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J. Biol. Chem., Vol. 279, Issue 27, 28149-28158, July 2, 2004

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Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT_3A Receptor^*

Sandip Panicker, Hans Cruz¶, Christine Arrabit, Ka Fai Suen, and Paul A. Slesinger||

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

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT_3A) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd²⁺) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd²⁺ when applied extracellularly in the open state. Cd²⁺ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd²⁺ in the pore. When applied intracellularly, Cd²⁺ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and –2' positions to coordinate Cd²⁺ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT_3A receptors maintains a narrow pore during channel gating.

Received for publication, March 31, 2004 , and in revised form, May 6, 2004.

^* This work was supported by a National Institutes of Health training grant (to S. P.), the Legler Benbough Foundation (to S. P.), the Sloan Foundation (to P. A. S.), the McKnight Endowment for Neuroscience (to P. A. S.), and the Fritz-Burns Foundation (to P. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Current address: Dept. of Basic Neurosciences, University of Geneva, Switzerland, CH-1211.

^|| To whom correspondence should be addressed: Peptide Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Rd. La Jolla, CA 92037. Tel.: 858-453-4100 (ext. 1560); Fax: 858-552-1546; E-mail: slesinger{at}salk.edu.

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