Ca2+-mediated activation of the skeletal-muscle ryanodine receptor ion channel

Cryo-electron micrograph studies recently have identified a Ca2+-binding site in the 2,200-kDa ryanodine receptor ion channel (RyR1) in skeletal muscle. To clarify the role of this site in regulating RyR1 activity, here we applied mutational, electrophysiological, and computational methods. Three amino acid residues that interact directly with Ca2+ were replaced, and these RyR1 variants were expressed in HEK293 cells. Single-site RyR1-E3893Q, -E3893V, -E3967Q, -E3967V, and -T5001A variants and double-site RyR1-E3893Q/E3967Q and -E3893V/E3967V variants displayed cellular Ca2+ release in response to caffeine, which indicated that they retained functionality as caffeine-sensitive, Ca2+-conducting channels in the HEK293 cell system. Using [3H]ryanodine binding and single-channel measurements of membrane isolates, we found that single- and double-site RyR1-E3893 and -E3967 variants are not activated by Ca2+. We also noted that RyR1-E3893Q/E3967Q and -E3893V/E3967V variants maintain caffeine- and ATP-induced activation and that RyR1-E3893Q/E3967Q is inhibited by Mg2+ and elevated Ca2+. RyR1-T5001A exhibited decreased Ca2+ sensitivity compared with WT-RyR1 in single-channel measurements. Computational methods suggested that electrostatic interactions between Ca2+ and negatively charged glutamate residues have a critical role in transducing the functional effects of Ca2+ on RyR1. We conclude that the removal of negative charges in the recently identified RyR1 Ca2+-binding site impairs RyR1 activation by physiological Ca2+ concentrations and results in loss of binding to Ca2+ or reduced Ca2+ affinity of the binding site.

Ryanodine receptor ion channels (RyRs) 2 release Ca 2ϩ from intracellular Ca 2ϩ -storing compartments to regulate multiple cellular functions (1)(2)(3)(4). There are three mammalian RyR iso-forms. RyR1 is present in skeletal muscle, RyR2 is present in heart muscle, and RyR3 is present at low levels in many tissues, including brain and slow-twitch skeletal muscle. Calcium ions play a predominant role in the regulation of RyRs. Activation by micromolar Ca 2ϩ and inhibition by millimolar Ca 2ϩ suggest the presence of high-affinity Ca 2ϩ activation and low-affinity Ca 2ϩ inactivation sites. Additional regulation is mediated by ATP and caffeine, which increase Ca 2ϩ -gated RyR activities. Mg 2ϩ inhibits Ca 2ϩ -activated RyRs by competing with Ca 2ϩ for high-affinity Ca 2ϩ activation sites and by binding to inhibitory low-affinity divalent cation sites (3).
Cryo-EM studies have provided detailed information about closed and open structures of the 2,200-kDa RyRs (5)(6)(7)(8)(9)(10)(11)(12). Des Georges et al. (9) determined the location of RyR1-binding sites for endogenous channel activators Ca 2ϩ and ATP and the exogenous activator caffeine (Fig. 1). Ca 2ϩ or ATP/caffeine alone induced structural changes in the large cytosolic domain and primed RyR1 to nearly full open conformation in the presence of the three channel activators (9).
Three conserved amino acid residues contribute to the primary coordination sphere of bound Ca 2ϩ in the large cytoplasmic domain of RyRs. Thr-5001 is conserved in RyRs, whereas RyR1 residues Glu-3893 and Glu-3967 are conserved in both RyRs and the related inositol trisphosphate receptor families. The RyRs and IP 3 receptors are expressed in intracellular membrane compartments containing up to millimolar concentrations of bound and free Ca 2ϩ . The release of Ca 2ϩ is triggered by a Ca 2ϩ -induced Ca 2ϩ release mechanism for the cardiac and brain RyR isoforms and by the action of IP 3 and Ca 2ϩ for the IP 3 receptors (3,13). A distinguishing feature of skeletal muscle is that voltage-sensing L-type Ca 2ϩ channels (DHPRs, Cav1.1s) open juxtaposed RyR1s through direct protein-protein interactions (14). Ca 2ϩ -induced Ca 2ϩ release is regulated on a slow time scale in mammalian skeletal muscle compared with physiological rates of Ca 2ϩ release (15,16). This suggests that while having a predominant role in the regulation of cardiac and brain RyR isoforms, Ca 2ϩ may have a more confined role in the activation of mammalian skeletal-muscle RyRs.
The present study tested the hypothesis that the Ca 2ϩ -binding site identified by cryo-EM (9) has a major role in transducing the functional effects of Ca 2ϩ in RyR1. Five single-site RyR1 variants (E3893Q, E3893V, E3967Q, E3967V, and T5001A) and two double-site RyR1 variants (E3893Q/E3967Q and E3893V/ E3967V) were expressed as caffeine-sensitive, Ca 2ϩ -conducting channels in HEK293 cells. Studies using membrane isolates suggested that Ca 2ϩ did not significantly activate single-and double-site RyR1-E3893 and -E3967 variants, whereas RyR1-T5001A exhibited altered Ca 2ϩ -dependent regulation compared with WT. Computational methods using cryo-EM micrograph densities provided structural information on the interaction between Ca 2ϩ and amino acid residues of the Ca 2ϩbinding sites of RyR1-WT and variant channels.

Results
Three conserved amino acid residues (Glu-3893, Glu-3967, and Thr-5001) were shown previously to directly interact with Ca 2ϩ in a Ca 2ϩ -binding site in RyR1 ( Fig. 1) (9). In the present study, we focused on the role of the two negatively charged glutamates in regulating RyR1 activity. Negative charges were removed by replacing one or both Glu residues with Gln or Val (RyR1-E3893Q, -E3893V, -E3967Q, -E3967V, -E3893Q/ E3967Q, and -E3893V/E3967V) while maintaining residue volumes (17). RyR1 Thr-5001 was mutated to Ala, reducing the size of the side chain at this position. SDS-PAGE and immunoblot analysis indicated that single-and double-site RyR1-E3893 and -E3967 variants were expressed at variable, elevated levels compared with WT and RyR1-T5001A at a level comparable with WT (Fig. 2, top and Table 1).
The expression of functional RyR1 variant channels was monitored in HEK293 cells using the RyR1 agonist caffeine and fluorescence Ca 2ϩ indicator Fluo-4. Millimolar caffeine activates RyR1 (18) and has little effect on Fluo-4 fluorescence in HEK293 cells transfected with the pCMV5 plasmid (Fig. 2, bottom). A variable caffeine-induced Ca 2ϩ release was observed in 30 -60% of HEK293 cells transfected with WT-RyR1. The variable response may have resulted from uneven exposure to caffeine and/or removal of released Ca 2ϩ by HEK293 cellular transport systems. The number of variant HEK293 cells showing caffeine-induced Ca 2ϩ release ranged from 46.5% for RyR1-E3967V to 119% for RyR1-E3893Q compared with WT (Table  1). These observations suggest that all variants expressed caffeine-sensitive, Ca 2ϩ -conducting channels in HEK293 cells.
Two methods were used to determine WT and variant activities. Regulation by Ca 2ϩ was directly measured using the lipid bilayer method (19) (see below). In a second widely used but less direct method, Ca 2ϩ -dependent RyR activity was determined using the RyR-specific plant alkaloid ryanodine (20).
We considered the possibility that the different [ 3 H]ryanodine binding levels of the variants in Fig. 3 could be accounted for by varying levels of expression in HEK293 cells and/or recovery of functional variants in the membrane isolates. We assessed this in recording single channels using the lipid bilayer method, which directly compares WT and variant activities. RyR1s were recorded as K ϩ -conducting channels using 0.25 M KCl on both sides of the bilayer at cytosolic Ca 2ϩ ranging from 0.01 M Ca 2ϩ to 10 mM Ca 2ϩ .
RyR1-WT showed a biphasic Ca 2ϩ activation/inhibition profile with a maximum averaged channel open probability (P o ) of 0.16 at ϳ20 M cytosolic Ca 2ϩ (Fig. 4). RyR1-T5001A resulted in a rightward shift of the P o -Ca 2ϩ activation response curve and reduced peak P o at ϳ100 M Ca 2ϩ compared with WT at 20 M Ca 2ϩ .
In agreement with the [ 3 H]ryanodine-binding data of Fig. 3, RyR1-E3893Q/E3967Q was not significantly activated by Ca 2ϩ . Averaged P o was 0.02 at 0.01 M Ca 2ϩ (Table 1) and half-maximal levels at ϳ6 M Ca 2ϩ (Fig. 4B) suggested that binding of elevated cytosolic Ca 2ϩ had an inhibitory effect (p Ͻ 0.05). One possibility we could not rule out is that the presence of Ca 2ϩinhibitory sites interfered with the activation of RyR1-E3893Q/ E3967Q by the binding of Ca 2ϩ to low-affinity sites. RyR1-E3893Q, -E3893V, -E3967Q, -E3967V, and -E3893V/E3967V had very low averaged P o values of 0.002 and less at Ca 2ϩ ranging from 0.01 to 2 M ( Table 1). The results suggest that the two negatively charged Glu-3893 and Glu-3967 residues have a critical role in transducing the functional effects of Ca 2ϩ in RyR1.
The amino acid mutations could have potentially modified the RyR1 ion permeation properties, resulting in reduced K ϩ conductances and loss of Ca 2ϩ conductances (3). However, measurement of the voltage dependence of variant channel currents indicated that K ϩ conductances were similar to WT (Table 1). In the presence of 10 mM luminal Ca 2ϩ , RyR1-E3893Q/E3967Q and T5001A variants conducted Ca 2ϩ and maintained a Ca 2ϩ /K ϩ permeability ratio similar to WT. The permeability ratio of the remaining variants could not be determined due to low open channel probabilities.
Des Georges et al. (9) determined single-channel activities of the purified RyR1-FK506-binding protein 12.6 (FKBP12.6, Calstabin2) complex reconstituted in lipid bilayer vesicles. FKBP12.6 and the related FKBP12 bind with nanomolar affinity to RyRs and are considered constitutive members of RyR ion channel complexes. Dissociation of FKBP12 from the homotetrameric RyR1 complex increased channel open probability and
RyR1 is activated by ATP and caffeine and inhibited by Mg 2ϩ (3). Single-channel traces (Fig. 5A) and averaged -fold changes of P o values (Fig. 5B) show that at 0.01 M cytosolic Ca 2ϩ , the initial addition of 5 mM caffeine increased RyR1-WT, RyR1-E3893Q/E3967Q, and E3893V/E3967V channel activities from their low P o values ( Table 1). The addition of caffeine signifi-  Table 1 were corrected for number of pCMV5-transfected cells that showed a caffeine signal (ϳ10% of WT transfected cells). A.U., arbitrary units.

