A Ca2+-binding domain in RyR1 that interacts with the calmodulin binding site and modulates channel activity

A fragment of RyR1 (amino acids 4064-4210) is predicted to fold to at least one lobe of calmodulin and to bind Ca(2+). This fragment of RyR1 (R4064-4210) was subcloned, expressed, refolded, and purified. Consistent with the predicted folding pattern, R4064-4210 was found to bind two molecules of Ca(2+) and undergo a structural change upon binding Ca(2+) that exposes hydrophobic amino acids. R4064-4210 also binds to RyR1, the L-type Ca(2+) channel (Cav(1.1)), and several synthetic calmodulin binding peptides. Both R4064-4210 and a peptide representing the calmodulin-binding region of RyR1 (R3614-3643) alter the Ca(2+) dependence of ((3)H)ryanodine binding to RyR1, suggesting that they may both be interfering with an intramolecular interaction between amino acids 4064-4210 and amino acids 3614-3643 in the native RyR1 to alter or regulate the response of the channel to changes in Ca(2+) concentration. The finding that a domain within RyR1 binds Ca(2+) and interacts with calmodulin-binding motifs may provide insights into the mechanism for calcium- and calmodulin-dependent regulation of this channel and perhaps for its regulation by the L-type Ca(2+) channel.     

Common allosteric mechanisms between ryanodine and inositol-1,4,5-trisphosphate receptors

Ryanodine receptors (RyRs) are calcium release channels found in the membrane of the endoplasmic reticulum (ER). We recently described the crystal structure of the RyR1 N-terminal disease hot spot. It is built up by three domains that show clear structural homology with the inositol-1,4,5-triphosphate (IP3) binding core and suppressor domain of IP3 receptors (IP3Rs) . Here we analyze the structural features of the domains in both calcium release channels, and propose a model for the closed state of the IP3R N-terminal region. This model explains the effect of the suppressor domain on the affinity for IP3 and is supported by mutational studies performed previously. We propose a mechanism whereby opening of both RyR and IP3R is allosterically coupled to a displacement of the N-terminal domain from the following two domains. This displacement can be affected by disease mutations, glutathionylation of a highly reactive cysteine residue, or ligand binding.     

Common allosteric mechanisms between ryanodine and inositol-1,4,5-trisphosphate receptors

Ryanodine receptors (RyRs) are calcium release channels found in the membrane of the endoplasmic reticulum (ER). We recently described the crystal structure of the RyR1 N-terminal disease hot spot. It is built up by three domains that show clear structural homology with the inositol-1,4,5-triphosphate (IP3) binding core and suppressor domain of IP3 receptors (IP3Rs) . Here we analyze the structural features of the domains in both calcium release channels, and propose a model for the closed state of the IP3R N-terminal region. This model explains the effect of the suppressor domain on the affinity for IP3 and is supported by mutational studies performed previously. We propose a mechanism whereby opening of both RyR and IP3R is allosterically coupled to a displacement of the N-terminal domain from the following two domains. This displacement can be affected by disease mutations, glutathionylation of a highly reactive cysteine residue, or ligand binding.     

The conserved sites for the FK506-binding proteins in ryanodine receptors and inositol 1,4,5-trisphosphate receptors are structurally and functionally different

We compared the interaction of the FK506-binding protein (FKBP) with the type 3 ryanodine receptor (RyR3) and with the type 1 and type 3 inositol 1,4,5-trisphosphate receptor (IP(3)R1 and IP(3)R3), using a quantitative GST-FKBP12 and GST-FKBP12.6 affinity assay. We first characterized and mapped the interaction of the FKBPs with the RyR3. GST-FKBP12 as well as GST-FKBP12.6 were able to bind approximately 30% of the solubilized RyR3. The interaction was completely abolished by FK506, strengthened by the addition of Mg(2+), and weakened in the absence of Ca(2+) but was not affected by the addition of cyclic ADP-ribose. By using proteolytic mapping and site-directed mutagenesis, we pinpointed Val(2322), located in the central modulatory domain of the RyR3, as a critical residue for the interaction of RyR3 with FKBPs. Substitution of Val(2322) for leucine (as in IP(3)R1) or isoleucine (as in RyR2) decreased the binding efficiency and shifted the selectivity to FKBP12.6; substitution of Val(2322) for aspartate completely abolished the FKBP interaction. Importantly, the occurrence of the valylprolyl residue as alpha-helix breaker was an important determinant of FKBP binding. This secondary structure is conserved among the different RyR isoforms but not in the IP(3)R isoforms. A chimeric RyR3/IP(3)R1, containing the core of the FKBP12-binding site of IP(3)R1 in the RyR3 context, retained this secondary structure and was able to interact with FKBPs. In contrast, IP(3)Rs did not interact with the FKBP isoforms. This indicates that the primary sequence in combination with the local structural environment plays an important role in targeting the FKBPs to the intracellular Ca(2+)-release channels. Structural differences in the FKBP-binding site of RyRs and IP(3)Rs may contribute to the occurrence of a stable interaction between RyR isoforms and FKBPs and to the absence of such interaction with IP(3)Rs.     

Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy

Using cryo-electron microscopy and single particle image processing techniques, we present the first three-dimensional reconstructions of isoform 3 of the ryanodine receptor/calcium release channel (RyR3). Reconstructions were carried out on images obtained from a purified, detergent-solubilized receptor for two different buffer conditions, which were expected to favor open and closed functional states of the channel. As for the heart (RyR2) and skeletal muscle (RyR1) receptor isoforms, RyR3 is a homotetrameric complex comprising two main components, a multidomain cytoplasmic assembly and a smaller ( approximately 20% of the total mass) transmembrane region. Although the isoforms show structural similarities, consistent with the approximately 70% overall sequence identity of the isoforms, detailed comparisons of RyR3 with RyR1 showed one region of highly significant difference between them. This difference indicated additional mass present in RyR1, and it likely corresponds to a region of the RyR1 sequence (residues 1303-1406, known as diversity region 2) that is absent from RyR3. The reconstructions of RyR3 determined under "open" and "closed" conditions were similar to each other in overall architecture. A difference map computed between the two reconstructions reveals subtle changes in conformation at several widely dispersed locations in the receptor, the most prominent of which is a approximately 4 degrees rotation of the transmembrane region with respect to the cytoplasmic assembly.     

Maurocalcine and domain A of the II-III loop of the dihydropyridine receptor Cav 1.1 subunit share common binding sites on the skeletal ryanodine receptor

Maurocalcine is a scorpion venom toxin of 33 residues that bears a striking resemblance to the domain A of the dihydropyridine voltage-dependent calcium channel type 1.1 (Cav1.1) subunit. This domain belongs to the II-III loop of Cav1.1, which is implicated in excitation-contraction coupling. Besides the structural homology, maurocalcine also modulates RyR1 channel activity in a manner akin to a synthetic peptide of domain A. Because of these similarities, we hypothesized that maurocalcine and domain A may bind onto an identical region(s) of RyR1. Using a set of RyR1 fragments, we demonstrate that peptide A and maurocalcine bind onto two discrete RyR1 regions: fragments 3 and 7 encompassing residues 1021-1631 and 3201-3661, respectively. The binding onto fragment 7 is of greater importance and was thus further investigated. We found that the amino acid region 3351-3507 of RyR1 (fragment 7.2) is sufficient for these interactions. Proof that peptide A and maurocalcine bind onto the same site is provided by competition experiments in which binding of fragment 7.2 to peptide A is inhibited by preincubation with maurocalcine. Moreover, when expressed in COS-7 cells, RyR1 carrying a deletion of fragment 7 shows a loss of interaction with both peptide A and maurocalcine. At the functional level, this deletion abolishes the maurocalcine induced stimulation of [3H]ryanodine binding onto microsomes of transfected COS-7 cells without affecting the caffeine and ATP responses.     

Three-dimensional localization of divergent region 3 of the ryanodine receptor to the clamp-shaped structures adjacent to the FKBP binding sites

Of the three divergent regions of ryanodine receptors (RyRs), divergent region 3 (DR3) is the best studied and is believed to be involved in excitation-contraction coupling as well as in channel regulation by Ca(2+) and Mg(2+). To gain insight into the structural basis of DR3 function, we have determined the location of DR3 in the three-dimensional structure of RyR2. We inserted green fluorescent protein (GFP) into the middle of the DR3 region after Thr-1874 in the sequence. HEK293 cells expressing this GFP-RyR2 fusion protein, RyR2(T1874-GFP,) were readily detected by their green fluorescence, indicating proper folding of the inserted GFP. RyR2(T1874-GFP) was further characterized functionally by assays of Ca(2+) release and [(3)H]ryanodine binding. These analyses revealed that RyR2(T1874-GFP) functions as a caffeine- and ryanodine-sensitive Ca(2+) release channel and displays Ca(2+) dependence and [(3)H]ryanodine binding properties similar to those of the wild type RyR2. RyR2(T1874-GFP) was purified from cell lysates in a single step by affinity chromatography using GST-FKBP12.6 as the affinity ligand. The three-dimensional structure of the purified RyR2(T1874-GFP) was then reconstructed using cryoelectron microscopy and single particle image analysis. Comparison of the three-dimensional reconstructions of wild type RyR2 and RyR2(T1874-GFP) revealed the location of the inserted GFP, and hence the DR3 region, in one of the characteristic domains of RyR, domain 9, in the clamp-shaped structure adjacent to the FKBP12 and FKBP12.6 binding sites. COOH-terminal truncation analysis demonstrated that a region between 1815 and 1855 near DR3 is essential for GST-FKBP12.6 binding. These results provide a structural basis for the role of the DR3 region in excitation-contraction coupling and in channel regulation.     

