Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Full Text Full Text (PDF, 3822K) PDF+supp data (3904K) PPT slides of all figures Supplemental Material Index Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via CrossRef Google Scholar Articles by Bannister, R. A. Articles by Beam, K. G. PubMed PubMed Citation Articles by Bannister, R. A. Articles by Beam, K. G. Social Bookmarking                            What's this?

Effects of inserting fluorescent proteins into the _1S II–III loop: insights into excitation–contraction coupling

¹ Department of Physiology and Biophysics, School of Medicine, University of Colorado Denver, Aurora, CO 80045
² Department of Biomedical Sciences, Neurosciences Division, Colorado State University, Fort Collins, CO 80523

Correspondence to Kurt G. Beam: kurt.beam{at}ucdenver.edu

In skeletal muscle, intermolecular communication between the 1,4-dihydropyridine receptor (DHPR) and RYR1 is bidirectional: orthograde coupling (skeletal excitation–contraction coupling) is observed as depolarization-induced Ca²⁺ release via RYR1, and retrograde coupling is manifested by increased L-type Ca²⁺ current via DHPR. A critical domain (residues 720–765) of the DHPR _1S II–III loop plays an important but poorly understood role in bidirectional coupling with RYR1. In this study, we examine the consequences of fluorescent protein insertion into different positions within the _1S II–III loop. In four constructs, a cyan fluorescent protein (CFP)–yellow fluorescent protein (YFP) tandem was introduced in place of residues 672–685 (the peptide A region). All four constructs supported efficient bidirectional coupling as determined by the measurement of L-type current and myoplasmic Ca²⁺ transients. In contrast, insertion of a CFP–YFP tandem within the N-terminal portion of the critical domain (between residues 726 and 727) abolished bidirectional signaling. Bidirectional coupling was partially preserved when only a single YFP was inserted between residues 726 and 727. However, insertion of YFP near the C-terminal boundary of the critical domain (between residues 760 and 761) or in the conserved C-terminal portion of the _1S II–III loop (between residues 785 and 786) eliminated bidirectional coupling. None of the fluorescent protein insertions, even those that interfered with signaling, significantly altered membrane expression or targeting. Thus, bidirectional signaling is ablated by insertions at two different sites in the C-terminal portion of the _1S II–III loop. Significantly, our results indicate that the conserved portion of the _1S II–III loop C terminal to the critical domain plays an important role in bidirectional coupling either by conveying conformational changes to the critical domain from other regions of the DHPR or by serving as a site of interaction with other junctional proteins such as RYR1.

C.S. Haarmann’s present address is Nanion Technologies GmbH, 80335 Munich, Germany.

Abbreviations used in this paper: CFP, cyan fluorescent protein; DHPR, 1,4-dihydropyridine receptor; EC, excitation–contraction; YFP, yellow fluorescent protein.

© 2009 Bannister et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents