HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 45, 30668-30676, November 7, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/45/30668    most recent M805216200v1 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 Google Scholar Google Scholar Articles by Bal, M. Articles by Shapiro, M. S. Search for Related Content PubMed PubMed Citation Articles by Bal, M. Articles by Shapiro, M. S. Social Bookmarking                      What's this?

Homomeric and Heteromeric Assembly of KCNQ (Kv7) K⁺ Channels Assayed by Total Internal Reflection Fluorescence/Fluorescence Resonance Energy Transfer and Patch Clamp Analysis^*

From the Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229

M-type K⁺ channels, consisting of KCNQ1–5 (Kv7.1–7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent protein- and yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.

Received for publication, July 9, 2008 , and in revised form, August 28, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant R01 NS043394 (to M. S. S.). This work was also supported by an American Heart Association-Texas affiliate grant-in-aid (to M. S. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Physiology, MS 7756, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229. Tel.: 210-567-4328; Fax: 210-567-4410; E-mail: shapirom{at}uthscsa.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?