Crystal structures of Ca ²⁺ –calmodulin bound to Na _V C-terminal regions suggest role for EF-hand domain in binding and inactivation

Abstract

Voltage-gated sodium (Na V ) and calcium channels (Ca V ) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the Cterminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca 2+occupied CaM, bound to the CT of Na V 1.5. The structure shows that the C-terminal lobe binds to a site ∼90°rotated relative to a previous site reported for an apoCaM complex with the Na V 1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca 2+ -occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca 2+ -free and Ca 2+ -occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related Na V 1.4 channel has been shown to undergo Ca 2+ -dependent inactivation (CDI) akin to Ca V s. We present a crystal structure of Ca 2+ /CaM bound to the Na V 1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EFhand domain and the IQ domain as a possible conformational switch that underlies CDI.

Bidirectional Modulation of the Voltage-Gated Sodium (Nav1.6) Channel by Rationally Designed Peptidomimetics

“…Theses similarities between the binding interactions of 12 and FGF14 with the Nav1.6 C-terminal tail suggest that 12 s truncated size relative to tetrapeptide analogs confers it with heightened mimicry of FGF14, thereby giving rise to its marked suppression of Nav1.6-mediated peak current density. Based upon theses structural analyses, and consistent with current models of Nav channel function [37][38][39], we propose that 12 and 19 exert opposite effects on Nav1.6-mediated currents as a result of differential interactions with the EF hand-like (EFL) and IQ domains of the C-terminal tail of the channel at sites that have established roles in channel trafficking and inactivation.…”

Arrhythmia mutations in calmodulin can disrupt cooperativity of Ca2+ binding and cause misfolding

“…2018; Gardill et al . 2019), a structural investigation of arrhythmogenic CaM variants of other cardiac ion channels should be possible.…”

“…An intrinsic limitation is that we are lacking the upstream EF-hand domain, which also plays a role in CDI (Ben Johny et al 2013). So far any Ca V construct containing this element has failed to produce properly folded protein, in contrast to the progress made for voltage-gated sodium channels (Wang et al 2012(Wang et al , 2014Gabelli et al 2014;Gardill et al 2019;Yoder et al 2019). Our structural studies of CaM disease mutants show that different mutations have distinct effects on the structure in the presence of the Ca V 1.2 IQ domain, despite the fact that most, but not all, have a similar functional outcome on CDI:…”

“…Thus far, all structural studies of CaM disease mutations have been performed in the context of the Ca V 1.2 IQ domain. However, with available cryo-EM structures of CaM bound to full-length RyR2 (Gong et al 2019) or KCNQ1 potassium channels (Sun & MacKinnon, 2017), as well as NMR and crystal structures of Na V 1.5 cytosolic fragments bound to apoCaM (Chagot & Chazin, 2011;Wang et al 2012Wang et al , 2014Gabelli et al 2014) or Ca 2+ -CaM (Sarhan et al 2012;Johnson et al 2018;Gardill et al 2019), a structural investigation of arrhythmogenic CaM variants of other cardiac ion channels should be possible.…”

Biophysical Investigation of Sodium Channel Interaction with β-Subunit Variants Associated with Arrhythmias

“…5 However, it is now well established that Na V channels do not function in isolation but form membrane-embedded macromolecular complexes that include auxiliary proteins such as b-subunits, 1,[6][7][8] fibroblast growth factor proteins, calmodulin, and others. [9][10][11][12][13][14][15][16] b-subunits are known to modify gating properties, regulate trafficking, and influence plasma membrane recruitment of Na V channels. 6 Four main b-subunit isoforms (b1-4) have been identified and despite a shared, single transmembrane-segment protein topology, each interacts distinctly with and exerts disparate effects on specific Na V channel subtypes (Na V 1.1-1.9).…”