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The Journal of Neuroscience, October 12, 2005, 25(41):9358-9366; doi:10.1523/JNEUROSCI.1045-05.2005

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Cellular/Molecular
A Role for the {beta}1-{beta}2 Loop in the Gating of 5-HT3 Receptors

David C. Reeves,1 Michaela Jansen,1 Moez Bali,1 Thomas Lemster,2 and Myles H. Akabas1

1Department of Physiology and Biophysics and Department of Neuroscience, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, and 2Department of Medicinal Chemistry, Institute of Pharmacy, Johannes Gutenberg University, D-55099 Mainz, Germany

Based on the Torpedo acetylcholine receptor structure, Unwin and colleagues (Miyazawa et al., 2003; Unwin, 2005) hypothesized that the transduction of agonist binding to channel gate opening involves a "pin-into-socket" interaction between {alpha}V46 at the tip of the extracellular {beta}1-{beta}2 loop and the transmembrane M2 segment and M2-M3 loop. We mutated to cysteine the aligned positions in the 5-HT3A and 5-HT3B subunit {beta}1-{beta}2 loops K81 and Q70, respectively. The maximal 5-HT-activated currents in receptors containing 5-HT3A/K81C or 5-HT3B/Q70C were markedly reduced compared with wild type. Desensitization of wild-type currents involved fast and slow components. Mutant currents desensitized with only the fast time constant. Reaction with several methanethiosulfonate reagents potentiated currents to wild-type levels, but reaction with other more rigid thiol-reactive reagents caused inhibition. Single-channel conductances of wild type, K81C, and K81C after modification were similar. We tested the proximity of K81C to the M2-M3 loop by mutating M2-M3 loop residues to cysteine in the K81C background. Disulfide bonds formed in 5-HT3A/K81C/A304C and 5-HT3A/K81C/I305C when coexpressed with 5-HT3B. We conclude that in the resting state, K81 is not in a hydrophobic pocket as suggested by the pin-into-socket hypothesis. K81 interacts with the extracellular end of M2 and plays a critical role in channel opening and in the return from fast desensitization. We suggest that during channel activation, {beta}1-{beta}2 loop movement moves M2 and the M2-M3 loop so that the M2 segments rotate/translate away from the channel axis, thereby opening the lumen. Recovery from fast desensitization requires the interaction between K81 and the extracellular end of M2.

Key words: ion channel; SCAM; acetylcholine; GABA; serotonin; cross-linking

Received March 17, 2005; revised August 25, 2005; accepted August 25, 2005.




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