In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on ␥-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.The Cys loop superfamily of neurotransmitter-gated ion channels plays a prominent role in mediating fast synaptic transmission. Receptors for acetylcholine (nicotinic ACh receptor, nAChR), 2 serotonin (5-HT 3 receptor), ␥-aminobutyric acid (GABA, types A and C receptors), and glycine are known, and the receptors are classified as excitatory (cation-conducting; nAChR and 5-HT 3 ) or inhibitory (anion-conducting; GABA and glycine). Malfunctions in these receptors are responsible for a number of "channelopathies," and the receptors are targets of pharmaceutical efforts toward treatments for a wide range of neurological disorders, including Alzheimer disease, Parkinson disease, addiction, schizophrenia, and depression (1, 2). The receptors share a common architecture, are significantly homologous, and are known to have evolved from a single ancestral gene that coded for an ACh receptor.The gating mechanism for the Cys loop superfamily is one of the most challenging questions in molecular neuroscience. At issue is how the binding of a small molecule neurotransmitter can induce a structural change in a large, multisubunit, integral membrane protein sufficient to open (gate) a previously closed ion channel contained within the receptor (3, 4). All evidence indicates that the neurotransmitter-binding site is quite remote (50 -60 Å) from the channel gate, the region that blocks the channel when the neurotransmitter is absent and that must move to open the channel.The quest for a gating mechanism has been greatly aided by several recent structural advances. First, crystal structures of the soluble acetylcholine-binding protein (AChBP) (5-7), which is homologous to the extracellular ...

Fig. 2. (A)Interaction web of top-down and bottom-up effects in the eelgrass study system. The top predator is the sea otter (E. lutris), the mesopredators are crabs (Cancer spp. and Pugettia producta), the epiphyte mesograzers are primarily an isopod (I. resecata) and a sea slug (P. taylori), and algal epiphyte competitors of eelgrass primarily consist of chain-forming diatoms, and the red alga Smithora naiadum. Solid arrows indicate direct effects, dashed arrows indicate indirect effects, and the plus and minus symbols indicate positive and/or negative effects on trophic guilds and eelgrass condition. C, competitive interaction; T, trophic interaction. (Original artwork by A. C. Hughes.) (B-E) Survey results testing for the effects of sea otter density on eelgrass bed community properties (Tables S2 and S3). Elkhorn Slough (sea otters present and high nutrients) eelgrass beds (n = 4) are coded in red, and the Tomales Bay reference site (no sea otters, low nutrients) beds (n = 4) are coded in blue. (B) Crab biomass and size structure of two species of Cancer crabs; (C) grazer biomass per shoot and large grazer density; (D) algal epiphyte loading; and (E) aboveground and belowground eelgrass biomass. DW, dry weight; FW, fresh weight.

Potent and selective antagonists of the voltage-gated sodium channel Na1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human Na1.7 channels with an IC of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other Na family members, including Na1.4 expressed in muscle and Na1.5 expressed in the heart, as well as TTX-resistant Na channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple Na1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective Na1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.

Neurotoxin receptor site-3 at voltage-gated Na(+) channels is recognized by various peptide toxin inhibitors of channel inactivation. Despite extensive studies of the effects of these toxins, their mode of interaction with the channel remained to be described at the molecular level. To identify channel constituents that interact with the toxins, we exploited the opposing preferences of LqhαIT and Lqh2 scorpion α-toxins for insect and mammalian brain Na(+) channels. Construction of the DIV/S1-S2, DIV/S3-S4, DI/S5-SS1, and DI/SS2-S6 external loops of the rat brain rNa(v)1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNa(v)1 channel (highly sensitive to LqhαIT), and examination of toxin activity on the channel chimera expressed in Xenopus oocytes revealed a substantial decrease in LqhαIT effect, whereas Lqh2 was as effective as at rNa(v)1.2a. Further substitutions of individual loops and specific residues followed by examination of gain or loss in Lqh2 and LqhαIT activities highlighted the importance of DI/S5-S6 (pore module) and the C-terminal region of DIV/S3 (gating module) of rNa(v)1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNa(v)1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNa(v)1.2a mutant channels has suggested that the toxin core domain interacts with the gating module of DIV. These results constitute the first step in better understanding of the way scorpion α-toxins interact with voltage-gated Na(+)-channels at the molecular level.

␥-Aminobutyric acid type A (GABA A ) receptors are members of the Cys-loop superfamily of ligand-gated ion channels. Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state, but the mechanism of gating is not well understood. Here we utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study the gating interface of the human homopentameric 1 GABA A receptor. We have identified an ion pair interaction between two conserved charged residues, Glu 92 in loop 2 of the extracellular domain and Arg 258 in the pre-M1 region. We hypothesize that the salt bridge exists in the closed state by kinetic measurements and free energy analysis. Several other charged residues at the gating interface are not critical to receptor function, supporting previous conclusions that it is the global charge pattern of the gating interface that controls receptor function in the Cys-loop superfamily.Fast inhibitory neurotransmission in the adult mammalian central nervous system is primarily mediated by the amino acid ␥-aminobutyric acid (GABA).2 So far, three types of GABA receptors have been identified, termed GABA A , GABA B , and the homopentameric 1 GABA A receptor, also known as GABA C (1, 2). Although GABA B is a G protein-coupled receptor, GABA A and GABA C receptors are homologous but distinct members of the Cys-loop superfamily of ligand-gated ion channels, which also includes the nicotinic acetylcholine (nAChR), serotonin, and glycine receptors. Members of this superfamily are composed of five subunits arranged around a central ionconducting pore, with each subunit consisting of a large extracellular domain, four transmembrane helices (M1-M4), and a large intracellular loop. The newest member of this family, the GABA C receptor (3), is expressed predominantly on retinal neurons, although recent studies indicate a wide distribution throughout the central nervous system (4 -6).The binding of agonist to a Cys-loop receptor triggers a complex structural transition that results in the opening of a "gate," allowing ions to flow through the channel (7). Identifying the linkage pathway has been limited by the lack of a complete atomic-resolution structure of any fast synaptic receptor. However, two breakthroughs have propelled the field into the structural age. The first is determination of the crystal structure of acetylcholine-binding protein (8), which is homologous to the extracellular domain of the nAChR and, by extension, all Cysloop receptors. This structural template provides critical insights into the nature of the binding site, but, of course, the ion channel and its gate are missing from such structures. Second, a refined electron microscopy structure of the Torpedo acetylcholine receptor by Unwin and co-workers (Protein Data Bank code 2BG9) has shed light onto the global structure and has suggested molecular determinants of functional mechanisms in Cys-loop receptors (9 -11).The available structural informati...