Molecular Events Beyond apoCAM Preassociation in the CAM Regulation of Cav1.3 Channels

Abstract

CaV channel regulation by calmodulin (CaM) is a central prototype for ion-channel modulation. Despite long study, relatively little is known mechanistically, beyond the initial preassociation of Ca2+-free CaM (apoCaM) with an IQ domain on the carboxy-terminus of channels. Most studies have focused on the IQ domain and immediate upstream ‘preIQ’ regions, despite hints that further upstream elements in the carboxy-terminus could be important. Accordingly, we here undertake alanine scanning mutagenesis of the entire carboxy-terminus upstream of the IQ domain (the proximal CI-region, PCI). For analysis, we chose CaV1.3 channels (highly homologous to classic CaV1.2), because they exhibit robust CaM-mediated inactivation (CDI), with particularly well-resolved profiles for both N- and C-lobe forms of inactivation. Several unexpected results were obtained. First, mutations throughout the preIQ domain left CDI essentially unchanged, at odds with functional hotspots in the homologous region of CaV1.2. Second, newly identified segments, situated upstream of the preIQ region, proved selectively critical for the C-lobe form of CDI. Specifically, we argue that the PCI region is the Ca2+/CaM effector site for C-lobe CDI, as revealed by quantitative comparison of the effect of PCI mutations on CDI, to their effect on PCI binding with Ca2+/CaM (‘Ψ-analysis’). Third, we further exploit Ψ-analysis to extend and confirm that the Ca2+/CaM effector site for the N-lobe form of CDI is structurally distinct, residing in the NSCaTE element of the channel amino terminus (Nature 451:830). Finally, while the IQ-domain is a primary site for apoCaM preassociation, our scan surprisingly reveals that PCI harbors additional preassociation sites, especially important for the N-lobe of apoCaM. Overall, this alanine scan of the CaV1.3, together with that of the IQ domain (companion abstract), outlines the long-sought molecular events beyond the initial apoCaM preassociation with the channel.