Activation of RyR1 by Ca 2؉
cantly increased channel activity using Student's t test. The subsequent addition of 2 mM ATP further increased channel activities (Fig. 5A). One-way but not two-way ANOVA (except for WT) indicated significant increases of P o after the addition of 5 mM caffeine and 2 mM ATP compared with control (Fig. 5B). RyR1-E3893Q/E3967Q was inhibited ϳ10-fold by 0.25 mM Mg 2ϩ (Fig. 5, C and D), whereas inhibition of RyR1-WT and RyR1-E3893V/E3967V was not determined due to very low open channel probability. The results suggest that RyR1-E3893Q/E3967Q and E3893V/E3967V maintained the ability of WT to be activated by caffeine and ATP and of E3893Q/ E3967Q to be inhibited by Mg 2ϩ .
Single RyR1-WT and RyR1-E3893Q/E3967Q, -E3893V/ E3967V, and -T5001A variant channel activities were also determined under conditions comparable with those of open RyR1 channels in the presence of the three channel activators Ca 2ϩ , ATP, and caffeine (9). The addition of 2 mM ATP and 5 mM caffeine to cytosolic 30 M Ca 2ϩ raised single-channel activities of WT and variant channels (Fig. 6A). Averaged P o values of RyR1-WT increased 2.9-fold from 0.20 to 0.57, those of RyR1-E3893Q/E3967Q increased 70-fold from 0.003 to 0.207, those of RyR1-E3893V/E3967V increased 46-fold from 0.0003 to 0.014, and those of RyR1-T5001A increased 11-fold from 0.029 to 0.32 (Fig. 6B), confirming that all three variants retained ATP/caffeine activation.

