The KCNQ2 channel can exist in multiple resting conformations.

ion that effectively acts as a "foot in the door." We infer that, upon deactivation, the cytoplasmic side of the pore of the AChserotonin receptor chimera constricts to close the channel. Eukaryotic pLGICs3 (also called Cys-loop receptors) constitute a superfamily of transmembrane receptors located at the cell surface of excitable neuronal and muscle cells. There, they mediate rapid transport of ions, such as Nadown their electrochemical gradients to alter the membrane potential or to enable a rise in intracellular Ca 2ϩ . The five subunits of pLGICs are radially aligned around the axis of ion permeation pathway to form a channel activatable by neurotransmitters such as ACh, serotonin, ␥-aminobutyric acid, glycine, glutamate, or histamine that bind to an extracellular ligandbinding domain (LigBD) (Fig. 1A) (1-7).Although it is well accepted that loops located at the LigBDchannel interface mediate movements of the pore-lining helices (M2s) (8 -16), the specific M2 motions that open and close pLGICs are widely debated. Cryo-electron microscopy images of open and closed conformations of the nicotinic ACh receptor (nAChR) from Torpedo marmorata gave rise to the concept of gating motions, in which rotations along the longitudinal axis of the M2 segments open or close a mid-pore hydrophobic barrier (17, 18). State-dependent accessibilities of cysteines, histidines, or lysines substituted along the M2s to methanethiosulfonates, Zn 2ϩ or protons, have excluded channel gating via rotations (19 -23). It was further suggested that rigid body tilting (21) or small scale dilation (23) motions of the M2s gate the pore of an ACh-serotonin receptor chimera or a muscle nAChR, respectively. Several computational simulations suggested that the opening and closing of pLGICs depend on "global quaternary twist" motions (24, 25), rotations (26, 27), or rotations combined with either tilting (28) or bending vibrations (29) of the M2s. In contrast to these computational simulations, recent x-ray crystal structures of two different prokaryotic pLGICs, one displaying a closed pore conformation (30) and another with a potentially open pore conformation (31, 32), suggest that the pore-lining helices rigidly tilt to gate the channel. Yet, a major difference between the tilting gating motions suggested for the prokaryotic channels and those suggested for a eukaryotic pLGIC (21) emerges. In the case of the prokaryotic channels, the putative gating process involves the opening of a barrier to ions at the extracellular side of the pore (30 -32), whereas functional studies in eukaryotic pLGICs indicate that activation involves opening of a barrier located at the cytoplasmic side of the pore (19 -21, 33).Here, we used eukaryotic pLGICs that have an engineered capacity to coordinate a metal ion at the cytoplasmic side of their pore (21). In a previous study the accessibility of the engineered histidines to Zn 2ϩ ions was probed before or after activation (21), but in this study we detect Zn 2ϩ ion trapping inside the pore upon agonist dissociation....

Many proteinaceous macromolecules selectively transport substrates across lipid bilayers and effectively serve as gated nanopores. Here, we engineered cleavage-site motifs for human matrix metalloprotease 7 (MMP-7) into the extracellular and pore-constricting loops of OprD, a bacterial substrate-specific transmembrane channel. Concurrent removal of two extracellular loops allowed MMP-7 to access and hydrolyze a cleavage-site motif engineered within the pore's major constricting loop, in both membrane-incorporated and detergent-solubilized OprDs. Import of antibiotics by the engineered OprDs into living bacteria pointed to their proper folding and integration in biological membranes. Purified engineered OprDs were also found to be properly folded in detergent. Hence, this study demonstrates the design of nanopores with a constriction cleavable by tumor-secreted enzymes (like MMP-7) for their potential incorporation in lipid-based nanoparticles to accelerate drug release at the tumor site.

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...