Biomedical subjects

Valérie Leuranguer

Publications and source records attributed to Valérie Leuranguer.

3 recordsLinked to original sources

PubMed2005

Organization of calcium channel beta1a subunits in triad junctions in skeletal muscle.

In skeletal muscle, dihydropyridine receptors (DHPRs) in the plasma membrane interact with the type 1 ryanodine receptor (RyR1) at junctions with the sarcoplasmic reticulum. This interaction organizes junctional DHPRs into groups of four termed tetrads. In addition to the principle alpha1S subunit, the beta1a subunit of the DHPR is also important for the interaction with RyR1. To probe this interaction, we measured fluorescence resonance energy transfer (FRET) of beta1a subunits labeled with cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP). Expressed in dysgenic (alpha1S-null) myotubes, YFP-beta1a-CFP and CFP-beta1a-YFP were diffusely distributed in the cytoplasm and highly mobile as indicated by fluorescence recovery after photobleaching. Thus, beta1a does not appear to bind to other cellular proteins in the absence of alpha1S. FRET efficiencies for these cytoplasmic beta1a subunits were approximately 6-7%, consistent with the idea that <10 nm separates the N and C termini. After coexpression with unlabeled alpha1S (in dysgenic or beta1-null myotubes), both constructs produced discrete fluorescent puncta, which correspond to assembled DHPRs in junctions and that did not recover after photobleaching. In beta1-null myotubes, FRET efficiencies of doubly labeled beta1a in puncta were similar to those of the same constructs diffusely distributed in the cytoplasm and appeared to arise intramolecularly, since no FRET was measured when mixtures of singly labeled beta1a (CFP or YFP at the N or C terminus) were expressed in beta1-null myotubes. Thus, DHPRs in tetrads may be arranged such that the N and C termini of adjacent beta1a subunits are located >10 nm from one another.

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PubMed2004

Mapping sites of potential proximity between the dihydropyridine receptor and RyR1 in muscle using a cyan fluorescent protein-yellow fluorescent protein tandem as a fluorescence resonance energy transfer probe.

Excitation-contraction coupling in skeletal muscle involves conformational coupling between the dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) at junctions between the plasma membrane and sarcoplasmic reticulum. In an attempt to find which regions of these proteins are in close proximity to one another, we have constructed a tandem of cyan and yellow fluorescent proteins (CFP and YFP, respectively) linked by a 23-residue spacer, and measured the fluorescence resonance energy transfer (FRET) of the tandem either in free solution or after attachment to sites of the alpha1S and beta1a subunits of the DHPR. For all of the sites examined, attachment of the CFP-YFP tandem did not impair function of the DHPR as a Ca2+ channel or voltage sensor for excitation-contraction coupling. The free tandem displayed a 27.5% FRET efficiency, which decreased significantly after attachment to the DHPR subunits. At several sites examined for both alpha1S (N-terminal, proximal II-III loop of a two fragment construct) and beta1a (C-terminal), the FRET efficiency was similar after expression in either dysgenic (alpha1S-null) or dyspedic (RyR1-null) myotubes. However, compared with dysgenic myotubes, the FRET efficiency in dyspedic myotubes increased from 9.9 to 16.7% for CFP-YFP attached to the N-terminal of beta1a, and from 9.5 to 16.8% for CFP-YFP at the C-terminal of alpha1S. Thus, the tandem reporter suggests that the C terminus of alpha1S and the N terminus of beta1a may be in close proximity to the ryanodine receptor.

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PubMed2003

Potentiated L-type Ca2+ channels rectify.

Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type alpha1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-alpha1C) or repeat III (E3K-alpha1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-alpha1C and E3K-alpha1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (-)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-alpha1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-alpha1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels.

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