pentamers. Homopentameric receptors assembled from the Caenorhabditis elegans (C. elegans) GluClα (GLC-1) subunit can inherently respond to IVM but not to glutamate (the neurotransmitter). In contrast, heteromeric GluClα/β (GLC-1/GLC-2) assemblies respond to both ligands, independently of each other. Glutamate and IVM bind at the interface between adjacent subunits, far away from each other; glutamate in the extracellular ligand-binding domain, and IVM in the ion-channel pore periphery. To understand the importance of putative intersubunit contacts located outside the glutamate and IVM binding sites, we introduced mutations at intersubunit interfaces, between these two binding-site types. Then, we determined the effect of these mutations on the activation of the heteromeric mutant receptors by glutamate and IVM. Amongst these mutations, we characterized an α-subunit point mutation located close to the putative IVM-binding pocket, in the extracellular end of the first transmembrane helix (M1). This mutation (αF276A) moderately reduced the sensitivity of the heteromeric GluClαF276A/βWT receptor to glutamate, and slightly decreased the receptor subunits’ cooperativity in response to glutamate. In contrast, the αF276A mutation drastically reduced the sensitivity of the receptor to IVM and significantly increased the receptor subunits’ cooperativity in response to IVM. We suggest that this mutation reduces the efficacy of channel gating, and impairs the integrity of the IVM-binding pocket, likely by disrupting important interactions between the tip of M1 and the M2-M3 loop of an adjacent subunit. We hypothesize that this physical contact between M1 and the M2-M3 loop tunes the relative orientation of the ion-channel transmembrane helices M1, M2 and M3 to optimize pore opening. Interestingly, pre-exposure of the GluClαF276A/βWT mutant receptor to subthreshold IVM concentration recovered the receptor sensitivity to glutamate. We infer that IVM likely retained its positive modulation activity by constraining the transmembrane helices in a preopen orientation sensitive to glutamate, with no need for the aforementioned disrupted interactions between M1 and the M2-M3 loop.

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...