Paper Report for: Williams_2011_J.Gen.Physiol_137_369

We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of alpha7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, whereas corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We coexpressed alpha7L119C subunits with pseudo wild-type alpha7C116S subunits, as well as ACh-insensitive alpha7Y188F subunits with wild-type alpha7 subunits in Xenopus laevis oocytes using varying ratios of cRNA. When mutant alpha7 cRNA was coinjected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 microM ACh were retained by alpha7L119C-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 fewer 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 ACh concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic ACh receptors. Single-channel patch clamp analysis revealed that the burst durations of fetal wild-type and alpha1beta1gammadeltaL121C receptors were equivalent until the alpha1beta1gammadeltaL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (<=2 ms). The longest duration events of the receptors modified at only one binding site were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists.