Biology subjects

Guichard, S.

Publications and source records attributed to Guichard, S..

8 recordsLinked to original sources

bioRxiv2025

Biochemical assessment of α-α-subunit interactions of Nav1.5 in a heterologous expression system

Heterologous overexpression of any protein, and especially of the large transmembrane channel Nav1.5, could be associated with the insufficiency of endoplasmic reticulum folding machinery, hence leading to aspecific protein aggregation indistinguishable from the genuine --subunit interactions. In this study, we show that the interactions between heterologous Nav1.5 proteins depend on nascent N-linked glycosylation, are supported by non-native intermolecular disulfide bonds, and are likely predisposed to hydrophobic "stickiness". Particularly, we show strong interactions between the full-length Nav1.5 and its truncated peptides: N-terminal domain, all four transmembrane domains, as well as the intracellular linker between domains I and II. Taken together, we conclude that the heterologous expression system is not optimal for the identification of --subunit interaction sites of Nav1.5, and this question needs to be further addressed in the native tissues. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/679760v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@b29b7dorg.highwire.dtl.DTLVardef@1fe5909org.highwire.dtl.DTLVardef@1877990org.highwire.dtl.DTLVardef@13e04b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

All-optical Diamond Heater-Thermometer enables versatile and reliable thermal modulation of ion channels at the single-cell level

A living cell is a nonequilibrium thermodynamic system where, nevertheless, a notion of local equilibria exists. This notion applies to all micro- and nanoscale aqueous volumes, each containing a large number of molecules. This allows one to define sets of local conditions, including thermodynamic ones; for instance, a defined temperature requires thermodynamic equilibrium by definition. Once such a condition is fulfilled, one can control local variables and their gradients to theoretically describe the thermodynamic state of living systems at the micro- and nanoscale. Performing ultralocal experimental manipulations has become possible thanks to the patch clamp technique to control the cell membrane potential, as well as fluorescent imaging to monitor molecular concentrations and their intracellular gradients. However, precise temperature gradient control at the micro-/nanoscale has yet lacked a reliable experimental realization in a living cell. Here, we present a new methodology - microscale control of a temperature gradient profile in aqueous media by a fully optical Diamond Heater-Thermometer in a plug-and-play configuration combined with the patch clamp technique. In particular, we demonstrate applications of the combined Diamond Heater-Thermometer-patch clamp approach for the fast and reproducible thermal modulation of ionic current from voltage-gated Nav1.5 sodium channels expressed in HEK293 cells and freshly isolated ventricular mouse cardiomyocytes. Such an approach of manipulating the ultra-local temperature down to the nanoscale has the potential to uncover previously inaccessible phenomena in various physiological intracellular processes related to the endogenous nanoscale heat sources, such as open ion channels capable of producing Joule heat.

biophysics↗

bioRxiv2024

Identification of a Binding Site for Small Molecule Inhibitors Targeting Human TRPM4

Transient receptor potential (TRP) melastatin 4 (TRPM4) protein is a calcium-activated monovalent cation channel associated with various genetic and cardiovascular disorders. The anthranilic acid derivative NBA is a potent and specific TRPM4 inhibitor, but its binding site in TRPM4 has been unknown, although this information is crucial for drug development targeting TRPM4. We determined three cryo-EM structures of full-length human TRPM4 embedded in native lipid nanodiscs without inhibitor, bound to NBA, and a new anthranilic acid derivative, IBA. We found that the small molecules NBA and IBA were bound in a pocket formed between the S3, S4, and TRP helices and the S4-S5 linker of TRPM4. Our structural data and results from patch clamp experiments enable validation of a binding site for small molecule inhibitors, paving the way for further drug development targeting TRPM4.

biochemistry↗

bioRxiv2023

Amino acid substitution in the S1 or CH1-CH2 linker domain of TRPM4: Two new TRPM4 variants found in complete heart block patients lead to gain of expression and gain of current

Withdrawal Statement"The authors have withdrawn their manuscript owing to the following: In an effort to conduct blinded reproducibility experiments, they encountered difficulties in reproducing a portion of the data pertaining to the pathogenicity of the variants. While the clinical data presented in the manuscript are not affected, they are actively working to address and resolve these issues. The authors will resubmit the manuscript once they have ensured the accuracy and integrity of all the experimental data. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors."

