The Role of EF-Hand in Calmodulin Binding of Voltage-Gated Cav2.1 and Cav2.2 Calcium Channels

摘要

Voltage-gated Cav2.1 (P/Q-type) and Cav2.2 (N-type) channels are two closely related calcium channels that play indispensable roles in signal transduction pathways by regulating neurotransmitter release. Despite having highly conserved amino acid sequences, they are differentially modulated by calmodulin, which mediate two important feedback mechanisms known as Ca2+-dependent inactivation (CDI) and Ca 2+-dependent facilitation (CDF). These dual regulatory mechanisms contribute to synaptic plasticity, but only CDI is observed in Cav2.2 channel, while both CDI and CDF are present in Cav2.1 channel. Previously, it was hypothesized that the lack of CDF in Cav2.2 channel is due to the pre-IQ-IQ domain of the channel's lower binding affinity for calmodulin compared to that of Cav2.1 channel. Now that the EF-hand domain of calcium channels is identified as one of the two minimally required molecular determinants that are responsible for supporting CDF in Cav2.1 channel and preventing CDF in Cav2.2 channel, it was necessary to determine the role of EF-hand domain in calmodulin binding of Cav2.1 and Cav2.2 channels. Using pull-down binding assays, this study finds that the EF-hand domain enhances calmodulin binding to the proximal C-terminal domain of Cav2.2 channel, which suggests that the lack of CDF in Cav2.2 does not result from the channel's weak interaction with CaM, but from the EF-pre-IQ-IQ domain of the channel's inability to allow calmodulin from fully exerting its effects.