Probing Structure on Well-defined Functional States of the Nicotinic Receptor Using Systematically-engineered Ionizable Residues and Proton-transfer Events

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The conformational changes that underlie the closed-open transition in members of the nicotinic-receptor superfamily remain elusive and controversial. To gain insight into the structural properties of the pore-domain of the muscle-nicotinic acetylcholine-receptor channel (AChR) in the open state, while retaining the advantages of studies on intact cells and in real time, we engineered basic residues along the M1, M2, and M3 transmembrane segments of all four types of subunit and recorded the individual proton-transfer events using single-channel patch-clamp electrophysiology. Proton binding-unbinding reactions to and from individual side chains were manifest as blocking-unblocking events of the passing cation current. Two observables, namely, the extent to which the current is attenuated upon side-chain protonation, and the pKa-shifts of the engineered ionizable groups relative to bulk water, were analyzed to reveal the electrostatic properties of the local microenvironment around the transmembrane segments in the open-channel conformation. In turn, these data were interpreted in terms of secondary and tertiary structure, and compared with existing structural models of the closed state in order to elucidate the change in conformation that opens the AChR. Our open-channel data suggests that the orientation of the M1, M2, and M3 transmembrane segments of the AChR with respect to the pore and each other is very similar to that in the closed-channel structural model developed on the basis of the cryo-EM images or Torpedo's receptor at 4-A resolution. To the extent that this structural model corresponds to the actual closed-channel conformation, our results indicate that the expansion of the pore that underlies channel opening involves only a limited rearrangement of these three helices. Such a modest change seems optimal to ensure rapid closed-open interconversion rates, and hence, a fast postsynaptic response upon neurotransmitter-binding.