molecular biology↗

bioRxiv2022

Role of 14-3-3 proteins in human cardiac sodium channel Nav1.5 regulation

Background14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial. AimsIn this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5. Methods and ResultsAmongst the seven 14-3-3 isoforms, only 14-3-3{eta} (encoded by YWHAH gene) weakly co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3{eta} overexpression or inhibition with difopein, and 14-3-3{eta} did not affect physical interaction between Nav1.5 - subunits. The current-voltage relationship and the amplitude of Nav1.5-mediated sodium peak current density were also not changed. ConclusionsOur findings illustrate that the direct implication of 14-3-3 proteins in regulating Nav1.5 is not evident in a transformed human kidney cell line tsA201. SummaryThis work shows that only 14-3-3{eta}, exhibits weak/transient interaction with Nav1.5, and does not modify its total protein expression, plasmalemmal trafficking, and basal biophysical properties of the whole-cell current. Furthermore, inhibition of endogenous 14-3-3/ligand interactions with difopein does not affect the dimerization of Nav1.5. Therefore, 14-3-3 proteins are suggested to be dispensable for the Nav1.5 regulation in a heterologous expression system.

biophysics↗

bioRxiv2022

Deletion of the ion channel Trpm4 increases cardiac inflammatory markers and fibrosis after myocardial infarction in mice

BACKGROUNDThe first cause of mortality worldwide is ischemic heart disease. In myocardial infarction (MI), the ischemic event causes cell death, which triggers a large inflammatory response responsible for removing necrotic material and inducing tissue repair. Endothelial cells, immune cells and fibroblasts play a key role in orchestrating this healing process. TRPM4 is a Ca2+-activated ion channel permeable to monovalent cations and its silencing or knocking out was shown to critically modify cellular functions of these non-myocytic cell types. OBJECTIVEOur aims were to 1) evaluate the role of TRPM4 on mice survival and cardiac function after MI; and 2) investigate the role of TRPM4 in the post-MI acute and chronic inflammatory response. METHODSWe performed ligation of the left anterior descending coronary artery or sham intervention on 154 Trpm4 WT or KO male mice and monitored survival for up to 5 weeks as well as cardiac function using echocardiography at 72h and five weeks. We drew blood at different acute time points (6h, 12h, 24h) and performed time-of-flight mass spectrometry to analyze the sera proteomes. Further, we sacrificed sub-groups of mice at 24h and 72h after surgery and performed single-cell RNA sequencing on the non-myocytic cells. Lastly, we assessed fibrosis and angiogenesis at five weeks using type I collagen and CD31 immunostaining respectively. RESULTSWe observed no significant differences in survival or cardiac function post-MI between both genotypes. However, our serum proteomics data showed significantly decreased tissue injury markers such as creatine kinase M and VE-Cadherin in KO compared to WT 12h after MI. On the other hand, inflammation characterized by serum amyloid P component in the serum, as well as higher number of recruited granulocytes, M1 macrophages, M1 monocytes, Mac-6 macrophages, and expression of pro-inflammatory genes such as Il1b, Lyz2 and S100a8/a9 was significantly higher in endothelial cells, macrophages and fibroblasts of KO than of WT. This correlated with increased cardiac fibrosis and angiogenesis 5 weeks after MI in KO. CONCLUSIONOur data suggest that knocking out Trpm4 drastically increases acute inflammation post-MI, is associated with increased chronic fibrosis and does not improve survival at 5 weeks post-MI. Thus, targeting TRPM4 in the context of MI should be pondered carefully and approaches that nuance the timing of the inhibition or cellular target may be required.

molecular biology↗

bioRxiv2019

A distinct pool of Nav1.5 channels at the lateral membrane of murine ventricular cardiomyocytes

