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.

Ryanodine receptors (RyRs) are huge ion channels that are responsible for the release of Ca 2؉ from the sarco/endoplasmic reticulum. RyRs form homotetramers with a mushroom-like shape, consisting of a large cytoplasmic head and transmembrane stalk. Ca 2؉ is a major physiological ligand that triggers opening of RyRs, but a plethora of modulatory proteins and small molecules in the cytoplasm and sarco/endoplasmic reticulum lumen have been recognized. Over 300 mutations in RyRs are associated with severe skeletal muscle disorders or triggered cardiac arrhythmias. With the advent of high-resolution structures of individual domains, many of these can be mapped onto the three-dimensional structure.The South American plant Ryania speciosa has long been recognized for its insecticidal properties (1). Its active compound, an alkaloid known as ryanodine, targets a eukaryotic membrane protein known as the ryanodine receptor (RyR). 2Long before their isolation, RyRs had already been visualized in thin section or negative stain electron microscopy studies of muscle ultrastructure. These images showed the presence of intracellular junctions between the sarcoplasmic reticulum (SR) and transverse tubular invaginations of the plasma membrane. Electron dense protrusions, named "feet," were found to span these junctions (2). Although the identities of the feet structures were initially unknown, later purification and electron microscopy studies confirmed them to be RyRs (3, 4).RyRs form homotetrameric assemblies and constitute the largest ion channels known to date, with molecular masses of ϳ2.2 MDa and each monomer consisting of ϳ5000 amino acid residues (3, 4). They are responsible for the release of Ca 2ϩ from the endoplasmic reticulum (ER) and SR and thus control many Ca 2ϩ -dependent processes within the cell. Ryanodine binds RyRs preferentially in the open state. At nanomolar concentrations, it "locks" the channel in a subconductance state, but at concentrations Ͼ100 M, it inhibits Ca 2ϩ release (5).In mammalian organisms, RyRs are found in a wide variety of cell types, including neurons, exocrine cells, epithelial cells, lymphocytes, and many more (6). They are known mostly for their involvement in excitation-contraction coupling, releasing Ca 2ϩ from the SR and thus driving muscle contraction. Three different isoforms (RyR1-3) have been found to date. RyR1 is widely expressed in skeletal muscle and was the first one to be cloned (7, 8). RyR2 is found primarily in the heart (9, 10), and RyR3 was originally identified in the brain (11), although each isoform is found in many different cell types (6). They share ϳ65% sequence identity, and the largest degree of difference is found in three "divergent regions" throughout the sequence, known as D1 (residues 4254 -4631 in RyR1), D2 (residues 1342-1403), and D3 (residues 1872-1923). Lower organisms express fewer RyR isoforms. Non-mammalian vertebrates express two isoforms, RyR␣ and RyR␤, whereas a single isoform was found to be expressed in lower organisms, including nematodes, f...

Ryanodine receptors are large channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of RyR mutations can cause cardiac and skeletal muscle disorders, yet detailed mechanisms explaining their effects have been lacking. Here we compare pseudo-atomic models and propose that channel opening coincides with widening of a cytoplasmic vestibule formed by the N-terminal region, thus altering an interface targeted by 20 disease mutations. We solve crystal structures of several disease mutants that affect intrasubunit domain–domain interfaces. Mutations affecting intrasubunit ionic pairs alter relative domain orientations, and thus couple to surrounding interfaces. Buried disease mutations cause structural changes that also connect to the intersubunit contact area. These results suggest that the intersubunit contact region between N-terminal domains is a prime target for disease mutations, direct or indirect, and we present a model whereby ryanodine receptors and inositol-1,4,5-trisphosphate receptors are activated by altering domain arrangements in the N-terminal region.

Voltage-gated sodium channels underlie the rapid regenerative upstroke of action potentials and are modulated by cytoplasmic calcium ions through a poorly understood mechanism. We describe the 1.35 Å crystal structure of Ca 2+ -bound calmodulin (Ca 2+ /CaM) in complex with the inactivation gate (DIII-IV linker) of the cardiac sodium channel (Na V 1.5). The complex harbors the positions of five disease mutations involved with long Q-T type 3 and Brugada syndromes. In conjunction with isothermal titration calorimetry, we identify unique inactivation-gate mutations that enhance or diminish Ca 2+ /CaM binding, which, in turn, sensitize or abolish Ca 2+ regulation of full-length channels in electrophysiological experiments. Additional biochemical experiments support a model whereby a single Ca 2+ /CaM bridges the C-terminal IQ motif to the DIII-IV linker via individual N and C lobes, respectively. The data suggest that Ca 2+ /CaM destabilizes binding of the inactivation gate to its receptor, thus biasing inactivation toward more depolarized potentials.crystallography | patch-clamp electrophysiology | structural biology | cardiac arrhythmia V oltage-gated sodium channels (Na V s) support excitability in the cardiovascular and nervous systems, where they contribute to the rhythm and rate of action potentials. These large (∼220-kDa) transmembrane protein complexes are expressed at a high density in excitable cells, where they conduct large macroscopic inward sodium currents. These channels are exquisitely sensitive to subtle changes in the transmembrane potential, and modest alterations in channel gating can fine-tune or disorder electrical signaling at the organ and systemic level. The α-subunit of the channel contains cytoplasmic amino and carboxyl termini and is composed of four homologous transmembrane domains (DI-DIV) that are connected by intracellular linkers. Each domain contains voltage-sensing (S1-S4) and pore-forming (S5 and S6) domains that form the selectivity filter and putative activation gates. A crystal structure of a bacterial Na V was recently described (1) showing a similar overall fold compared with potassium channels. However, this bacterial variant is homotetrameric, and seems to lack a conserved fast-inactivation mechanism. As such, it has no homology with several relevant domains in mammalian Na V channels, and no crystal structure of any eukaryotic Na V region has yet been reported.Calcium ions (Ca 2+ ) are universal second messengers, and in the heart they form the electrochemical link between plasma membrane depolarization and myocyte contraction. Consequently, their cytoplasmic levels oscillate between nanomolar and micromolar levels with each excitation-contraction cycle (2). Sodium channel steady-state inactivation, a process that controls channel availability at a given transmembrane potential, is modulated through interactions with Ca 2+ and calmodulin (CaM) (3-10). The mechanistic details of Ca 2+ modulation of sodium channel inactivation are sparse, but the C-terminal region of the cha...

Ryanodine Receptors (RyRs) are huge Ca²⁺ release channels in the endoplasmic reticulum membrane and form targets for phosphorylation and disease mutations. We present crystal structures of a domain in three RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. The RyR1 domain is the target for 11 disease mutations. Several of these are clustered near the phosphorylation sites, suggesting that phosphorylation and disease mutations may affect the same interface. The L2867G mutation causes a drastic thermal destabilization and aggregation at room temperature. Crystal structures for other disease mutants show that they affect surface properties and intradomain salt bridges. In vitro phosphorylation experiments show that up to five residues in one long loop of RyR2 can be phosphorylated by PKA or CaMKII. Docking into cryo-electron microscopy maps suggests a putative location in the clamp region, implying that mutations and phosphorylation may affect the allosteric motions within this area.