Functional determination of calcium-binding sites required for the activation of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca2+) signals in response to various external cues. Notably, IP3R channel activity is determined by several obligatory factors, including IP3, Ca2+, and ATP. The critical basic amino acid residues in the N-terminal IP3-binding core (IBC) region that facilitate IP3 binding are well characterized. In contrast, the residues conferring regulation by Ca2+ have yet to be ascertained. Using comparative structural analysis of Ca2+-binding sites identified in two main families of intracellular Ca2+-release channels, ryanodine receptors (RyRs) and IP3Rs, we identified putative acidic residues coordinating Ca2+ in the cytosolic calcium sensor region in IP3Rs. We determined the consequences of substituting putative Ca2+ binding, acidic residues in IP3R family members. We show that the agonist-induced Ca2+ release, single-channel open probability (P0), and Ca2+ sensitivities are markedly altered when the negative charge on the conserved acidic side chain residues is neutralized. Remarkably, neutralizing the negatively charged side chain on two of the residues individually in the putative Ca2+-binding pocket shifted the Ca2+ required to activate IP3R to higher concentrations, indicating that these residues likely are a component of the Ca2+ activation site in IP3R. Taken together, our findings indicate that Ca2+ binding to a well-conserved activation site is a common underlying mechanism resulting in increased channel activity shared by IP3Rs and RyRs.

RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice.

Mice with a malignant hyperthermia mutation (Y522S) in the ryanodine receptor (RyR1) display muscle contractures, rhabdomyolysis, and death in response to elevated environmental temperatures. We demonstrate that this mutation in RyR1 causes Ca(2+) leak, which drives increased generation of reactive nitrogen species (RNS). Subsequent S-nitrosylation of the mutant RyR1 increases its temperature sensitivity for activation, producing muscle contractures upon exposure to elevated temperatures. The Y522S mutation in humans is associated with central core disease. Many mitochondria in the muscle of heterozygous Y522S mice are swollen and misshapen. The mutant muscle displays decreased force production and increased mitochondrial lipid peroxidation with aging. Chronic treatment with N-acetylcysteine protects against mitochondrial oxidative damage and the decline in force generation. We propose a feed-forward cyclic mechanism that increases the temperature sensitivity of RyR1 activation and underlies heat stroke and sudden death. The cycle eventually produces a myopathy with damaged mitochondria.

Gating machinery of InsP3R channels revealed by electron cryomicroscopy.

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are ubiquitous ion channels responsible for cytosolic Ca(2+) signalling and essential for a broad array of cellular processes ranging from contraction to secretion, and from proliferation to cell death. Despite decades of research on InsP3Rs, a mechanistic understanding of their structure-function relationship is lacking. Here we present the first, to our knowledge, near-atomic (4.7 Å) resolution electron cryomicroscopy structure of the tetrameric mammalian type 1 InsP3R channel in its apo-state. At this resolution, we are able to trace unambiguously ∼85% of the protein backbone, allowing us to identify the structural elements involved in gating and modulation of this 1.3-megadalton channel. Although the central Ca(2+)-conduction pathway is similar to other ion channels, including the closely related ryanodine receptor, the cytosolic carboxy termini are uniquely arranged in a left-handed α-helical bundle, directly interacting with the amino-terminal domains of adjacent subunits. This configuration suggests a molecular mechanism for allosteric regulation of channel gating by intracellular signals.

Structural Insights into IP3R Function.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ubiquitously expressed intracellular ligand-gated Ca2+ channels present on the endoplasmic reticulum of virtually all eukaryotic cells. These channels mediate the Ca2+ release from intracellular stores in response to activation by the signaling molecule IP3, which functions to transmit diverse signals received by the cell, e.g. from hormones, neurotransmitters, growth factors and hypertrophic stimuli, to various signaling pathways within the cell. Thus, IP3R channels can be conceptualized as highly dynamic scaffold membrane protein complexes, where binding of ligands can change the scaffold structure leading to cellular Ca2+ signals that direct markedly different cellular actions. Although extensively characterized in physiological and biochemical studies, the detailed mechanisms of how IP3Rs produce highly controlled Ca2+ signals in response to diversified extra- and intracellular stimuli remains unknown and requires high-resolution knowledge of channel molecular architecture. Recently, single-particle electron cryomicroscopy (cryo-EM) has yielded a long-awaited near-atomic resolution structure of the entire full-length type 1 IP3R. This structure provides important insights into the molecular underpinnings of ligand-mediated activation and regulation of IP3R. In this chapter, we evaluate available information and research progress on the structure of IP3R channel in an attempt to shed light on its function.

MCTP2 is a dosage-sensitive gene required for cardiac outflow tract development.

Coarctation of the aorta (CoA) and hypoplastic left heart syndrome (HLHS) have been reported in rare individuals with large terminal deletions of chromosome 15q26. However, no single gene important for left ventricular outflow tract (LVOT) development has been identified in this region. Using array-comparative genomic hybridization, we identified two half-siblings with CoA with a 2.2 Mb deletion on 15q26.2, inherited from their mother, who was mosaic for this deletion. This interval contains an evolutionary conserved, protein-coding gene, MCTP2 (multiple C2-domains with two transmembrane regions 2). Using gene-specific array screening in 146 individuals with non-syndromic LVOT obstructive defects, another individual with HLHS and CoA was found to have a de novo 41 kb intragenic duplication within MCTP2, predicted to result in premature truncation, p.F697X. Alteration of Mctp2 gene expression in Xenopus laevis embryos by morpholino knockdown and mRNA overexpression resulted in the failure of proper OT development, confirming the functional importance of this dosage-sensitive gene for cardiogenesis. Our results identify MCTP2 as a novel genetic cause of CoA and related cardiac malformations.

Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity.

A wide variety of factors influence inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) activity resulting in modulation of intracellular Ca 2+ release. This regulation is thought to define the spatio-temporal patterns of Ca 2+ signals necessary for the appropriate activation of downstream effectors. The binding of both IP 3 and Ca 2+ are obligatory for IP 3 R channel opening, however, Ca 2+ regulates IP 3 R activity in a biphasic manner. Mutational studies have revealed that Ca 2+ binding to a high-affinity pocket formed by the ARM3 domain and linker domain promotes IP 3 R channel opening without altering the Ca 2+ dependency for channel inactivation. These data suggest a distinct low-affinity Ca 2+ binding site is responsible for the reduction in IP 3 R activity at higher [Ca 2+ ]. We determined the consequences of mutating a cluster of acidic residues in the ARM2 and central linker domain reported to coordinate Ca 2+ in cryo-EM structures of the IP 3 R type 3. This site is termed the "CD Ca 2+ binding site" and is well-conserved in all IP 3 R sub-types. We show that the CD site Ca 2+ binding mutants where the negatively charged glutamic acid residues are mutated to alanine exhibited enhanced sensitivity to IP 3 -generating agonists. Ca 2+ binding mutants displayed spontaneous elemental Ca 2+ events (Ca 2+ puffs) and the number of IP 3 -induced Ca 2+ puffs was significantly augmented in cells stably expressing Ca 2+ binding site mutants. When measured with "on-nucleus" patch clamp, the inhibitory effect of high [Ca 2+ ] on single channel-open probability (P o ) was reduced in mutant channels and this effect was dependent on [ATP]. These results indicate that Ca 2+ binding to the putative CD Ca 2+ inhibitory site facilitates the reduction in IP 3 R channel activation when cytosolic [ATP] is reduced and suggest that at higher [ATP], additional Ca 2+ binding motifs may contribute to the biphasic regulation of IP 3 -induced Ca 2+ release.

Understanding IP3R channels: From structural underpinnings to ligand-dependent conformational landscape.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ubiquitously expressed large-conductance Ca2+-permeable channels predominantly localized to the endoplasmic reticulum (ER) membranes of virtually all eukaryotic cell types. IP3Rs work as Ca2+ signaling hubs through which diverse extracellular stimuli and intracellular inputs are processed and then integrated to result in delivery of Ca2+ from the ER lumen to generate cytosolic Ca2+ signals with precise temporal and spatial properties. IP3R-mediated Ca2+ signals control a vast repertoire of cellular functions ranging from gene transcription and secretion to the more enigmatic brain activities such as learning and memory. IP3Rs open and release Ca2+ when they bind both IP3 and Ca2+, the primary channel agonists. Despite overwhelming evidence supporting functional interplay between IP3 and Ca2+ in activation and inhibition of IP3Rs, the mechanistic understanding of how IP3R channels convey their gating through the interplay of two primary agonists remains one of the major puzzles in the field. The last decade has seen much progress in the use of cryogenic electron microscopy to elucidate the molecular mechanisms of ligand binding, ion permeation, ion selectivity and gating of the IP3R channels. The results of these studies, summarized in this review, provide a prospective view of what the future holds in structural and functional research of IP3Rs.

Disrupted Ca2+ homeostasis and immunodeficiency in patients with functional IP3 receptor subtype 3 defects.

Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca2+) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP3R), a homo- or heterotetramer of the IP3R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP3R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP3R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP3R3 in IP3R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype-phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca2+ channels and immunodeficiency and identify IP3Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca2+-associated immunodeficiency from store-operated entry to impaired Ca2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca2+ signaling.

Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca2+* calmodulins.

