The molecular mechanisms that couple agonist binding to the gating of Cys-loop ionotropic receptors are not well understood. The crystal structure of the acetylcholine (ACh) binding protein has provided insights into the structure of the extracellular domain of nicotinic receptors and a framework for testing mechanisms of activation. Key ligand binding residues are located at the C-terminal end of the ␤9 strand. At the N-terminal end of this strand (loop 9) is a conserved glutamate [E 172 in chick ␣7 nicotinic acetylcholine receptors (nAChRs)] that is important for modulating activation. We hypothesize that agonist binding induces the movement of loop 9. To test this, we used the substituted-cysteine accessibility method to examine agonist-dependent changes in the modification of cysteines introduced in loop 9 of L 247 T ␣7 nAChRs. In the absence of agonist, ACh-evoked responses of E 172 C/L 247 T ␣7 nAChRs were inhibited by 2-trimethylammonioethylmethane thiosulfonate (MTSET). Agonist coapplication with MTSET reduced the extent and rate of modification. The dose-dependence of ACh activation was nearly identical with that of ACh-dependent protection from modification. ACh increased the inhibition by methanethiosulfonate reagents of N 170 C and did not change inhibition of G 171 C receptors. The antagonist dihydro-␤-erythroidine did not mimic the effects of ACh. Combined with a structural model, the data suggest that receptor activation includes subunit rotation and/or intrasubunit conformational changes that move N 170 to a more accessible position and E 172 to a more protected position away from the vestibule. Thus, loop 9, located near the junction between the extracellular and transmembrane domains, participates in conformational changes triggered by ligand binding.

Neuronal alpha(7) nicotinic acetylcholine receptors (nAChRs) are permeable to Ca(2+) and other divalent cations. We characterized the modulation of the pharmacological properties of nondesensitizing mutant (L(247)T and S(240)T/L(247)T) alpha(7) nAChRs by permeant (Ca(2+), Ba(2+), and Sr(2+)) and impermeant (Cd(2+) and Zn(2+)) divalent cations. alpha(7) receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh, whereas impermeant divalent cations decreased potency and maximal efficacy. The antagonist dihydro-beta-erythroidine (DHbetaE) was a strong partial agonist of L(247)T and S(240)T/L(247)T alpha(7) receptors in the presence of divalent cations but was a weak partial agonist in the presence of impermeant divalent cations. Mutation of the "intermediate ring" glutamates (E(237)A) in L(247)T alpha(7) nAChRs eliminated Ca(2+) conductance but did not alter the Ca(2+)-dependent increase in ACh potency, suggesting that site(s) required for modulation are on the extracellular side of the intermediate ring. The difference between permeant and impermeant divalent cations suggests that sites within the pore are important for modulation by divalent cations.

. These permeant divalent cations interact with slowly desensitizing L 247 T ␣7 nAChRs to increase the potency and maximal efficacy of ACh, increase the efficacy of dihydro-␤-erythroidine (DH␤E), and increase agonistindependent activity. Mutation of glutamate 172 (E 172 ) to glutamine or cysteine eliminated these effects of permeant divalent cations. 2-(Trimethylammonium)ethyl methanethiosulfonate (MTSET), a cysteine-modifying reagent directed at water-accessible thiols, inhibited ACh-evoked currents of E 172 C/L 247 T ␣7 nAChRs by Ͼ90%, demonstrating that E 172 was accessible to permeant ions. The data are consistent with a model of ␣7 receptors, derived from the crystal structure of the ACh binding protein (AChBP) from Lymnaea stagnalis, in which E 172 projects toward the lumen of the extracellular vestibule. The observations that E 172 was essential for divalent cation modulation of L 247 T ␣7 nAChRs and was accessible to permeating ions suggest that this residue participates in coupling ion permeation with modulation of receptor activity.

The ␣7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that modulates neurotransmitter release in the central nervous system. We show here that functional, homo-oligomeric ␣7 nAChRs can be synthesized in vitro with a rabbit reticulocyte lysate translation system supplemented with endoplasmic reticulum microsomes, reconstituted into planar lipid bilayers, and evaluated using single-channel recording techniques. Because wild-type ␣7 nAChRs desensitize rapidly, we used a nondesensitizing form of the ␣7 receptor with mutations in the second transmembrane domain (S2T and L9T) to record channel activity in the continuous presence of agonist. Endoglycosidase H treatment of microsomes containing nascent ␣7 S2T/L9T nAChRs indicated that the receptors were glycosylated. A proteinase K protection assay revealed a 36-kDa fragment in the ER lumen, consistent with a large extracellular domain predicted by most topological models, indicating that the protein was folded integrally through the ER membrane. ␣7 S2T/L9T receptors reconstituted into planar lipid bilayers had a unitary conductance of ϳ50 pS, were highly selective for monovalent cations over Cl ؊ , were nonselective between K ؉ and Na ؉ , and were blocked by ␣-bungarotoxin. This is the first demonstration that a functional ligand-gated ion channel can be synthesized using an in vitro expression system. The nicotinic acetylcholine receptor (nAChR)1 is a member of a superfamily of ligand-gated ion channels that also includes GABA A receptors, serotonin (5-HT 3 ) receptors, glycine receptors, and an invertebrate glutamate-gated chloride channel (1). Nicotinic receptors are located at the neuromuscular junction and in the central and peripheral nervous systems. Muscletype nAChRs are pentamers of homologous subunits in the stoichiometry of ␣ 2 ␤␥␦ (or ␣ 2 ␤␦⑀) arranged around a central pore (2). Neuronal nAChRs also form pentameric complexes (3, 4) from various combinations of the 11 neuronal nAChR genes (␣2-␣9 and ␤2-␤4) that have been identified to date (5). ␣7, ␣8, and ␣9 nAChRs are blocked by the snake peptide toxin ␣-bungarotoxin (␣-BTX), which also blocks muscle and Torpedo nAChRs but not other subtypes of neuronal nAChRs (5). ␣7 nAChRs are the most abundantly expressed nicotinic receptor subunit in the central nervous system (6) and are important in neuronal development, hippocampal function, and the modulation of fast neurotransmission (7,8). ␣7 nAChRs are highly calcium-permeable (9, 10), and calcium influx through presynaptic ␣7 nAChRs modulates the release of excitatory neurotransmitters (11,12).During biosynthesis of nAChRs, the polypeptide is translocated into the endoplasmic reticulum (ER) membrane. The ER contains enzymes necessary for signal sequence cleavage (13) and other post-translational modifications required for correct subunit folding, assembly, and ligand-binding site formation. These modifications include core glycosylation (13) and disulfide bond formation (14, 15). In addition, ER and cytoplasmic chaperone proteins are tho...

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