Biology subjects

Lesage, A.

Publications and source records attributed to Lesage, A..

5 recordsLinked to original sources

bioRxiv2023

FHF2 phosphorylation and regulation of native myocardial NaV1.5 channels

Phosphorylation of the cardiac NaV1.5 channel pore-forming subunit is extensive and critical in modulating channel expression and function, yet the regulation of NaV1.5 by phosphorylation of its accessory proteins remains elusive. Using a phosphoproteomic analysis of NaV channel complexes purified from mouse left ventricles, we identified nine phosphorylation sites on Fibroblast growth factor Homologous Factor 2 (FHF2). To determine the roles of phosphosites in regulating NaV1.5, we developed two models from neonatal and adult mouse ventricular cardiomyocytes in which FHF2 expression is knockdown and rescued by WT, phosphosilent or phosphomimetic FHF2-VY. While the increased rates of closed-state and open-state inactivation of NaV channels induced by the FHF2 knockdown are completely restored by the FHF2-VY isoform in adult cardiomyocytes, sole a partial rescue is obtained in neonatal cardiomyocytes. The FHF2 knockdown also shifts the voltage-dependence of activation towards hyperpolarized potentials in neonatal cardiomyocytes, which is not rescued by FHF2-VY. Parallel investigations showed that the FHF2-VY isoform is predominant in adult cardiomyocytes, while expression of FHF2-VY and FHF2-A is comparable in neonatal cardiomyocytes. Similar to WT FHF2-VY, however, each FHF2-VY phosphomutant restores the NaV channel inactivation properties in both models, preventing identification of FHF2 phosphosite roles. FHF2 knockdown also increases the late Na+ current in adult cardiomyocytes, which is restored similarly by WT and phosphosilent FHF2-VY. Together, our results demonstrate that ventricular FHF2 is highly phosphorylated, implicate differential roles for FHF2 in regulating neonatal and adult mouse ventricular NaV1.5, and suggest that the regulation of NaV1.5 by FHF2 phosphorylation is highly complex. eTOC SummaryLesage et al. identify the phosphorylation sites of FHF2 from mouse left ventricular NaV1.5 channel complexes. While no roles for FHF2 phosphosites could be recognized yet, the findings demonstrate differential FHF2-dependent regulation of neonatal and adult mouse ventricular NaV1.5 channels.

physiology↗

bioRxiv2021

Assessing the polymer coil-globule state from the very first spectral modes

The determination of the coil-globule transition of a polymer is generally based on the reconstruction of scaling laws, implying the need for samples from a rather wide range of different polymer lengths N. The spectral point of view developed in this work allows for a very parsimonious description of all the aspects of the finite-size coil-globule transition on the basis of the first two Rouse (cosine) modes only, shedding new light on polymer theory. Capturing the relevant configuration path features, the proposed approach enables to determine the state of a polymer without the need of any information about the polymer length or interaction strength. Importantly, we propose an experimental implementation of our analysis that can be easily performed with modern fluorescent imaging techniques, and would allow differentiation of coil or globule conformations by simply recording the positions of three discernible loci on the polymer.

biophysics↗

bioRxiv2020

Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation reveals multisite regulation of surface expression and gating

Phosphorylation of NaV1.5 channels regulates cardiac excitability, yet the phosphorylation sites regulating channel function and the underlying mechanisms remain largely unknown. Using a systematic quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from non-failing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines-664 and -667 regulate the voltage-dependence of channel activation in a cumulative manner, whereas phosphorylation of the nearby serine-671, which is increased in failing hearts, decreases cell surface NaV1.5 expression and peak Na+ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, the results demonstrate that ventricular NaV1.5 is highly phosphorylated, and that the phosphorylation-dependent regulation of NaV1.5-encoded channels is highly complex, site-specific and dynamic. AbbreviationsA, alanine; E, glutamate; HEK-293, Human Embryonic Kidney 293 cells; INa, peak Na+ current; INaL, late Na+ current; IP, immunoprecipitation; mNaVPAN, anti-NaV channel subunit mouse monoclonal antibody; MS, Mass Spectrometry; MS1, mass spectrum of peptide precursors; MS2 or MS/MS, fragmentation mass spectrum of peptides selected in narrow mass range (2 Da) from MS1 scan; NaV, voltage-gated Na+ channel; pS, phosphoserine; pT, phosphothreonine; S, serine; T, threonine; TAC, Transverse Aortic Constriction; TMT, Tandem Mass Tag.

physiology↗

bioRxiv2020

Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

The patch-clamp technique has contributed to major advances in the characterization of ion channels. The recent development of high throughput patch-clamp provides a new momentum to the field. However, whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that current amplitude profoundly impacts the precision of the analyzed characteristics of the ion current under study. For voltagegated channels, the higher the current amplitude is, the less precise the characteristics of voltagedependence are. Similarly, in ion channel pharmacology, the characteristics of dose-response curves are hindered by high current amplitudes. In addition, the recent development of high throughput patch-clamp technique is often associated with the generation of stable cell lines demonstrating high current amplitudes. It is therefore critical to set the limits for current amplitude recordings to avoid inaccuracy in the characterization of channel properties or drug actions, such limits being different from one channel to another. In the present study, we use kinetic models of a voltage-gated sodium channel and a voltage-gated potassium channel to edict simple guidelines for good practice of whole-cell voltage-clamp recordings.

biophysics↗

bioRxiv2018

Polymer coil-globule phase transition is a universal folding principle of Drosophila epigenetic domains

BackgroundLocalized functional domains within chromosomes, known as topologically associating domains (TADs), have been recently highlighted. In Drosophila, TADs are biochemically defined by epigenetic marks, this suggesting that the 3D arrangement may be the \"missing link\" between epigenetics and gene activity. Recent observations (Boettiger et al., Nature 2016) provide access to structural features of these domains with unprecedented resolution thanks to super-resolution experiments. In particular, they give access to the distribution of the radii of gyration for domains of different linear length and associated with different transcriptional activity states: active, inactive or repressed. Intriguingly, the observed scaling laws lack consistent interpretation in polymer physics.\n\nResultsWe develop a new methodology conceived to extract the best information from such super-resolution data by exploiting the whole distribution of gyration radii, and to place these experimental results on a theoretical framework. We show that the experimental data are compatible with the finite-size behavior of a self-attracting polymer. The same generic polymer model leads to quantitative differences between active, inactive and repressed domains. Active domains behave as pure polymer coils, while inactive and repressed domains both lie at the coil-globule crossover. For the first time, the \"colo-specificity\" of both the persistence length and the mean interaction energy are estimated, leading to important differences between epigenetic states.\n\nConclusionsThese results point toward a crucial role of criticality to enhance the system responsivity, resulting in both energy transitions and structural rearrangements. We get strong indications that epigenetically induced changes in nucleosome-nucleosome interaction can cause chromatin to shift between different activity states.

biophysics↗