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Effects of inserting fluorescent proteins into the alpha1S II-III loop: insights into excitation-contraction coupling.

ABSTRACT: 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 Ca(2+) release via RYR1, and retrograde coupling is manifested by increased L-type Ca(2+) current via DHPR. A critical domain (residues 720-765) of the DHPR alpha(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 alpha(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 Ca(2+) 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 alpha(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 alpha(1S) II-III loop. Significantly, our results indicate that the conserved portion of the alpha(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.

SUBMITTER: Bannister RA

PROVIDER: S-EPMC2712974 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Effects of inserting fluorescent proteins into the alpha1S II-III loop: insights into excitation-contraction coupling.

Bannister Roger A RA Papadopoulos Symeon S Haarmann Claudia S CS Beam Kurt G KG

The Journal of general physiology 20090701 1

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 Ca(2+) release via RYR1, and retrograde coupling is manifested by increased L-type Ca(2+) current via DHPR. A critical domain (residues 720-765) of the DHPR alpha(1S) II-III loop plays an important but poorly understood role in bidirectional coupling with RYR1. In this study ...[more]

PMID: 19564426

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Project description:In skeletal muscle, excitation-contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca(2+) channel (Ca(V)1.1) to be communicated to the type 1 ryanodine-sensitive Ca(2+) release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein-protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca(V)1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing ?(1S) subunit of Ca(V)1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary ?(1a) subunit of Ca(V)1.1 is a conduit for this intermolecular communication. However, a direct role for ?(1a) has been difficult to test because ?(1a) serves two other functions that are prerequisite for conformational coupling between Ca(V)1.1 and RYR1. Specifically, ?(1a) promotes efficient membrane expression of Ca(V)1.1 and facilitates the tetradic ultrastructural arrangement of Ca(V)1.1 channels within plasma membrane-SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established ? subunit-interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca(2+) transients without greatly affecting Ca(V)1.1 targeting, intramembrane gating charge movement, or releasable SR Ca(2+) store content. In contrast, a ?(1a)-binding-deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca(2+) release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca(V)1.1 from RYR1-mediated SR Ca(2+) release via its ability to interact with ?(1a). Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the ?(1a) subunit in skeletal-type EC coupling.

Dataset Information

Effects of inserting fluorescent proteins into the alpha1S II-III loop: insights into excitation-contraction coupling.

Publications

Effects of inserting fluorescent proteins into the alpha1S II-III loop: insights into excitation-contraction coupling.

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