Structural insights into the regulatory mechanism of IP3 receptor

Inositol 1,4,5-trisphosphate receptors (IP(3)R) are intracellular Ca(2+) release channels whose opening requires binding of two intracellular messengers IP(3) and Ca(2+). The regulation of IP(3)R function has also been shown to involve a variety of cellular proteins. Recent biochemical and structural analyses have deepened our understanding of how the IP(3)-operated Ca(2+) channel functions. Specifically, the atomic resolution structure of the IP(3)-binding region has provided a sound structural basis for the receptor interaction with the natural ligand. Electron microscopic studies have also shed light on the overall shape of the tetrameric receptor. This review aims to provide comprehensive overview of the current information available on the structure and function relationship of IP(3)R.     

Structural characterization of the RyR1-FKBP12 interaction

The 12 kDa FK506-binding protein (FKBP12) constitutively binds to the calcium release channel RyR1. Removal of FKBP12 using FK506 or rapamycin causes an increased open probability and an increase in the frequency of sub-conductance states in RyR1. Using cryo-electron microscopy and single-particle image processing, we have determined the 3D difference map of FKBP12 associated with RyR1 at 16 A resolution that can be fitted with the atomic model of FKBP12 in a unique orientation. This has allowed us to better define the surfaces of close apposition between FKBP12 and RyR1. Our results shed light on the role of several FKBP12 residues that had been found critical for the specificity of the RyR1-FKBP12 interaction. As predicted from previous immunoprecipitation studies, our results suggest that Gln3 participates directly in this interaction. The orientation of RyR1-bound FKBP12, with part of its FK506 binding site facing towards RyR1, allows us to propose how FK506 is involved in the dissociation of FKBP12 from RyR1.     

Toward decrypting the allosteric mechanism of the ryanodine receptor based on coarse‐grained structural and dynamic modeling

The ryanodine receptors (RyRs) are a family of calcium (Ca) channels that regulate Ca release by undergoing a closed‐to‐open gating transition in response to action potential or Ca binding. The allosteric mechanism of RyRs gating, which is activated/regulated by ligand/protein binding >200 Å away from the channel gate, remains elusive for the lack of high‐resolution structures. Recent solution of the closed‐form structures of the RyR1 isoform by cryo‐electron microscopy has paved the way for detailed structure‐driven studies of RyRs functions. Toward elucidating the allosteric mechanism of RyRs gating, we performed coarse‐grained modeling based on the newly solved closed‐form structures of RyR1. Our normal mode analysis captured a key mode of collective motions dominating the observed structural variations in RyR1, which features large outward and downward movements of the peripheral domains with the channel remaining closed, and involves hotspot residues that overlap well with key functional sites and disease mutations. In particular, we found a key interaction between a peripheral domain and the Ca‐binding EF hand domain, which may allow for direct coupling of Ca binding to the collective motions as captured by the above mode. This key mode was robustly reproduced by the normal mode analysis of the other two closed‐form structures of RyR1 solved independently. To elucidate the closed‐to‐open conformational changes in RyR1 with amino‐acid level of details, we flexibly fitted the closed‐form structures of RyR1 into a 10‐Å cryo‐electron microscopy map of the open state. We observed extensive structural changes involving the peripheral domains and the central domains, resulting in the channel pore opening. In sum, our findings have offered unprecedented structural and dynamic insights to the allosteric mechanism of RyR1 via modulation of the key collective motions involved in RyR1 gating. The predicted hotspot residues and open‐form conformation of RyR1 will guide future mutational and functional studies. Proteins 2015; 83:2307–2318. © 2015 Wiley Periodicals, Inc.     

Type 2 Ryanodine Receptor Domain A Contains a Unique and Dynamic α-Helix That Transitions to a β-Strand in a Mutant Linked with a Heritable Cardiomyopathy

Ryanodine receptors (RyRs) are large tetrameric calcium (Ca^2 +) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca^2 + sensitivity, propagating sarcoplasmic reticulum luminal Ca^2 + release in the process of excitation–contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1–559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R169Q and delta exon 3 (Δ3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile α-helix, unique to type 2; further, this α2 helix rescues the β-strand lost in RyR2A Δ3 but remains dynamic in the hot-spot loop (HS-loop) P164S, R169Q and R176Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A α2 helix is in close proximity to dense “columns” projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain.     

