Lynn Kimlicka

The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule

Many physiological events require transient increases in cytosolic Ca2+ concentrations. Ryanodine receptors (RyRs) are ion channels that govern the release of Ca2+ 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.

Disease mutations in the ryanodine receptor N-terminal region couple to a mobile intersubunit interface.

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.

The structural biology of ryanodine receptors

Ryanodine receptors are ion channels that allow for the release of Ca2+ from the endoplasmic or sarcoplasmic reticulum. They are expressed in many different cell types but are best known for their predominance in skeletal and cardiac myocytes, where they are directly involved in excitation-contraction coupling. With molecular weights exceeding 2 MDa, Ryanodine Receptors are the largest ion channels known to date and present major challenges for structural biology. Since their discovery in the 1980s, significant progress has been made in understanding their behaviour through multiple structural methods. Cryo-electron microscopy reconstructions of intact channels depict a mushroom-shaped structure with a large cytoplasmic region that presents many binding sites for regulatory molecules. This region undergoes significant motions during opening and closing of the channel, demonstrating that the Ryanodine Receptor is a bona fide allosteric protein. High-resolution structures through X-ray crystallography and NMR currently cover ∼11% of the entire protein. The combination of high- and low-resolution methods allows us to build pseudo-atomic models. Here we present an overview of the electron microscopy, NMR, and crystallographic analyses of this membrane protein giant.

The Deletion of Exon 3 in the Cardiac Ryanodine Receptor Is Rescued by β Strand Switching

Mutations in the cardiac Ryanodine Receptor (RYR2) are linked to triggered arrhythmias. Removal of exon 3 results in a severe form of catecholaminergic polymorphic ventricular tachycardia (CPVT). This exon encodes secondary structure elements that are crucial for folding of the N-terminal domain (NTD), raising the question of why the deletion is neither lethal nor confers a loss of function. We determined the 2.3 Å crystal structure of the NTD lacking exon 3. The removal causes a structural rescue whereby a flexible loop inserts itself into the β trefoil domain and increases thermal stability. The exon 3 deletion is not tolerated in the corresponding RYR1 domain. The rescue shows a novel mechanism by which RYR2 channels can adjust their Ca²⁺ release properties through altering the structure of the NTD. Despite the rescue, the deletion affects interfaces with other RYR2 domains. We propose that relative movement of the NTD is allosterically coupled to the pore region.

The cardiac ryanodine receptor N-terminal region contains an anion binding site that is targeted by disease mutations.

Ryanodine receptors (RyRs) are calcium release channels located in the membrane of the endoplasmic and sarcoplasmic reticulum and play a major role in muscle excitation-contraction coupling. The cardiac isoform (RyR2) is the target for >150 mutations that cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and other conditions. Here, we present the crystal structure of the N-terminal region of RyR2 (1-547), an area encompassing 29 distinct disease mutations. The protein folds up in three individual domains, which are held together via a central chloride anion that shields repulsive positive charges. Several disease mutant versions of the construct drastically destabilize the protein. The R420Q disease mutant causes CPVT and ablates chloride binding. The mutation results in reorientations of the first two domains relative to the third domain. These conformational changes likely activate the channel by destabilizing intersubunit interactions that are disrupted upon channel opening.

The CPVT-associated RyR2 mutation G230C enhances store overloadinduced Ca2+ release and destabilizes the N-terminal domains

CPVT (catecholaminergic polymorphic ventricular tachycardia) is an inherited life-threatening arrhythmogenic disorder. CPVT is caused by DADs (delayed after-depolarizations) that are induced by spontaneous Ca2+ release during SR (sarcoplasmic reticulum) Ca2+ overload, a process also known as SOICR (store-overload-induced Ca2+ release). A number of mutations in the cardiac ryanodine receptor RyR2 are linked to CPVT. Many of these CPVT-associated RyR2 mutations enhance the propensity for SOICR and DADs by sensitizing RyR2 to luminal or luminal/cytosolic Ca2+ activation. Recently, a novel CPVT RyR2 mutation, G230C, was found to increase the cytosolic, but not the luminal, Ca2+ sensitivity of single RyR2 channels in lipid bilayers. This observation led to the suggestion of a SOICR-independent disease mechanism for the G230C mutation. However, the cellular impact of this mutation on SOICR is yet to be determined. To this end, we generated stable inducible HEK (human embryonic kidney)-293 cell lines expressing the RyR2 WT (wild-type) and the G230C mutant. Using single-cell Ca2+ imaging, we found that the G230C mutation markedly enhanced the propensity for SOICR and reduced the SOICR threshold. Furthermore, the G230C mutation increased the sensitivity of single RyR2 channels to both luminal and cytosolic Ca2+ activation and the Ca2+-dependent activation of [3H]ryanodine binding. In addition, the G230C mutation decreased the thermal stability of the N-terminal region (amino acids 1-547) of RyR2. These data suggest that the G230C mutation enhances the propensity for SOICR by sensitizing the channel to luminal and cytosolic Ca2+ activation, and that G230C has an intrinsic structural impact on the N-terminal domains of RyR2.

