Effects of Disease-Associated Calmodulin Variants on Intracellular Calcium Signaling: A Calcium Imaging Study in SH-SY5Y Cells
Translated title
Effekter af sygdomsassocierede calmodulinvarianter på intracellulær calciumsignalering: Et calciumimaging-studie i SH-SY5Y-celler
Author
Hansen, Michele Bostrup Wittendorff
Term
4. term
Education
Publication year
2026
Submitted on
2026-06-01
Pages
62
Abstract
Calmodulin (CaM) is a calcium-binding protein that regulates many processes in nerve cells and works with voltage-gated calcium channels (VGCCs), which open when the cell membrane’s electrical state changes. CaM mutations have been linked to neurological and cardiac diseases, but their effects on calcium (Ca2+) signaling in neurons remain unclear. This thesis examined four CaM variants in the EF-hand IV calcium-binding site (E140V, E141V, E141G, and F142L) using the human nerve-like SH-SY5Y cell line. We created a stable SH-SY5Y line that expresses the fluorescent Ca2+ sensor GCaMP6m-Xc for live calcium imaging and introduced the CaM variants transiently to test their effects. We also used immunocytochemistry (ICC) to quantify CaM expression after transfection. We successfully established the GCaMP6m-Xc cell line and confirmed CaM overexpression in transiently transfected cells. CaM expression levels were influenced by both the chosen CALM gene and the expression vector backbone. Stimulation with carbachol produced reproducible Ca2+ responses, whereas KCl-induced depolarization gave highly variable responses, limiting the assessment of variant-specific effects on VGCC-mediated Ca2+ signaling. Overall, this study provides a cell-based platform to investigate disease-associated CaM variants and highlights the need to optimize depolarization-based assays to reliably evaluate their impact on neuronal Ca2+ signaling.
Calmodulin (CaM) er et calciumbindende protein, der styrer mange processer i nerveceller og samarbejder med strømstyrede calciumkanaler (VGCC'er), som åbner ved elektriske ændringer i cellemembranen. Mutationer i CaM er sat i forbindelse med neurologiske og hjertesygdomme, men deres betydning for calcium (Ca2+)-signalering i neuroner er stadig uklar. Dette speciale undersøgte fire CaM-varianter i det calciumbindende område EF-hand IV (E140V, E141V, E141G og F142L) i den menneskelige nerve-lignende cellelinje SH-SY5Y. Vi etablerede en stabil SH-SY5Y-cellelinje, der producerer den fluorescerende Ca2+-sensor GCaMP6m-Xc, så vi kunne måle Ca2+-signaler i levende celler, og vi indførte CaM-varianterne midlertidigt (transient ekspression) for at teste deres effekter. Derudover anvendte vi immunocytokemi (ICC) til at kvantificere CaM-ekspressionsniveauer efter transfektion. Vi fandt, at den GCaMP6m-Xc-stabile cellelinje var vellykket, og at CaM blev overekspresseret i de transient transficerede celler. CaM-niveauerne blev påvirket af både det valgte CALM-gen og den anvendte ekspressionsvektor. Stimulering med carbachol gav reproducerbare Ca2+-responser, mens depolarisering med KCl gav meget varierende responser. Det begrænsede vores mulighed for at vurdere variantspecifikke effekter på VGCC-medieret Ca2+-signalering. Samlet set præsenterer studiet en cellebaseret platform til at undersøge sygdomsassocierede CaM-varianter og peger på behovet for at optimere depolariseringsbaserede assays for pålideligt at evaluere deres indflydelse på neuronal Ca2+-signalering.
[This apstract has been rewritten with the help of AI based on the project's original abstract]