Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent Ca²⁺ channel

In the nerve terminals, the active zone protein CAST/ERC2 forms a protein complex with the other active zone proteins ELKS, Bassoon, Piccolo, RIM1 and Munc13-1, and is thought to play an organizational and functional role in neurotransmitter release. However, it remains obscure how CAST/ERC2 regulates the Ca²⁺-dependent release of neurotransmitters. Here, we show an interaction of CAST with voltage-dependent Ca²⁺ channels (VDCCs), which are essential for regulating neurotransmitter release triggered by depolarization-induced Ca²⁺ influx at the active zone. Using a biochemical assay, we showed that CAST was coimmunoprecipitated with the VDCC β₄-subunit from the mouse brain. A pull-down assay revealed that the VDCC β₄-subunit interacted directly with at least the N-and C-terminal regions of CAST. The II-III linker of VDCC α1-subunit also interacted with C-terminal regions of CAST; however, the interaction was much weaker than that of β₄-subunit. Furthermore, coexpression of CAST and VDCCs in baby hamster kidney cells caused a shift in the voltage dependence of activation towards the hyperpolarizing direction. Taken together, these results suggest that CAST forms a protein complex with VDCCs, which may regulate neurotransmitter release partly through modifying the opening of VDCCs at the presynaptic active zones.