Discussion
Three amino acid residues that directly interact with Ca 2ϩ in the high-affinity Ca 2ϩ -binding site were mutated and characterized. The results suggest that the high-affinity Ca 2ϩ -binding site plays a critical role in Ca 2ϩ -dependent activation of RyR1. Neutralization of negatively charged Glu-3893 and Glu-3967 resulted in loss of Ca 2ϩ -dependent activation of RyR1. Loss of activation appeared to be specific because other regulatory mechanisms, such as RyR1 ion permeation, inhibition by Mg 2ϩ and elevated levels of Ca 2ϩ , and activation by ATP and caffeine, were maintained in single-channel measurements. RyR1-T5001A differentially affected RyR1 Ca 2ϩ activation by shifting the Ca 2ϩ activation/inactivation curve rightward relative to WT in single-channel measurements. Computational analysis using cryo-EM densities determined the structure of the Ca 2ϩbinding sites of nominally Ca 2ϩ -free and Ca 2ϩ /ATP/caffeineactivated variant channels.
The Ca 2ϩ -binding site is located in the large cytoplasmic side of RyR1 ϳ70 nm from the transmembrane effector channel sites. This suggests that transition from the nominally Ca 2ϩfree closed to the Ca 2ϩ -activated open channel involves additional sites. RyR1-E4032A mutation outside the Ca 2ϩ -binding site (9) exhibited a reduced Ca 2ϩ -dependent channel activity in lipid bilayers (24) and depolarization-induced Ca 2ϩ transients in myotubes (25). Activation of the RyR1-⌬183-4006 deletion variant by micromolar Ca 2ϩ suggested a Ca 2ϩ -activation site different from the one identified by cryo-EM (26). Other regions shown to be involved in activation and inactivation of RyR1 by Ca 2ϩ include residues in the transmembrane helix S2 (27), S4 -S5 linker (28,29), and pore-lining S6 helix of skeletalmuscle and cardiac-muscle RyR isoforms (30,31).
Ca 2ϩ -induced Ca 2ϩ release has been suggested to contribute little to the depolarization-induced Ca 2ϩ release in adult mammalian skeletal muscle (15,16). This raises the question of the physiological significance of Ca 2ϩ activation of RyR1 seen with isolated RyR1s. We reported that the RyR1-G4941K mutation

