rat 42 and mouse ( 1)21 ion channels were expressed in xenopus laevis oocytes . for 42 receptors , subunit stoichiometry was controlled by varying the 4:2 subunit ratio and verified by voltage jump experiments .
dose - response measurements for these channels were performed with a holding potential of 60 mv .
unnatural amino acids and -hydroxy acids were prepared , coupled to dca and ligated to 74-mer thg73 as described previously15 .
single - channel recording was performed in the cell - attached configuration with a pipette potential of + 100 mv as described previously27 .
popen values were calculated from event - detected data using clampfit 9.2 single - channel search .
full methods and any associated references are available in the online version of the paper at www.nature.com/nature .
rat 42 and mouse ( 1)21 ion channels were expressed in xenopus laevis oocytes . for 42 receptors , subunit stoichiometry was controlled by varying the 4:2 subunit ratio and verified by voltage jump experiments .
dose - response measurements for these channels were performed with a holding potential of 60 mv .
unnatural amino acids and -hydroxy acids were prepared , coupled to dca and ligated to 74-mer thg73 as described previously15 .
single - channel recording was performed in the cell - attached configuration with a pipette potential of + 100 mv as described previously27 .
popen values were calculated from event - detected data using clampfit 9.2 single - channel search .
full methods and any associated references are available in the online version of the paper at www.nature.com/nature .
 nicotine addiction begins with high - affinity binding of nicotine to acetylcholine ( ach ) receptors in the brain . 
 the end result is over 4,000,000 smoking - related deaths annually worldwide and the largest source of preventable mortality in developed countries . 
 stress reduction , pleasure , improved cognition , and other cns effects are strongly associated with smoking . 
 but , if nicotine activated ach receptors found in muscle as potently as it does brain receptors , smoking would cause intolerable and perhaps fatal muscle contractions . despite extensive pharmacological , functional , and structural studies of ach receptors , 
 the basis for the differential action of nicotine on brain vs. muscle ach receptors has not been determined . 
 here we show that at the 42 brain receptors thought to underlie nicotine addiction , the high affinity of nicotine is the result of a strong cation- interaction to a specific aromatic amino acid of the receptor , trpb . 
 in contrast , the low affinity of nicotine at the muscle - type receptor is largely due to the fact that this key interaction is absent , even though the immediate binding site residues , including the key trpb , are identical in the brain and muscle receptors . at the same time 
 a hydrogen bond from nicotine to the backbone carbonyl of trpb is enhanced in the neuronal receptor relative to the muscle - type . a point mutation near trpb that differentiates 42 and muscle - type receptors appears to influence the shape of the binding site , allowing nicotine to interact more strongly with trpb in the neuronal receptor . 
 ach receptors are established therapeutic targets for alzheimer s disease , schizophrenia , parkinson s disease , smoking cessation , pain , attention deficit - hyperactivity disorder , epilepsy , autism , and depression1 . along with solving a chemical mystery in nicotine addiction , 
 our results provide guidance for efforts to develop drugs that target specific types of nicotinic receptors . 
Methods Summary
Whole-cell electrophysiological characterization of agonist-induced responses
Unnatural amino acid / -hydroxy acid incorporation
Single-channel characterization of 42
Supplementary Material