The role of the S4-S5 linker and C-terminal tail in inositol 1,4,5-trisphosphate receptor function

In previous studies we have suggested that spatial proximity of the C- and N-terminal domains of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) may be critical for the channel gating mechanism. In the present study we have examined the sites of C-N interaction in more detail. We report that deletion mutations within the S4-S5 linker (amino acids 2418-2437) prevent co-immunoprecipitation of the C- and N-terminal domains, inhibit channel activity and enhance IP(3) binding. We also show that a region of the C-terminal tail (amino acids 2694-2721), predicted to be a coiled-coil, is also required for channel activity. Circular dichroism spectroscopy and gel filtration studies confirm that this region has a helical structure with the ability to form tetramers. We propose a model in which IP(3)-induced conformational changes in the N-terminal domain are mechanically transmitted to the opening of the pore through an attachment to the S4-S5 linker. The coiled-coil domain in the C-terminal tail may play a critical role in maintaining the structural integrity of the channel.     

Three-dimensional localization of serine 2808, a phosphorylation site in cardiac ryanodine receptor

Type 2 ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. Phosphorylation of RyR2 by cAMP-dependent protein kinase A and by calmodulin-dependent protein kinase II modulates channel activity. Hyperphosphorylation at a single amino acid residue, Ser-2808, has been proposed to directly disrupt the binding of a 12.6-kDa FK506-binding protein (FKBP12.6) to RyR2, causing a RyR2 malfunction that triggers cardiac arrhythmias in human heart failure. To determine the structural basis of the interaction between Ser-2808 and FKBP12.6, we have employed two independent approaches to map this phosphorylation site in RyR2 by three-dimensional cryo-electron microscopy. In one approach, we inserted a green fluorescent protein (GFP) after amino acid Tyr-2801, and mapped the GFP three-dimensional location in the RyR2 structure. In another approach, the binding site of monoclonal antibody 34C was mapped in the three-dimensional structure of skeletal muscle RyR1. The epitope of antibody 34C has been mapped to amino acid residues 2,756 through 2,803 of the RyR1 sequence, corresponding to residues 2,722 through 2,769 of the RyR2 sequence. These locations of GFP insertion and antibody binding are adjacent to one another in domain 6 of the cytoplasmic clamp region. Importantly, the three-dimensional location of the Ser-2808 phosphorylation site is 105-120 A distance from the FKBP12.6 binding site mapped previously, indicating that Ser-2808 is unlikely to be directly involved in the binding of FKBP12.6 to RyR2, as had been proposed previously.     

Characterization of the binding sites for the interactions between FKBP12 and intracellular calcium release channels

FKBP12, an FK506 binding protein, interacts with type 1 ryanodine receptor (RyR1) and modulates its calcium channel activity. However, there are many opposing reports of FKBP12's interaction with other related calcium channels, such as type 1 IP(3) receptor and type 3 ryanodine receptor (IP(3)R1 and RyR3). In addition, the involvement of the prolyl-dipeptide motif in the calcium channels and the corresponding binding residues in FKBP12 remain controversial. Through pulldown assays with recombinant proteins, we provide biochemical evidence of the interaction between FKBP12 and RyR1, RyR3 and IP(3)R1. Using NMR chemical shift mapping, we show that the important binding residues in FKBP12 are located in its hydrophobic FK506 binding region. Consistently, we demonstrate that FK506 can competitively inhibit the interaction between FKBP12 and the dipeptide motifs of the calcium channels. We believe our results shed lights on the binding mechanism of calcium channel-FKBP12 interaction.     

A skeletal muscle ryanodine receptor interaction domain in triadin

Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200-232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200-232 or 200-231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49±0.11-fold and 1.8±0.15-fold respectively, when added to its luminal side. A similar increase in [(3)H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95's 200-231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction.     