Voltage-dependent calcium channels (Ca(V)) open in response to changes in membrane potential, but their activity is modulated by Ca(2+) binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca(V)1.2 alpha(1) subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca(2+).CaM in 2 distinct binding modes. The structure of the Ca(V)1.2 fragment is an unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca(2+).CaMs interacting with the pre-IQ regions and a canonical Ca(V)1-IQ-Ca(2+).CaM complex. Native Ca(V)1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca(2+)-dependent inactivation of heterologously expressed Ca(V)1.2 channels.

Conformational motions and ligand-binding underlying gating and regulation in IP3R channel.

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are activated by IP3 and Ca2+ and their gating is regulated by various intracellular messengers that finely tune the channel activity. Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP3R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP3, Ca2+ and ATP, revealing conformational changes that couple ligand-binding to channel opening. Using a deep-learning approach and 3D variability analysis we extracted molecular motions of the key protein domains from cryo-EM density data. We find that IP3 binding relies upon intrinsic flexibility of the ARM2 domain in the tetrameric channel. Our results highlight a key role of dynamic side chains in regulating gating behavior of IP3R channels. This work represents a stepping-stone to developing mechanistic understanding of conformational pathways underlying ligand-binding, activation and regulation of the channel.

Orbital and In-Situ Investigation of Periodic Bedrock Ridges in Glen Torridon, Gale Crater, Mars.

Gale crater, the field site for NASA's Mars Science Laboratory Curiosity rover, contains a diverse and extensive record of aeolian deposition and erosion. This study focuses on a series of regularly spaced, curvilinear, and sometimes branching bedrock ridges that occur within the Glen Torridon region on the lower northwest flank of Aeolis Mons, the central mound within Gale crater. During Curiosity's exploration of Glen Torridon between sols ∼2300-3080, the rover drove through this field of ridges, providing the opportunity for in situ observation of these features. This study uses orbiter and rover data to characterize ridge morphology, spatial distribution, compositional and material properties, and association with other aeolian features in the area. Based on these observations, we find that the Glen Torridon ridges are consistent with an origin as wind-eroded bedrock ridges, carved during the exhumation of Mount Sharp. Erosional features like the Glen Torridon ridges observed elsewhere on Mars, termed periodic bedrock ridges (PBRs), have been interpreted to form transverse to the dominant wind direction. The size and morphology of the Glen Torridon PBRs are consistent with transverse formative winds, but the orientation of nearby aeolian bedforms and bedrock erosional features raise the possibility of PBR formation by a net northeasterly wind regime. Although several formation models for the Glen Torridon PBRs are still under consideration, and questions persist about the nature of PBR-forming paleowinds, the presence of PBRs at this site provides important constraints on the depositional and erosional history of Gale crater.

The dynamic atmospheric and aeolian environment of Jezero crater, Mars.

Despite the importance of sand and dust to Mars geomorphology, weather, and exploration, the processes that move sand and that raise dust to maintain Mars' ubiquitous dust haze and to produce dust storms have not been well quantified in situ, with missions lacking either the necessary sensors or a sufficiently active aeolian environment. Perseverance rover's novel environmental sensors and Jezero crater's dusty environment remedy this. In Perseverance's first 216 sols, four convective vortices raised dust locally, while, on average, four passed the rover daily, over 25% of which were significantly dusty ("dust devils"). More rarely, dust lifting by nonvortex wind gusts was produced by daytime convection cells advected over the crater by strong regional daytime upslope winds, which also control aeolian surface features. One such event covered 10 times more area than the largest dust devil, suggesting that dust devils and wind gusts could raise equal amounts of dust under nonstorm conditions.

Cryo-EM structure of type 1 IP3R channel in a lipid bilayer.

Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is the predominant Ca2+-release channel in neurons. IP3R1 mediates Ca2+ release from the endoplasmic reticulum into the cytosol and thereby is involved in many physiological processes. Here, we present the cryo-EM structures of full-length rat IP3R1 reconstituted in lipid nanodisc and detergent solubilized in the presence of phosphatidylcholine determined in ligand-free, closed states by single-particle electron cryo-microscopy. Notably, both structures exhibit the well-established IP3R1 protein fold and reveal a nearly complete representation of lipids with similar locations of ordered lipids bound to the transmembrane domains. The lipid-bound structures show improved features that enabled us to unambiguously build atomic models of IP3R1 including two membrane associated helices that were not previously resolved in the TM region. Our findings suggest conserved locations of protein-bound lipids among homotetrameric ion channels that are critical for their structural and functional integrity despite the diversity of structural mechanisms for their gating.

Bcl-2-Protein Family as Modulators of IP3 Receptors and Other Organellar Ca2+ Channels.

