Title The effective opening of nicotinic acetylcholine receptors with single agonist binding sites Running title nAChR activation with single binding sites Authors and addresses

We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of α7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, while corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We co-expressed α7L119C subunits with pseudo-wild-type α7C116S subunits, as well as ACh-insensitive α7Y188F subunits with wild-type α7 subunits in Xenopus oocytes using varying ratios of cRNA. When mutant α7 cRNA was co-injected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 µM ACh were retained by α7L119C-containing mutants after MTSEA modification and by the ACh-insensitive Y188F-containing mutants, even though the expected number of ACh-sensitive wild-type binding sites would on average be less than two per receptor. Responses of muscle-type receptors with one MTSEA-sensitive subunit were reduced at low ACh concentrations, but much less of an effect was observed when acetylcholine concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic acetylcholine receptors. Single-channel patch-clamp analysis revealed the burst-durations of fetal wild-type and α1β1γδL121C receptors were equivalent until the α1β1γδL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (≤ 2 ms). The longest duration events of the modified receptors with single activatible binding sites were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists. clamp Williams et al.