Biology subjects

Marionneau, C.

Publications and source records attributed to Marionneau, C..

3 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↗

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↗