The pro- and antiapoptotic proteins belonging to the B-cell lymphoma-2 (Bcl-2) family exert a critical control over cell-death processes by enabling or counteracting mitochondrial outer membrane permeabilization. Beyond this mitochondrial function, several Bcl-2 family members have emerged as critical modulators of intracellular Ca2+ homeostasis and dynamics, showing proapoptotic and antiapoptotic functions. Bcl-2 family proteins specifically target several intracellular Ca2+-transport systems, including organellar Ca2+ channels: inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), Ca2+-release channels mediating Ca2+ flux from the endoplasmic reticulum, as well as voltage-dependent anion channels (VDACs), which mediate Ca2+ flux across the mitochondrial outer membrane into the mitochondria. Although the formation of protein complexes between Bcl-2 proteins and these channels has been extensively studied, a major advance during recent years has been elucidating the complex interaction of Bcl-2 proteins with IP3Rs. Distinct interaction sites for different Bcl-2 family members were identified in the primary structure of IP3Rs. The unique molecular profiles of these Bcl-2 proteins may account for their distinct functional outcomes when bound to IP3Rs. Furthermore, Bcl-2 inhibitors used in cancer therapy may affect IP3R function as part of their proapoptotic effect and/or as an adverse effect in healthy cells.

Cryo-EM reveals ligand induced allostery underlying InsP3R channel gating.

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are cation channels that mobilize Ca2+ from intracellular stores in response to a wide range of cellular stimuli. The paradigm of InsP3R activation is the coupled interplay between binding of InsP3 and Ca2+ that switches the ion conduction pathway between closed and open states to enable the passage of Ca2+ through the channel. However, the molecular mechanism of how the receptor senses and decodes ligand-binding signals into gating motion remains unknown. Here, we present the electron cryo-microscopy structure of InsP3R1 from rat cerebellum determined to 4.1 Å resolution in the presence of activating concentrations of Ca2+ and adenophostin A (AdA), a structural mimetic of InsP3 and the most potent known agonist of the channel. Comparison with the 3.9 Å-resolution structure of InsP3R1 in the Apo-state, also reported herein, reveals the binding arrangement of AdA in the tetrameric channel assembly and striking ligand-induced conformational rearrangements within cytoplasmic domains coupled to the dilation of a hydrophobic constriction at the gate. Together, our results provide critical insights into the mechanistic principles by which ligand-binding allosterically gates InsP3R channel.

Structure of IP3R channel: high-resolution insights from cryo-EM.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ubiquitously expressed intracellular Ca2+ channels and the major mediators of cellular Ca2+ signals generated by the release of Ca2+ ions from intracellular stores in response to a variety of extracellular stimuli. Despite established physiological significance and proven involvements of IP3R channels in many human diseases, detailed structural basis for signal detection by these ion channels and their gating remain obscure. Recently, single particle electron cryomicroscopy (cryo-EM) has yielded a long-awaited near-atomic resolution structure of the entire full-length type 1 IP3R. This structure provided exciting mechanistic insights into the molecular assembly of IP3R, revealing the pronounced structural conservation of Ca2+ release channels and raising many fundamental and controversial questions on their activation and gating. Here we summarize the major technological advances that propelled our cryo-EM analysis of IP3R to near-atomic resolution and discuss what the future holds for structural biology of Ca2+ release channels.

Single-Particle Cryo-EM of the Ryanodine Receptor Channel in an Aqueous Environment.

Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca(2+) release channels that are responsible for the increase of cytosolic Ca(2+) concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca(2+) release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryo-microscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryo-EM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structure-function characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants.

Single-particle cryo-EM of the ryanodine receptor channel in an aqueous environment.

Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca2+ release channels that are responsible for the increase of cytosolic Ca2+ concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca2+ release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryo-microscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryo-EM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structure-function characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants.

Constructing and validating initial Cα models from subnanometer resolution density maps with pathwalking.

A significant number of macromolecular structures solved by electron cryo-microscopy and X-ray crystallography obtain resolutions of 3.5-6Å, at which direct atomistic interpretation is difficult. To address this, we developed pathwalking, a semi-automated protocol to enumerate reasonable Cα models from near-atomic resolution density maps without a structural template or sequence-structure correspondence. Pathwalking uses an approach derived from the Traveling Salesman Problem to rapidly generate an ensemble of initial models for individual proteins, which can later be optimized to produce full atomic models. Pathwalking can also be used to validate and identify potential structural ambiguities in models generated from near-atomic resolution density maps. In this work, examples from the EMDB and PDB are used to assess the broad applicability and accuracy of our method. With the growing number of near-atomic resolution density maps from cryo-EM and X-ray crystallography, pathwalking can become an important tool in modeling protein structures.