Voltage-gated calcium channels (Ca V s) govern muscle contraction, hormone and neurotransmitter release, neuronal migration, activation of calcium-dependent signalling cascades, and synaptic input integration 1 . An essential Ca V intracellular protein, the β-subunit (Ca V β)1 ,2 , binds a conserved domain (the α-interaction domain, AID) between transmembrane domains I and II of the pore-forming α 1 subunit 3 and profoundly affects multiple channel properties such as voltagedependent activation 2 , inactivation rates 2 , G-protein modulation 4 , drug sensitivity 5 and cell surface expression 6,7 . Here, we report the high-resolution crystal structures of the Ca V β 2a conserved core, alone and in complex with the AID. Previous work suggested that a conserved region, the β-interaction domain (BID), formed the AID-binding site 3, 8; however, this region is largely buried in the Ca V β core and is unavailable for protein-protein interactions. The structure of the AID-Ca V β 2a complex shows instead that Ca V β 2a engages the AID through an extensive, conserved hydrophobic cleft (named the α-binding pocket, ABP). The ABP-AID interaction positions one end of the Ca V β near the intracellular end of a pore-lining segment, called IS6, that has a critical role in Ca V inactivation 9,10 . Together, these data suggest that Ca V βs influence Ca V gating by direct modulation of IS6 movement within the channel pore. The 1.97 Å resolution structure of the Ca V β 2a core shows that Ca V βs comprise two wellconserved domains (Fig. 1a). The first, an SH3 fold, contains five antiparallel β-strands (β1-β5), a 3 10 helix (η1), and two α-helices (α1 and α2) that lie amino-terminal to β1 and carboxy-terminal to β4, respectively. The strand that completes the SH3 fold, β5 (residues 217-224), is separated in the primary structure from the core of the SH3 domain by approximately 70 residues (variable domain 2, V2, a site of splice variation and amino acid insertions and deletions2) that are absent from the structure (Fig. 1b). The second conserved domain consists of a five-stranded parallel β-sheet (β6-β10), surrounded by six α-helices (α3-α8) and two 3 10 helices (η2 and η3), and is related to the core of nucleotide kinase enzymes.Ca V βs share structural features with membrane-associated guanylate kinases (MAGUKs), a protein scaffold family that organizes signalling components near membranes 11 Comparison of Ca V β 2a with a representative MAGUK, 13), reveals other differences. Superposition of the nucleotide kinase domains shows that the relative orientations of the SH3 and nucleotide kinase domains differ by approximately 90°, an arrangement that makes Ca V β 2a a more elongated structure (Fig. 2a). The nucleotide kinase domain of MAGUKs is homologous to guanylate kinases and retains guanosine monophosphate (GMP) binding, but key residues for enzymatic function are missing 12 . The four-stranded β-sheet nucleotide kinase subdomain that binds GMP in MAGUKs is absent in Ca v β 2a (Fig. 2a). Furthermore, two Ca V β 2a loops (b...

