Biology subjects

Arullampalam, P.

Publications and source records attributed to Arullampalam, P..

6 recordsLinked to original sources

bioRxiv2025

Endothelial LRRC8C associates with LRRC8A and LRRC8B to regulate vascular reactivity and blood pressure

Vascular tone is impacted by the endotheliums ability to detect mechanical and chemical stimulation. Leucine-Rich Repeat-Containing protein 8A, (LRRC8A), was previously identified as a required component of the mechanoresponsive endothelial LRRC8 complex regulating AKT-endothelial nitric oxide synthase (eNOS) signaling and vascular function. While LRRC8A is broadly expressed, LRRC8B, C, D and E have tissue-restricted expression. Here, we identified 2 single nucleotide polymorphisms (SNPs) in LRRC8C highly associated with elevated diastolic and systolic blood pressure in human genetic studies, implicating LRRC8C as a regulator of vascular function. While LRRC8A/B/C/D/E are expressed in endothelium, co-immunoprecipitation experiments from lung endothelium using Lrrc8a-3xFlag knock-in mice, Lrrc8c-HA knock-in mice and endothelium-specific Lrrc8a-3xFlag overexpression mice demonstrate the endothelial LRRC8 complex to be composed largely of LRRC8A/B/C heteromers. Lrrc8a/b/c knock-out studies in mice and knock-down studies in human umbilical vein endothelial cells show co-dependent expression of LRRC8A/B/C proteins, but not LRRC8D. Functionally, LRRC8A and LRRC8C depletion reduces endothelial volume regulatory anion channel (VRAC) currents, inhibits AKT-eNOS signaling, increases myogenic tone, impairs eNOS dependent vasodilation, and exacerbates angiotensin-induced hypertension. These data identify LRRC8A, LRRC8B and LRRC8C as components of the endothelial LRRC8 complex and reveal LRRC8C as having a non-redundant role in regulating endothelial AKT-eNOS, vascular relaxation and susceptibility to hypertension.

cell biology↗

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

Knockdown of the TRPM4 channel alters cardiac electrophysiology and hemodynamics in a sex- and age-dependent manner in mice

BackgroundTRPM4 is a calcium-activated, voltage-modulated, non-selective ion channel widely expressed in various types of cells and tissues. TRPM4 regulates the influx of sodium ions, thus playing a role in regulating the membrane potential. In the heart, TRPM4 is expressed in both cardiomyocytes and cells of the conductive pathways. Clinical studies have linked TRPM4 mutations to several cardiac disorders. While data from experimental studies have demonstrated TRPM4s functional significance in cardiac physiology, its exact roles in the heart remain unclear. AimTo investigate the role of TRPM4 in cardiac physiology in a newly generated knockdown Trpm4 mouse model. Methods and resultsMale and female Trpm4 knockdown (Trpm4 -/-) and wild-type mice 5- to 12-weeks-old (young) or 24-week-old or more (adult) were characterized using a multimodal approach, encompassing surface electrocardiograms (ECG), echocardiography recordings, pseudo and intracardiac ECGs, western blots, and mRNA quantifications. The assessment of cardiac electrophysiology by surface ECGs revealed no significant differences between wild type and Trpm4 -/- 5- to 12-weeks-old mice of either sex. Above 24 weeks of age, adult male Trpm4 -/- mice showed significantly reduced heart rate and increased heart rate variability. Echocardiography reveals that only adult male Trpm4 -/- mice exhibited slight left ventricular hypertrophic alterations with an alteration of the mitral valve pressure half time, the mitral valve E/A ratio, the isovolumetric relaxation time, and the mitral valve deceleration. In addition, an assessment of the right ventricular systolic function by scanning the pulmonary valve highlighted an alteration in pulmonary valve peak velocity and pressure in male Trpm4 -/- adult mice. Finally, intracardiac ECG recordings showed that the application of 5 {micro}M NBA triggered a third-degree atrioventricular block on 40% of wild-type hearts only. ConclusionsThese results confirm the important role of TRPM4 in the proper structure and electrical function of the heart. It also reveals significant differences between male and female animals that have never been reported before. In addition, the investigation of the effects of NBA on heart function highlights the role of TRPM4 in atrioventricular conduction and provides the first evidence showing the efficacy of this compound on native cardiac tissues.

physiology↗

bioRxiv2021

Species-specific effects of cation channel TRPM4 small-molecule inhibitors

BackgroundThe Transient Receptor Potential Melastatin member 4 (TRPM4) gene encodes a calcium-activated non-selective cation channel expressed in several tissues. Mutations in TRPM4 have been reported in patients with different types of cardiac conduction defects. It is also linked to immune response and cancers, but the associated molecular mechanisms are still unclear. Thus far, 9-phenanthrol is the most common pharmacological compound used to investigate TRPM4 function. We recently identified two promising aryloxyacyl-anthranilic acid compounds (abbreviated CBA and NBA) inhibiting TRPM4. However, all afore-mentioned compounds were screened using assays expressing human TRPM4, whereas the efficacy on mouse TRPM4 has not been assessed. Mouse models are essential to investigate ion channel physiology and chemical compound efficacy. AimIn this study, we performed comparative electro-physiology experiments to assess the effect of these TRPM4 inhibitors on human and mouse TRPM4 channels heterologously expressed in TsA-201 cells. Methods and ResultsWe identified striking species-dependent differences in TRPM4 responses. NBA inhibited both human and mouse TRPM4 currents when applied intracellularly and extracellularly using excised membrane patches. CBA inhibited human TRPM4, both intracellularly and extracellularly. Unexpectedly, the application of CBA had no inhibiting effect on mouse TRPM4 current when perfused on the extracellular side. Instead, it increased mouse TRPM4 current at negative holding potentials. In addition, CBA on the intracellular side altered the outward rectification component of the mouse TRPM4 current. Application of 9-phenanthrol, both intracellularly and extracellularly, inhibited human TRPM4. For mouse TRPM4, 9-phenanthrol perfusion led to opposite effects depending on the site of application. With intracellular 9-phenanthrol, we observed a tendency towards potentiation of mouse TRPM4 outward current at positive holding potentials. ConclusionAltogether, these results suggest that pharmacological compounds screened using "humanised assays" should be extensively characterised before application in in vivo mouse models.

pharmacology and toxicology↗

bioRxiv2020

Deletion of Trpm4 alters the function and expression of NaV1.5 channel in murine cardiac myocytes

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca+ -activated non-selective cation channel that is functionally expressed in several tissues including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases including conduction block and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunohistochemistry studies on isolated atrial and ventricular cardiac myocytes and surface, pseudo and intracardiac ECGs either in vivo or on Langendorff-perfused explanted mouse hearts. We observed that Trpm4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in the myocytes. Hearts from Trpm4-/- mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of peak Na+ current observed in the isolated cardiac myocytes. This reduction in Na+ current is explained by the observed decrease in protein expression of NaV1.5 in Trpm4-/- mice. This study suggests that Trpm4 expression impacts Na+ current in murine cardiac myocytes and points towards a novel function of Trpm4 regulating the NaV1.5 expression in murine cardiac myocytes.

physiology↗