Design of an imaging probe to monitor real-time redistribution of L-type voltage gated calcium channels in astrocytic glutamate signalling

Abstract

PurposeIn the brain, astrocytes are non-excitable cells that undergo rapid morphological changes when stimulated by the excitatory neurotransmitter glutamate. We developed a chemical probe to monitor how glutamate affects the density and distribution of astrocytic L-type voltage-gated calcium channels (LTCC).ProceduresThe imaging probe FluoBar1 was created from a barbiturate ligand modified with a fluorescent coumarin moiety. The probe selectivity was examined with colocalization analyses of confocal fluorescence imaging in U118-MG and transfected COS-7 cells. Living cells treated with 50 nM FluoBar1 were imaged in real time to reveal changes in density and distribution of astrocytic LTCCs upon exposure to glutamate.ResultsFluoBar1 was synthesized in ten steps. The selectivity of the probe was demonstrated with immunoblotting and confocal imaging of immunostained cells expressing the CaV1.2 isoform of LTCCs proteins. Applying FluoBar1 to astrocyte model cells U118-MG allowed us to measure a 5-fold increase in fluorescence density of LTCCs upon glutamate exposure.ConclusionsImaging probe FluoBar1 allows the real-time monitoring of LTCCs in living cells, revealing for first time that glutamate causes a rapid increase of LTCC membranar density in astrocyte model cells. FluoBar1 may help tackle previously intractable questions about LTCC dynamics in cellular events.

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

“…Colocalization experiments via confocal microscopy showed good specificity of FluoBar1 for Ca V 1.2 in U118‐MG cells, confirmed by control experiments in non‐transfected and transfected COS‐7 cells. Application of FluoBar1 in real‐time imaging of live cells allowed to study the redistribution of Ca V 1.2 in response to glutamate stimulation in U118‐MG cells [86a] …”