Cavβ3 Contributes to the Maintenance of the Blood-Brain Barrier and Alleviates Symptoms of Experimental Autoimmune Encephalomyelitis.

Abstract

Background: Tight control of cytoplasmic Ca²⁺ concentration in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavβ3, a subunit of voltage-gated Ca²⁺ (Cav) channels, in modulating Ca²⁺ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier.

Methods: We investigated the function of Cavβ3 in BMECs by Ca²⁺ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavβ3^-/- (Cavβ3-deficient) mice as controls.

Results: We identified Cavβ3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavβ3. After induction of experimental autoimmune encephalomyelitis, Cavβ3^-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavβ3^-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca²⁺ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca²⁺-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavβ3^-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavβ3^-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavβ3 with inositol 1,4,5-trisphosphate receptor proteins.

Conclusions: Independent of its function as a subunit of Cav channels, Cavβ3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca²⁺ concentration and Ca²⁺-dependent MLC phosphorylation in BMECs, and this role of Cavβ3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.