Dose–response relations for nicotine-induced ΔF in HeLa cells. Exemplar data for iNicSnFR3a_PM and iNicSnFR3a_ER expressed in transfected HeLa cells. (A and C) 20-s nicotine dose application followed by 20-s wash in HBSS. The average response for three cells at each dose is overlaid in a 30-s window for the PM and ER traces in A and C, respectively. The SEM is shown as colored bands. (B and D) The averaged ΔF/F₀ at each response in A and C, respectively, is plotted against the logarithmic concentration scale with SEM given as error bars. (E) Comparisons among nicotine itself, N’MeNic, and ACh; experiments in transfected HeLa cells. The iNicSnFR3a_PM responses to ACh and to N’MeNic were normalized to NicSnFR3a_PM responses for nicotine (100 µM). The iNicSnFR3b_ER responses to ACh and to N’MeNic were normalized to iNicSnFR3b_ER responses for nicotine (100 µM). Note that iNIcSnFR3a_PM responds robustly to all three ligands, but only iNicSnFR3b_ER responds robustly only to nicotine, the only permeant molecule among the three tested. (F) Mutating a probable cation-π interacting residue, Tyr357, eliminates nicotine-induced ΔF. Exemplar data from cells transfected in parallel and tested on the same day.