Abstract
Ca(2+)-binding protein-1 (CaBP1) and calmodulin (CaM) are highly related Ca(2+)-binding proteins that directly interact with, and yet differentially regulate, voltage-gated Ca(2+) channels. Whereas CaM enhances inactivation of Ca(2+) currents through Ca(v)1.2 (L-type) Ca(2+) channels, CaBP1 completely prevents this process. How CaBP1 and CaM mediate such opposing effects on Ca(v)1.2 inactivation is unknown. Here, we identified molecular determinants in the alpha(1)-subunit of Ca(v)1.2 (alpha(1)1.2) that distinguish the effects of CaBP1 and CaM on inactivation. Although both proteins bind to a well characterized IQ-domain in the cytoplasmic C-terminal domain of alpha(1)1.2, mutations of the IQ-domain that significantly weakened CaM and CaBP1 binding abolished the functional effects of CaM, but not CaBP1. Pulldown binding assays revealed Ca(2+)-independent binding of CaBP1 to the N-terminal domain (NT) of alpha(1)1.2, which was in contrast to Ca(2+)-dependent binding of CaM to this region. Deletion of the NT abolished the effects of CaBP1 in prolonging Ca(v)1.2 Ca(2+) currents, but spared Ca(2+)-dependent inactivation due to CaM. We conclude that the NT and IQ-domains of alpha(1)1.2 mediate functionally distinct interactions with CaBP1 and CaM that promote conformational alterations that either stabilize or inhibit inactivation of Ca(v)1.2.
Highlights
Calmodulin (CaM)1 is the primordial member of a superfamily of EF-hand Ca2ϩ-binding proteins (CaBPs) that confer Ca2ϩdependent regulation to a vast array of enzymes, ion channels, and neurotransmitter receptors [1,2,3]
We found that the cytoplasmic N-terminal domain of ␣11.2 is essential for Cav1.2 modulation by Ca2؉-binding protein-1 (CaBP1) but not by CaM, the IQ-domain is important for Ca2ϩ-dependent association of CaBP1 with the channel
IQ mutations did not prevent the effects of CaBP1 in that Ires/Ipk was still significantly increased in cells cotransfected with Cav1.2IQ-AA (ϳ234%, p Ͻ0.01, compared with Cav1.2IQ-AA alone) and Cav1.2IQ-EE (ϳ153%, p Ͻ0.02, compared with Cav1.2IQ-EE alone)
Summary
Calmodulin (CaM)1 is the primordial member of a superfamily of EF-hand Ca2ϩ-binding proteins (CaBPs) that confer Ca2ϩdependent regulation to a vast array of enzymes, ion channels, and neurotransmitter receptors [1,2,3]. We found that the cytoplasmic N-terminal domain of ␣11.2 is essential for Cav1.2 modulation by CaBP1 but not by CaM, the IQ-domain is important for Ca2ϩ-dependent association of CaBP1 with the channel.
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