Ivermectin (IVM) is a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. By activating invertebrate pentameric glutamate-gated chloride channels (GluCl receptors; GluClRs), IVM induces sustained chloride influx and long-lasting membrane hyperpolarization that inhibit neural excitation in nematodes. Although IVM activates the C. elegans heteromeric GluClα/β receptor, it cannot activate a homomeric receptor composed of the C. elegans GluClβ subunits. To understand this incapability, we generated a homopentameric α7-GluClβ chimeric receptor that consists of an extracellular ligand-binding domain of an α7 nicotinic acetylcholine receptor known to be potentiated by IVM, and a chloride-selective channel domain assembled from GluClβ subunits. Application of IVM prior to acetylcholine inhibited the responses of the chimeric α7-GluClβR. Adding IVM to activated α7-GluClβRs, considerably accelerated the decline of ACh-elicited currents and stabilized the receptors in a non-conducting state. Determination of IVM association and dissociation rate constants and recovery experiments suggest that, following initial IVM binding to open α7-GluClβRs, the drug induces a conformational change and locks the ion channel in a closed state for a long duration. We further found that IVM also inhibits the activation by glutamate of a homomeric receptor assembled from the C. elegans full-length GluClβ subunits.

Receptors belonging to the Cys-loop gene superfamily of neurotransmittergated ion channels (e.g. acetylcholine, serotonin, GABA, and glycine recep-

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...