Neurotransmitter receptors from the Cys-loop superfamily couple the binding of agonist to the opening of an intrinsic ion pore in the final step in rapid synaptic transmission. Although atomic resolution structural data have recently emerged for individual binding and pore domains, how they are linked into a functional unit remains unknown. Here we identify structural requirements for functionally coupling the two domains by combining acetylcholine (ACh)-binding protein, whose structure was determined at atomic resolution, with the pore domain from the serotonin type-3A (5-HT3A) receptor. Only when amino-acid sequences of three loops in ACh-binding protein are changed to their 5-HT3A counterparts does ACh bind with low affinity characteristic of activatable receptors, and trigger opening of the ion pore. Thus functional coupling requires structural compatibility at the interface of the binding and pore domains. Structural modelling reveals a network of interacting loops between binding and pore domains that mediates this allosteric coupling process.

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The receptor chimera ␣7-5HT 3A has served as a prototype for understanding the pharmacology of ␣7 neuronal nicotinic receptors, yet its low single channel conductance has prevented studies of the activation kinetics of single receptor channels. In this study, we show that introducing mutations in the M3-M4 cytoplasmic linker of the chimera alters neither the apparent affinity for the agonist nor the EC 50 but increases the amplitude of agonist-evoked single channel currents to enable kinetic analysis. Channel events appear as single brief openings flanked by long closings or as bursts of several openings in quick succession. Both the open and closed time distributions are described as the sum of multiple exponential components, but these do not change over a wide range of acetylcholine (ACh), nicotine, or choline concentrations. Bursts elicited by a saturating concentration of ACh contain brief and long openings and closings, and a cyclic scheme containing two open and two closed states is found to adequately describe the data. The analysis indicates that once fully occupied, the receptor opens rapidly and efficiently, and closes and reopens several times before it desensitizes. Channel closing and desensitization occur at similar rates and account for the invariant open and closed time distributions.The Cys-loop superfamily of neurotransmitter receptors includes receptors activated by acetylcholine (ACh), GABA, glycine, and 5-HT. Each of these neurotransmitters activates a corresponding pentameric receptor composed of identical subunits (i.e., a homopentamer). Because homopentameric receptors diverged least from the common ancestral receptor, they are expected to share fundamental mechanisms common to all members of the receptor superfamily and thus serve as prototypes. The homomeric ␣7 ACh receptor contributes to a wide range of neurophysiological processes, has been implicated in neurological diseases, and is a target for pharmacological agents (Gotti and Clementi, 2004). Nevertheless, little is known about the mechanism by which ACh activates ␣7 receptors, mainly because they are difficult to express in mammalian cells. The limited cell surface expression was traced to decreased palmitoylation of ␣7 (Drisdel et al., 2004); more recently, however, the chaperone protein ric-3 was found to promote cell-surface expression. As a result, ACh-evoked whole cell currents have been recorded from human embryonic kidney cells coexpressing ␣7 and ric-3 (Williams et al., 2005), but single channel currents have not been reported. Furthermore, although ␣7 receptors express in oocytes, few studies have reported single-channel currents, and analysis of single channel current kinetics has not been reported. In addition, several conductance states of channels were observed in both wild-type and mutant ␣7 AChRs expressed in oocytes (Palma et al., 1999;Fucile et al., 2000), complicating kinetic analysis.Chimeric receptors, composed of ␣7 sequence from the N terminus to the start of M1 followed by 5HT 3A receptor sequence,...

Caenorhabditis elegans muscle contains seven different nicotinic receptor (AChR) subunits, five of which have been shown to be components of adult levamisole-sensitive AChRs (LAChRs). To elucidate the reason for such subunit diversity, we explore their functional roles in larva 1 (L1) muscle cells. Singlechannel and macroscopic current recordings reveal that the ␣-type LEV-8 subunit is a component of native L1 L-AChRs but behaves as a nonessential subunit. It plays a key role in maintaining a low rate and extent of desensitization of L-AChRs. In the absence of the ␣-type ACR-8 subunit, L-AChR channel properties are not modified, thus indicating that ACR-8 is not a component of L1 L-AChRs. Together with our previous findings, this study reveals that L1 muscle cells express a main L-AChR type composed of five different subunits: UNC-38, UNC-63, UNC-29, LEV-1, and LEV-8. Analysis of a double lev-8; acr-8-null mutant, which shows an uncoordinated and levamisole-resistant phenotype, reveals that ACR-8 can replace LEV-8 in its absence, thus attributing a functional role to this subunit. Docking into homology modeled L-AChRs proposes that ACh forms the typical cation-interaction, suggests why levamisole is less efficacious than ACh, and shows that ACR-8 can form activatable binding-sites, thus opening doors for elucidating subunit arrangement and anthelmintic selectivity.

Nicotinic acetylcholine receptors (nAChRs) are homo-or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in singlechannel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the 'levamisole-sensitive' nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER.