Strategy of the experiments. (A) Nicotine (pKa, 7.5–8.1) and varenicline (pKa, 9.5–10) are weak bases. They interconvert on a millisecond time scale between protonated and deprotonated forms; these are respectively membrane impermeant and permeant. (B) The tactic of confining a genetically encoded fluorescent nicotinic drug sensor to the PM or the ER. (C) Choline, ACh, and N’MeNic exist only as charged, membrane-impermeant forms near physiological pH.