The Calmodulin-Binding, Short Linear Motif, NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels Valentina Taiakina 2 , Adrienne N. Boone 1 , Julia Fux 1 , Adriano Senatore 1 , Danielle Weber-Adrian 1 , J. Guy Guillemette 2. , J. David Spafford 1 * . 1 Department of Biology, University of Waterloo, Waterloo, Canada, 2 Department of Chemistry, University of Waterloo, Waterloo, Canada Abstract NSCaTE is a short linear motif of (x Wxxx( I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca 2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Ca v 1.1 and Ca v 1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCa v 1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca 2+ -CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels. Citation: Taiakina V, Boone AN, Fux J, Senatore A, Weber-Adrian D, et al. (2013) The Calmodulin-Binding, Short Linear Motif, NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels. PLoS ONE 8(4): e61765. doi:10.1371/journal.pone.0061765 Editor: Zhe Zhang, Virginia Commonwealth University, United States of America Received January 9, 2013; Accepted March 11, 2013; Published April 23, 2013 Copyright: ß 2013 Taiakina et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Heart and Stroke Foundation of Canada, Grant-in-Aid to JDS, and Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Operating grants to JDS and JGG. VT received support from an NSERC Canadian Graduate Scholarship Doctoral Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Corresponding author JDS is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: spafford@uwaterloo.ca . These authors contributed equally to this work. Introduction Changes in intracellular Ca 2+ concentrations induce conforma- tional shifts in the ubiquitous Ca 2+ sensor protein, calmodulin (CaM) [1]. CaM is able to bind up to 300 different known target proteins to date, altering cellular functions [2]. Calmodulin has a particularly unique relationship with calcium-permeant ion channels such as InsP3 receptors [3], ryanodine receptors [4], transient receptor potential channels [5], and high voltage- activated calcium channels [6]. Calcium permeant channels self- regulate their own channel gating when calcium increases are sensed by the CaM sensor located at their intracellular surface, altering their refractoriness or inactivation and preventing excessive calcium entry, or in some cases, facilitate the current [6,7]. CaM binds to the IQ motifs of C-terminal tails of L-type calcium channels in a parallel orientation with the flexible CaM linker separating its two globular domains (N- and C- terminal domains with doublet pairs of heterogenous, Ca 2+ -binding EF hands) [8,9]. The CaM N-terminal domain has lower Ca 2+ affinity and is sensitive to high intracellular buffering of Ca 2+ , while the C- terminal domain has higher affinity to Ca 2+ and is buffer resistant [10]. L-type channel homologs from single cell protozoans [11–13] and mammalian tissues [6], have calcium-dependent inactivation regulated by a CaM-like protein and shared a conserved C- terminal IQ motif. A second CaM binding motif, NSCaTE in the N-terminus was more recently identified for vertebrate Cav1.2 and Cav1.3 channels [14]. We mined available genomes to determine that NSCaTE appears in L-type calcium channels of all coelomate animals. NSCaTE is a short linear motif composed of residues with high helix-forming propensities, with a key tryptophan (W) residue separated from an isoleucine or leucine (I/L) residue (x-W- xxx (I or L)-xxxx). Binding of NSCaTE to CaM is dependent on rises of intracellular calcium that is sensitive to high intracellular buffering with 10 mM EGTA. A conserved methionine down- stream of NSCaTE suggests that there is an alternative translation site for L-type calcium channels for generating L-type channels with and without NSCaTE. The consequence of an optional NSCaTE motif is a much faster calcium-dependent inactivation (CDI) than that contributed by the more ancient IQ motif of L- type calcium channels. PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e61765