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The Journal of Neuroscience, March 1, 2002, 22(5):1629-1639

Evidence for a Centrally Located Gate in the Pore of a Serotonin-Gated Ion Channel

Sandip Panicker^{2, *}, Hans Cruz^{1, *}, Christine Arrabit¹, and Paul A. Slesinger^{1, 2}

¹ The Salk Institute for Biological Studies, La Jolla, California 92037, and ² Neurosciences Graduate Program, University of California, San Diego, La Jolla, California 92093

Serotonin-gated ion channels (5-HT₃) are members of the ligand-gated channel family, which includes channels that are opened directly by the neurotransmitter acetylcholine, GABA, glycine, or glutamate. Although there is general agreement that the second transmembrane domain (M2) lines the pore, the position of the gate in the M2 is less certain. Here, we used substituted cysteine accessibility method (SCAM) to provide new evidence for a centrally located gate that moves during channel activation. In the closed state, three cysteine substitutions, located on the extracellular side of M2, were modified by methanethiosulfonate (MTS) reagents. In contrast, 13 cysteine substitutions were modified in the open state with MTS reagents. The pattern of inhibition (every three to four substitutions) was consistent with an  helical structure for the middle and cytoplasmic segments of the M2 transmembrane domain. Unexpectedly, open-state modification of two amino acids in the center of M2 with three different MTS reagents prevented channels from fully closing in the absence of neurotransmitter. Our results are consistent with a model in which the central region of the M2 transmembrane domain is inaccessible in the closed state and moves during channel activation.

Key words: 5-HT₃R; substituted cysteine accessibility method; serotonin; gating; ligand-gated ion channel; ion channel structure; ionotropic receptor

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^* S.P. and H.C. contributed equally to this work.

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Copyright © 2002 Society for Neuroscience  0270-6474/02/2251629-11$05.00/0

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