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J. Biol. Chem., Vol. 283, Issue 28, 19301-19313, July 11, 2008

This Article Free via Author's Choice: AC AC Full Text Full Text (PDF) Supplemental Data AC All Versions of this Article: 283/28/19301    most recent M802406200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Livesey, M. R. Articles by Peters, J. A. PubMed PubMed Citation Articles by Livesey, M. R. Articles by Peters, J. A. Social Bookmarking                      What's this?

Structural Determinants of Ca²⁺ Permeability and Conduction in the Human 5-Hydroxytryptamine Type 3A Receptor^*

Matthew R. Livesey¹, Michelle A. Cooper, Tarek Z. Deeb, Jane E. Carland, Janna Kozuska, Tim. G. Hales^¶, Jeremy J. Lambert², and John A. Peters²³

From the Neurosciences Institute, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom and the Departments of Pharmacology and Physiology and ^¶Anesthesiology and Critical Care Medicine, George Washington University, Washington, D. C. 20037

Cation-selective cysteine (Cys)-loop transmitter-gated ion channels provide an important pathway for Ca²⁺ entry into neurones. We examined the influence on Ca²⁺ permeation of amino acids located at intra- and extracellular ends of the conduction pathway of the human 5-hydroxytryptamine type 3A (5-HT_3A) receptor. Mutation of cytoplasmic arginine residues 432, 436, and 440 to glutamine, aspartate, and alanine (the aligned residues of the human 5-HT_3B subunit (yielding 5-HT_3A(QDA)) increased P_Ca/P_Cs from 1.4 to 3.7. The effect was attributable to the removal of an electrostatic influence of the Arg-436 residue. Despite its relatively high permeability to Ca²⁺, the single channel conductance of the 5-HT_3A(QDA) receptor was depressed in a concentration-dependent and voltage-independent manner by extracellular Ca²⁺. A conserved aspartate, located toward the extracellular end of the conduction pathway and known to influence ionic selectivity, contributed to the inhibitory effect of Ca²⁺ on macroscopic currents mediated by 5-HT_3A receptors. We introduced a D293A mutation into the 5-HT_3A(QDA) receptor (yielding the 5-HT_3A(QDA D293A) construct) to determine whether the aspartate is required for the suppression of single channel conductance by Ca²⁺. The D293A mutation decreased the P_Ca/P_Cs ratio to 0.25 and reduced inwardly directed single channel conductance from 41 to 30 pS but did not prevent suppression of single channel conductance by Ca²⁺. The D293A mutation also reduced P_Ca/P_Cs when engineered into the wild-type 5-HT_3A receptor. The data helped to identify key residues in the cytoplasmic domain (Arg-436) and extracellular vestibule (Asp-293) that markedly influence P_Ca/P_Cs and additionally directly demonstrated a depression of single channel conductance by Ca²⁺.

Received for publication, March 27, 2008 , and in revised form, May 8, 2008.

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^* This work was supported in part by grants from the Wellcome Trust (to J. J. L. and J. A. P.), from Tenovus Scotland and the Anonymous Trust (to J. A. P.), and from the National Science Foundation (to T. G. H.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Supported by a Biotechnology and Biological Sciences Research Council-Case studentship in conjunction with Eli Lilly and Co.

² Supported by a grant from Eli Lilly and Co.

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³ To whom correspondence should be addressed: Neurosciences Inst., Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, The University of Dundee, Dundee DD19SY, Scotland, United Kingdom. Tel.: 44-1382-660111; Fax: 44-1382-667120; E-mail: j.a.peters{at}dundee.ac.uk.

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