Molecular determinants of Ca(2+)/calmodulin-dependent regulation of Ca(v)2.1 channels

Ca2+-dependent facilitation and inactivation (CDF and CDI) of Cav2.1 channels modulate presynaptic P/Q-type Ca2+ currents and contribute to activity-dependent synaptic plasticity. This dual feedback regulation by Ca2+ involves calmodulin (CaM) binding to the alpha1 subunit (alpha12.1). The molecular determinants for Ca2+-dependent modulation of Cav2.1 channels reside in CaM and in two CaM-binding sites in the C-terminal domain of alpha12.1, the CaM-binding domain (CBD) and the IQ-like domain. In transfected tsA-201 cells, CDF and CDI were both reduced by deletion of CBD. In contrast, alanine substitution of the first two residues of the IQ-like domain (IM-AA) completely prevented CDF but had little effect on CDI, and glutamate substitutions (IM-EE) greatly accelerated voltage-dependent inactivation but did not prevent CDI. Mutational analyses of the Ca2+ binding sites of CaM showed that both the N- and C-terminal lobes of CaM were required for full development of facilitation, but only the N-terminal lobe was essential for CDI. In biochemical assays, CaM12 and CaM34 were unable to bind CBD, whereas CaM34 but not CaM12 retained Ca2+-dependent binding to the IQ-like domain. These findings support a model in which Ca2+ binding to the C-terminal EF-hands of preassociated CaM initiates CDF via interaction with the IQ-like domain. Further Ca2+ binding to the N-terminal EF-hands promotes secondary CaM interactions with CBD, which enhance facilitation and cause a conformational change that initiates CDI. This multifaceted mechanism allows positive regulation of Cav2.1 in response to local Ca2+ increases (CDF) and negative regulation during more global Ca2+ increases (CDI).