Activation of RyR1 by Ca 2؉
in the pore-lining S6 helix increased RyR1 sensitivity to luminal Ca 2ϩ (30). This suggested that luminal Ca 2ϩ activates RyR1 by accessing the cytosolic Ca 2ϩ -binding site in the open channel.
Thus an intriguing possibility is that in skeletal muscle, voltagedependent activation of Cav1.1 renders the Ca 2ϩ -binding site accessible to SR luminal Ca 2ϩ passing through the channel and amplifies depolarization-induced Ca 2ϩ release via a Ca 2ϩ -induced Ca 2ϩ release mechanism. Murayama et al. (32) reported results using RyR2 variants that correspond to RyR1-E3893 and -E3967 while this paper was under revision. Alanine substitution of glutamates caused loss of [ 3 H]ryanodine binding to RyR2-E3847A and -E3921A. Decreasing size but not charge resulted in minimal [ 3 H]ryano-dine binding to RyR2-E3847D, whereas RyR2-E3921D had a Ca 2ϩ -binding profile similar to RyR2-WT. The results suggest that, as in RyR1, the corresponding Ca 2ϩ -binding site of RyR2 has a critical role in the regulation by Ca 2ϩ .
Replacement of negatively charged glutamate residues Glu-3893 and Glu-3967 with glutamine and valine resulted in loss of Ca 2ϩ -dependent activation of RyR1. Glu, Gln, and Val residues have a similar residue volume of 140, 147, and 139 Å 3 , respectively (17), suggesting that electrostatic interactions between Ca 2ϩ and Glu residues contributed to transducing the functional effects of Ca 2ϩ .
We determined single-channel and structural properties of the Ca 2ϩ -binding site of RyR1-T5001A, -E3893Q/E3967Q, and -E3893V/E3967V variants under conditions similar to those reported by des Georges et al. (9). Single-channel activities and structure of the purified RyR1-FKBP12.6 (Calstabin2) complex were determined in the absence of Ca 2ϩ , the presence of Ca 2ϩ , the presence of ATP/caffeine, and the presence of Ca 2ϩ /ATP/ caffeine (9). Cryo-EM data sets were divided into four classes in an attempt to account for conformational heterogeneity. Ca 2ϩ or ATP/caffeine alone resulted in constricted closed-pore conformations comparable with the Ca 2ϩ -free closed states, even though a partial opening of channels was observed in the presence of 30 M cytosolic Ca 2ϩ or 2 mM ATP/5 mM caffeine in lipid bilayer studies. In contrast, in the combined presence of the three channel activators Ca 2ϩ , ATP, and caffeine, channels were nearly fully activated in single-channel recordings. Analysis of cryo-EM micrographs of Ca 2ϩ /ATP/caffeine-activated