A negatively charged region of the skeletal muscle ryanodine receptor is involved in Ca(2+)-dependent regulation of the Ca(2+) release channel

The ryanodine receptor/Ca(2+) release channels from skeletal (RyR1) and cardiac (RyR2) muscle cells exhibit different inactivation profiles by cytosolic Ca(2+). D3 is one of the divergent regions between RyR1 (amino acids (aa) 1872-1923) and RyR2 (aa 1852-1890) and may contain putative binding site(s) for Ca(2+)-dependent inactivation of RyR. To test this possibility, we have deleted the D3 region from RyR1 (DeltaD3-RyR1), residues 1038-3355 from RyR2 (Delta(1038-3355)-RyR2) and inserted the skeletal D3 into Delta(1038-3355)-RyR2 to generate sD3-RyR2. The channels formed by DeltaD3-RyR1 and Delta(1038-3355)-RyR2 are resistant to inactivation by mM [Ca(2+)], whereas the chimeric sD3-RyR2 channel exhibits significant inactivation at mM [Ca(2+)]. The DeltaD3-RyR1 channel retains its sensitivity to activation by caffeine, but is resistant to inactivation by Mg(2+). The data suggest that the skeletal D3 region is involved in the Ca(2+)-dependent regulation of the RyR1 channel.     

Further characterization of the type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm.

We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RyR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RyR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RyR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RyR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (Po), RyR3 displayed a homogeneous and steeply Ca2+-dependent activity with Po approximately 1. RyR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RyR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RyR3 between [3H]ryanodine binding and channel activity.     

Amino acid residues Gln4020 and Lys4021 of the ryanodine receptor type 1 are required for activation by 4-chloro-m-cresol

The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.     

Localization of an NH(2)-terminal disease-causing mutation hot spot to the "clamp" region in the three-dimensional structure of the cardiac ryanodine receptor

A region between residues 414 and 466 in the cardiac ryanodine receptor (RyR2) harbors more than half of the known NH(2)-terminal mutations associated with cardiac arrhythmias and sudden death. To gain insight into the structural basis of this NH(2)-terminal mutation hot spot, we have determined its location in the three-dimensional structure of RyR2. Green fluorescent protein (GFP), used as a structural marker, was inserted into the middle of this mutation hot spot after Ser-437 in the RyR2 sequence. The resultant GFP-RyR2 fusion protein, RyR2(S437-GFP,) was expressed in HEK293 cells and characterized using Ca(2+) release, [(3)H]ryanodine binding, and single cell Ca(2+) imaging studies. These functional analyses revealed that RyR2(S437-GFP) forms a caffeine- and ryanodine-sensitive Ca(2+) release channel that possesses Ca(2+) and caffeine dependence of activation indistinguishable from that of wild type (wt) RyR2. HEK293 cells expressing RyR2(S437-GFP) displayed a propensity for store overload-induced Ca(2+) release similar to that in cells expressing RyR2-wt. The three-dimensional structure of the purified RyR2(S437-GFP) was reconstructed using cryo-electron microscopy and single particle image processing. Subtraction of the three-dimensional reconstructions of RyR2-wt and RyR2(S437-GFP) revealed the location of the inserted GFP, and hence the NH(2)-terminal mutation hot spot, in a region between domains 5 and 9 in the clamp-shaped structure. This location is close to a previously mapped central disease-causing mutation site located in a region between domains 5 and 6. These results, together with findings from previous studies, suggest that the proposed interactions between the NH(2)-terminal and central regions of RyR2 are likely to take place between domains 5 and 6 and that the clamp-shaped structure, which shows substantial conformational differences between the closed and open states, is highly susceptible to disease-causing mutations.     

Structural insights into excitation—contraction coupling by electron cryomicroscopy

In muscle, excitation-contraction coupling is defined as the process linking depolarization of the surface membrane with Ca²⁺ release from cytoplasmic stores, which activates contraction of striated muscle. This process is primarily controlled by interplay between two Ca²⁺ channels—the voltage-gated L-type Ca²⁺ channel (dihydropyridine receptor, DHPR) localized in the t-tubule membrane and the Ca²⁺-release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum membrane. The structures of both channels have been extensively studied by several groups using electron cryomicroscopy and single particle reconstruction techniques. The structures of RyR, determined at resolutions of 22–30 Å, reveal a characteristic mushroom shape with a bulky cytoplasmic region and the membrane-spanning stem. While the cytoplasmic region exhibits a complex structure comprising a multitude of distinctive domains with numerous intervening cavities, at this resolution no definitive statement can be made about the location of the actual pore within the transmembrane region. Conformational changes associated with functional transitions of the Ca²⁺ release channel from closed to open states have been characterized. Further experiments determined localization of binding sites for various channel ligands. The structural studies of the DHPR are less developed. Although four 3D maps of the DHPR were reported recently at 24–30 Å resolution from studies of frozen-hydrated and negatively stained receptors, there are some discrepancies between reported structures with respect to the overall appearance and dimensions of the channel structure. Future structural studies at higher resolution are needed to refine the structures of both channels and to substantiate a proposed molecular model for their interaction.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1506–1514.Original Russian Text Copyright © 2004 by Serysheva.