Running title: Hydrogen bonding in 7 nAChR function

A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on the human 7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles) or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand binding domain residue Q57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Q57 and replace it with hydrogen bond donating, hydrogen bond accepting, or non-hydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or pyrroles than for their 3-substituted isomers, whereas the non-hydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596-agonist co-application data revealed that for wild-type 7 nAChR, the 3furan desensitized state was relatively stabilized compared to that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.