Changes in activity-dependent calcium flux through voltage-gated calcium channels (Ca V s) drive two self-regulatory calcium-dependent feedback processes that require interaction between Ca 2+ / calmodulin (Ca 2+ /CaM) and a Ca V channel consensus isoleucine-glutamine (IQ) motif: calciumdependent inactivation (CDI) and calcium-dependent facilitation (CDF). Here, we report the highresolution structure of the Ca 2+ /CaM-Ca V 1.2 IQ domain complex. The IQ domain engages hydrophobic pockets in the N-terminal and C-terminal Ca 2+ /CaM lobes through sets of conserved 'aromatic anchors.' Ca 2+ /N lobe adopts two conformations that suggest inherent conformational plasticity at the Ca 2+ /N lobe-IQ domain interface. Titration calorimetry experiments reveal competition between the lobes for IQ domain sites. Electrophysiological examination of Ca 2+ /N lobe aromatic anchors uncovers their role in Ca V 1.2 CDF. Together, our data suggest that Ca V subtype differences in CDI and CDF are tuned by changes in IQ domain anchoring positions and establish a framework for understanding CaM lobe-specific regulation of Ca V s.Voltage-gated calcium channels are the ion channels that define excitable cells 1 . These channels control cellular calcium entry in response to changes in membrane potential and are pivotal in the generation of cardiac action potentials, excitation-contraction coupling, hormone and neurotransmitter release and activity-dependent transcription initiation 1,2 . Ca V s are multisubunit complexes composed of three essential channel subunits 2 , Ca V α 1 , Ca V β and Ca V α 2 δ, plus the ubiquitous intracellular calcium sensor calmodulin (CaM) 3 . An additional subunit, Ca V γ, is associated with skeletal muscle channels, but its general importance in other tissues is unsettled 4 .The Ca V α 1 subunits are single polypeptide chains of ∼1,800-2,200 residues in which the ion-conducting pore is formed from four homologous repeats that each bear six transmembrane segments 2 . There are three Ca V subfamilies, which have diverse physiological and pharmacological properties that depend largely on the Ca V α 1 -subunit: Ca V 1.x (L-type), Ca V 2.x (2.1, P/Q-type; 2.2, N-type; 2.3, R-type) and Ca V 3.x (T-type) 1 . Large interdomain intracellular loops bridge the four transmembrane repeats of the Ca V α 1 subunit and serve as docking sites for auxiliary subunits and regulatory molecules that Competing Interests Statement:The authors declare that they have no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access control channel activity and connect Ca V channels to larger macromolecular complexes and cellular signaling pathways 5,6 .Calcium influx is a potent activator of intracellular signaling pathways but is toxic in excess 1,7 . Because Ca V s are major sources of calcium influx, Ca V activity is strongly controlled by both self-regulatory and extrinsic mechanisms that tune channel action in response to electrical...

Many physiological events require transient increases in cytosolic Ca(2+) concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca(2+) from the endoplasmic and sarcoplasmic reticulum. Mutations in RyRs can lead to severe genetic conditions that affect both cardiac and skeletal muscle, but locating the mutated residues in the full-length channel structure has been difficult. Here we show the 2.5 Å resolution crystal structure of a region spanning three domains of RyR type 1 (RyR1), encompassing amino acid residues 1-559. The domains interact with each other through a predominantly hydrophilic interface. Docking in RyR1 electron microscopy maps unambiguously places the domains in the cytoplasmic portion of the channel, forming a 240-kDa cytoplasmic vestibule around the four-fold symmetry axis. We pinpoint the exact locations of more than 50 disease-associated mutations in full-length RyR1 and RyR2. The mutations can be classified into three groups: those that destabilize the interfaces between the three amino-terminal domains, disturb the folding of individual domains or affect one of six interfaces with other parts of the receptor. We propose a model whereby the opening of a RyR coincides with allosterically coupled motions within the N-terminal domains. This process can be affected by mutations that target various interfaces within and across subunits. The crystal structure provides a framework to understand the many disease-associated mutations in RyRs that have been studied using functional methods, and will be useful for developing new strategies to modulate RyR function in disease states.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca2+-release channels with each subunit containing a binding site for IP3 in the N-terminus. We provide evidence that four IP3 molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca2+ release suggested that IP3Rs are maximally occupied by IP3 before substantial Ca2+ release occurs. We showed that ligand binding–deficient subunits acted in a dominant-negative manner when coexpressed with wild-type monomers in the chicken immune cell line DT40-3KO, which lacks all three genes encoding IP3R subunits, and confirmed the same effect in an IP3R-null human cell line (HEK-3KO) generated by CRISPR/Cas9 technology. Using dimeric and tetrameric concatenated IP3Rs with increasing numbers of binding-deficient subunits, we addressed the obligate ligand stoichiometry. The concatenated IP3Rs with four ligand-binding sites exhibited Ca2+ release and electrophysiological properties of native IP3Rs. However, IP3 failed to activate IP3Rs assembled from concatenated dimers consisting of one binding-competent and one binding-deficient mutant subunit. Similarly, IP3Rs containing two monomers of IP3R2short, an IP3 binding-deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding competent ligand-binding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP3-induced Ca2+ release only occurs when each IP3R monomer within the tetramer is occupied by IP3, thereby ensuring fidelity of Ca2+ release.