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J. Biol. Chem., Vol. 283, Issue 44, 29740-29752, October 31, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/44/29740    most recent M802856200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted 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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lo, W.-y. Articles by Macdonald, R. L. Search for Related Content PubMed PubMed Citation Articles by Lo, W.-y. Articles by Macdonald, R. L. Social Bookmarking                      What's this?

A Conserved Cys-loop Receptor Aspartate Residue in the M3-M4 Cytoplasmic Loop Is Required for GABA_A Receptor Assembly^*

Wen-yi Lo, Emmanuel J. Botzolakis, Xin Tang, and Robert L. Macdonald^¶||1

From the Program in Neuroscience and Departments of Neurology, ^¶Molecular Physiology and Biophysics, and ^||Pharmacology, Vanderbilt University, Nashville, Tennessee 37232

Members of the Cys-loop superfamily of ligand-gated ion channels, which mediate fast synaptic transmission in the nervous system, are assembled as heteropentamers from a large repertoire of neuronal subunits. Although several motifs in subunit N-terminal domains are known to be important for subunit assembly, increasing evidence points toward a role for C-terminal domains. Using a combination of flow cytometry, patch clamp recording, endoglycosidase H digestion, brefeldin A treatment, and analytic centrifugation, we identified a highly conserved aspartate residue at the boundary of the M3-M4 loop and the M4 domain that was required for binary and ternary -aminobutyric acid type A receptor surface expression. Mutation of this residue caused mutant and partnering subunits to be retained in the endoplasmic reticulum, reflecting impaired forward trafficking. Interestingly although mutant and partnering wild type subunits could be coimmunoprecipitated, analytic centrifugation studies demonstrated decreased formation of pentameric receptors, suggesting that this residue played an important role in later steps of subunit oligomerization. We thus conclude that C-terminal motifs are also important determinants of Cys-loop receptor assembly.

Received for publication, April 14, 2008 , and in revised form, July 17, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant NS33300 to (R. L. M.). 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 To whom correspondence should be addressed: Vanderbilt University Medical Center, 6140 Medical Research Bldg. III, 465, 21st Ave., Nashville, TN 37232-8552. Tel.: 615-936-2287; Fax: 615-322-5517; E-mail: robert.macdonald{at}vanderbilt.edu.

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