Calmodulin-Like Region of CaV1.3 Harbors Novel Structural Determinants Underlying CaM-Mediated Channel Regulation

Abstract

The regulation of CaV channels by calmodulin (CaM) is both biologically critical and mechanistically rich, rendering this system a central prototype for ion-channel modulation. Despite a decade of study, little is known of the structural mechanisms underlying such modulation, beyond the initial preassociation of Ca2+-free CaM (apoCaM) with an IQ domain on the carboxy-terminus of channels. We have recently argued that the ultimate end-point of channel regulation is allosteric modulation of S6 cytoplasmic gates (Biophys J96:222a). However, the transduction events®ˆ'those linking calcification of apoCaM to this allosteric effect®ˆ'remain essentially unknown. The majority of structure-function analyses have narrowly focused on the IQ domain and immediately upstream ‘preIQ’ regions, despite hints that further upstream elements in the carboxy-terminus could be important. Additionally, deletions and non-conservative mutations have often been employed in these analyses, confounding interpretation with the potential for backbone fold disruption. Here, we undertook exhaustive alanine scanning mutagenesis of the carboxy-terminus, up to the IQ domain of CaV1.3 channels. Importantly, the substitution of alanines likely preserves backbone fold throughout. Moreover, CaV1.3 (highly homologous to classic CaV1.2 channels) exhibits robust CaM-mediated inactivation (CDI) that enhances structure-function analysis. Surprisingly, alanine substitutions throughout the preIQ domain left CDI essentially unchanged, at odds with functional hotspots in the homologous region of CaV1.2 (1582NEE1585, 1572IKTEG1576, and 1600LLDQV1605). Instead, newly identified and critical segments were situated upstream, in a region predicted to resemble a lobe of CaM by structural modeling (Rosetta). Intriguingly, homologous residues of NaV channels are linked to mutations underlying heritable LQT syndromes, hinting at conserved modulatory mechanisms across NaV and CaV channels. Overall, this alanine scan of the CaV1.3, together with that of the IQ domain in a companion abstract, lays the groundwork for understanding the structure-function mechanisms underlying CaM/channel regulation.