The clinical and genetic spectrum of catecholaminergic polymorphic ventricular tachycardia: findings from an international multicentre registry

Aims Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channelopathy characterized by ventricular arrhythmia during exertion or stress. Mutations in RYR2-coded Ryanodine Receptor-2 (RyR2) and CASQ2-coded Calsequestrin-2 (CASQ2) genes underlie CPVT1 and CPVT2, respectively. However, prognostic markers are scarce. We sought to better characterize the phenotypic and genotypic spectrum of CPVT, and utilize molecular modelling to help account for clinical phenotypes. Methods and results This is a Pediatric and Congenital Electrophysiology Society multicentre, retrospective cohort study of CPVT patients diagnosed at <19 years of age and their first-degree relatives. Genetic testing was undertaken in 194 of 236 subjects (82%) during 3.5 (1.4-5.3) years of follow-up. The majority (60%) had RyR2-associated CPVT1. Variant locations were predicted based on a 3D structural model of RyR2. Specific residues appear to have key structural importance, supported by an association between cardiac arrest and mutations in the intersubunit interface of the N-terminus, and the S4-S5 linker and helices S5 and S6 of the RyR2 C-terminus. In approximately one quarter of symptomatic patients, cardiac events were precipitated by only normal wakeful activities. Conclusion This large, multicentre study identifies contemporary challenges related to the diagnosis and prognostication of CPVT patients. Structural modelling of RyR2 can improve our understanding severe CPVT phenotypes. Wakeful rest, rather than exertion, often precipitated life-threatening cardiac events.

β Strand Switching: A Novel Structural Rescue Mechanism in a Δexon3 Cardiac Ryanodine Receptor Mutant

The contraction of cardiac muscle requires release of Ca2+ from the sarcoplasmic reticulum through the cardiac ryanodine receptor (RyR2). Several mutations in RyR2 are linked to inherited disorders, including triggered cardiac arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT) that may lead to sudden cardiac death. A severe form of CPVT is caused by removal of an entire third exon (Δexon3) of RyR2. The 35 deleted residues form secondary structure elements which are crucial in folding of the N-terminal domain, raising the question of why the deletion is neither lethal nor confers a loss-of-function phenotype. A 2.3A crystal structure shows that the removal results in a structural rescue: an otherwise flexible loop compensates for the loss by inserting itself into the β trefoil domain and increases the thermal stability. The other β strands in the domain show increased mobility to accommodate a sequence that bears no similarity to the deleted exon. The exon3 deletion is not tolerated in the corresponding RyR1 domain. The rescue shows a novel mechanism by which RyR2 channels can adjust their Ca2+ release properties through altering the structure of an individual domain.

The N-Terminal Disease Hot Spot of Ryanodine Receptors Forms a Cytoplasmic Vestibule

Ryanodine receptors (RyR) are ion channels that govern the release of Ca2+ from the endoplasmic reticulum. They thus regulate the contraction of skeletal and cardiac muscle. Mutations in RyR can lead to severe genetic conditions, including (but not limited to) malignant hyperthermia (MH) and catecholaminergic polymorphic ventricular tachycardia (CPVT). Despite the detailed investigation of the functional effects of the mutations, locating their position in the full-length channel structure has traditionally proven to be difficult. Here we present the 2.5 Angstrom resolution crystal structure of a region spanning most of the N-terminal disease hot spot (residues 1-559), containing over 50 disease mutations in RyR1 and RyR2. The hot spot consists of three domains that interact through a predominantly hydrophilic interface. We have been able to dock the position of this hot spot into various RyR1 cryo electron microscopy maps, allowing its unambiguous positioning in the cytoplasmic portion of the channel, forming a 240-kDa ring around the fourfold symmetry axis. The disease mutations can be grouped into three different categories, either destabilizing the interfaces between the three N-terminal domains, affecting the folding of individual domains, or affecting one of six interfaces with other RyR parts. We propose a model whereby the opening of RyR coincides with allosterically coupled motions within the N-terminal domains. This can be affected by mutations that target various interfaces within and across subunits. We also propose a mechanism whereby RyRs are activated by redox modification through the destabilization of observed domain-domain interfaces. The structure provides a framework to understand the many disease mutations that have been studied using functional methods.

Crystallographic Investigation of Several Malignant Hyperthermia and CPVT Mutations in Ryanodine Receptors

Mutations in the Ryanodine Receptor (RYR) are known to underlie many genetic diseases. In particular, the skeletal muscle isoform (RyR1) is involved in malignant hyperthermia (MH) and central core disease (CCD), whereas mutations in the cardiac isoform (RyR2) are known to cause catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD). Despite an extensive analysis of disease mutations on the functional level, very little is known about the structural changes induced by the mutations. Here we analyze the structures and stability of over 10 different disease mutants. We present crystal structures of mutant versions of the RyR1 N-terminal disease hot spot, encoding three domains, and of the RyR2 N-terminal domain, all solved between 2.0 and 3.0 Angstrom, and compare them to the wild type structures. The observed effects on structure and stability differ substantially among the mutants. Whereas some cause a major destabilization of the overall fold, others mainly cause relative domain-domain movements or confer large conformational changes within individual domains. We discuss the likely implications of the disease mutations on the overall structure and gating properties of the intact RyR.