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).
Lee
Amy
A
Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195-7280, USA.
Zhou
Hong
H
Scheuer
Todd
T
Catterall
William A
WA
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013569:Synapses / Q000502:physiology
D002118:Calcium; D020864:Calcium Channels, N-Type; D002147:Calmodulin; D011994:Recombinant Proteins