BackgroundIn cardiac ventricular muscle cells, the presence of voltage-gated sodium channels Nav1.5 at the lateral membrane depends in part on the interaction between the dystrophin-syntrophin complex and the Nav1.5 C-terminal PDZ-domain-binding sequence Ser-Ile-Val (SIV motif). 1-Syntrophin, a PDZ-domain adaptor protein, mediates the interaction between Nav1.5 and dystrophin at the lateral membrane of cardiac cells. Using the cell-attached patch-clamp approach on cardiomyocytes expressing Nav1.5 in which the SIV motif is deleted ({Delta}SIV), sodium current (INa) recordings from the lateral membrane revealed an SIV-motif-independent INa. Since immunostainings have suggested that Nav1.5 is expressed in transverse (T-) tubules, this remaining INa might be conducted by channels in the T-tubules. Of note, a recent study using heterologous expression systems showed that 1-syntrophin also interacts with the Nav1.5 N-terminus, which may explain the SIV-motif independent INa at the lateral membrane of cardiomyocytes.\n\nAimTo address the role of 1-syntrophin in regulating the INa at the lateral membrane of cardiac cells.\n\nMethods and resultsPatch-clamp experiments in cell-attached configuration were performed on the lateral membranes of wild-type, 1-syntrophin knock-down, and {Delta}SIV ventricular mouse cardiomyocytes. Compared to wild-type, a reduction of the lateral INa was observed in myocytes from 1-syntrophin knockdown hearts. However, similar to {Delta}SIV myocytes, a remaining INa was still recorded. In addition, cell-attached INa recordings from lateral membrane did not differ significantly between non-detubulated and detubulated {Delta}SIV cardiomyocytes. Lastly, we obtained evidence suggesting that cell-attached patch-clamp experiments on the lateral membrane cannot record currents conducted by channels in T-tubules such as calcium channels.\n\nConclusionAltogether, these results suggest the presence of a sub-pool of sodium channels at the lateral membrane of cardiomyocytes that is independent of 1-syntrophin and the PDZ-binding motif of Na 1.5, located in membrane domains outside of T-tubules. The question of a T-tubular pool of Nav1.5 channels however remains open.

physiology↗

bioRxiv2019

Dystrophin and calcium current are decreased in cardiomyocytes expressing Cre enzyme driven by αMHC but not TNT promoter

BackgroundThe Cre/lox system is a potent technology to control gene expression in mouse tissues. However, cardiac alterations following cardiac-specific Cre enzyme expression in non-loxP-flanked genome heart have been reported. Recently, many loxP like sites have been identified in the wild-type mouse genome. Interestingly one of them is localized in the Dmd gene encoding the dystrophin protein known to be crucial for stabilization of cardiac voltage-gated ion channels Nav1.5 and Cav1.2. AimHere, we studied the potential alteration of dystrophin expression in adult alpha-myosin heavy chain (MHC)-Cre mice, which are extensively used for cardiac-specific recombination, and investigated Troponin T (TNT)-Cre mice as a potential alternative. MethodsCardiac-specific MHC-Cre and TNT-Cre mouse lines expressing Cre recombinase under the control of the cardiac-specific alpha-myosin-heavy chain, and rat cardiac troponin T2 promoter respectively were used. Western blots, quantitative RT-PCR, immunostainings, and patch-clamp experiments were performed to characterize MHC-Cre and TNT-Cre mouse hearts and cardiomyocytes. ResultsDystrophin protein level was decreased in hearts from 12-week-old MHC-Cre+ mice compared to MHC-Cre-. Reduction of dystrophin was more pronounced with age. No significant difference was observed between 8-week-old MHC-Cre+ mice and MHC-Cre-. Immunostainings performed on cardiac sections showed reduced dystrophin signal at the lateral membrane of MHC-Cre+ cardiomyocytes. Quantitative RT-PCR showed decreased mRNA levels of Dmd gene encoding dystrophin. Finally, patch-clamp experiments showed a significant decrease in calcium current (ICaL) in adult MHC-Cre+ cardiomyocytes compared to MHC-Cre-. Neither dystrophin nor ICaL was reduced in adult TNT-Cre+ mouse hearts compared to TNT-Cre-. ConclusionIn contrary to TNT-Cre+ mice, the sole expression of Cre recombinase can alter the cardiac phenotype of MHC-Cre+ mice. Thus, researchers should include the "Cre-only" condition as control condition when designing experiments with Cre mouse strains.

physiology↗