Activation of RyR1 by Ca 2؉
channels yielded two conformations with a dilated pore (class 1 and 2, PDB code 5TAL) and two with a constricted pore (class 3 and 4, PDB code 5TAQ), which suggested an equal number of open and closed channels. The data suggested that Ca 2ϩ or ATP/caffeine alone primed RyR1 to open in the presence of the three channel activators (9). An alternative explanation was that in the presence of Ca 2ϩ or ATP/caffeine, the number of open channels was too low to be scored as a separate class. In the combined presence of the three channel activators, the number of open channels was proposed to increase, as the equilibrium of channel openings/closings shifted toward the open state(s) (see Fig. S5 of des Georges et al. (9)).
In our single-channel studies, membrane-bound RyR1s were incorporated into lipid bilayers. Fig. 7 shows the structure of the Ca 2ϩ -binding site of RyR1-WT and the in silico structures of RyR1-T5001A, -E3893Q/E3967Q, and -E3893V/E3967V variants. Low P o values and closely related Ca 2ϩ -binding structures were obtained for nominally Ca 2ϩ -free RyR1-WT and RyR1-E3893V/E3967V and -T5001A variants. A weak interaction between the Gln-3893 carboxyamide group and Thr-5001 carbonyl oxygen may have contributed to elevated P o of RyR1-E3893Q/E3967Q compared with WT and the two other variants (Fig. 7 and Table 2). The relative high P o values of RyR1-E3893Q/E3967Q compared with WT and single E3893Q and E3967Q variants also suggested the presence of distinct structural changes among the single-and double-site RyR1-E3893Q and -E3967Q variants.
In the closed RyR1-WT channel in the presence of the three activating ligands Ca 2ϩ , ATP, and caffeine, Ca 2ϩ strongly interacted with Glu-3893 and Glu-3967 carboxyl oxygens and Thr-5001 carbonyl oxygen and weakly with Glu-3893 carbonyl oxygen ( Fig. 7 and Table 2). Small rearrangements in the interaction of Ca 2ϩ with the three amino acid residues, including loss of weak interaction with Glu-3893 carbonyl oxygen, appeared to stabilize open WT channel states.
In RyR1-E3893V/E3967V closed-and open-channel structures, loss of a strong interaction between Ca 2ϩ and Val resulted in a profound decrease of channel activity, reducing P o ϭ 0.57 of WT to 0.014 for RyR1-E3893V/E3967V, which suggested a shift of the equilibrium of channel opening/closing toward the closed state(s). In the RyR1-E3893Q/E3967Q closed-channel structure, Ca 2ϩ ceased to interact with the Gln carboxyamides. However, in contrast to E3893V/E3967V, a strong interaction with Gln carboxyamide oxygens was maintained in the open-channel states. Additionally, hydrogen bonds between three pairs of amino acid residues in the closedchannel state and one pair in the open-channel state were predicted ( Fig. 7 and Table 2). We suggest that the additional interactions in RyR1-E3893Q/E3967Q stabilized the open state(s), decreasing P o ϭ 0.57 of WT to 0.21 for RyR1-E3893Q/E3967Q rather than to 0.014 for RyR1-E3893V/E3967V (Fig. 6B, ϩA/C). Removal of the hydroxyl group and a change in side-chain volume of RyR1-T5001A did not noticeably alter the open and closed Ca 2ϩ -binding structures compared with WT. A shift of the Ca 2ϩ activation/inactivation curve rightward from WT indicated that the T5001A mutation caused secondary structural changes in the presence of the three activating ligands Ca 2ϩ , ATP, and caffeine. Together, the results suggest that electrostatic interactions between Ca 2ϩ and glutamate residues of the RyR1 Ca 2ϩ -binding site have a major role in transducing the functional effects of Ca 2ϩ in RyR1.
In conclusion, our studies show that the removal of negative charges in a RyR1 Ca 2ϩ -binding site impairs activation of RyR1 by physiological concentrations of Ca 2ϩ and suggests loss of binding to or reduced Ca 2ϩ affinity of the site. Ca 2ϩ binding to inhibitory sites is expected to interfere with any low-affinity Ca 2ϩ activation of RyR1. Hence, a more detailed understanding of the mechanism(s) resulting in dysfunctional Ca 2ϩ activation may depend on identifying and eliminating low-affinity RyR1 Ca 2ϩ -inactivation sites.

Materials
[ 3 H]Ryanodine was obtained from PerkinElmer Life Sciences, protease and phosphatase inhibitors from Sigma-Aldrich, and phospholipids from Avanti Polar Lipids.

Preparation of variant channels
RyR1-E3893 and -E3967 single-and double-site variants were prepared using Pfu polymerase-based chain reaction, mutagenic oligonucleotides, and the QuikChange II site-directed mutagenesis kit (Agilent, Santa Clara, CA). RyR1-T5001A was prepared using a gene synthesis method (Genewiz, Inc., South Plainfield, NJ). WT and variant RyR1s were transiently expressed in HEK293 cells using jetPRIME (Polyplus, New York) according to the manufacturer's instructions. Transfected cells were harvested, and crude membrane isolates were prepared as described (33) in the presence of 1 mM GSSG.

SDS-PAGE and immunoblot analyses
Proteins in crude membrane isolates (20 g of protein/lane) were separated using 3-12% acrylamide gradient SDS-PAGE, transferred overnight to nitrocellulose membranes, and probed using primary rabbit anti-RyR1 polyclonal antibody 6425 (30). Immunoblots were developed using peroxidase-conjugated anti-rabbit IgG, enhanced chemiluminescence, and quantified using the Bio-Rad ChemiDoc MP Imaging System and Image-QuantTL analysis software. Intensity of RyR1 variant bands on immunoblots was normalized to RyR1-WT intensities.

Cellular Ca 2؉ release
Release of stored Ca 2ϩ was determined as described (34). Briefly, Ca 2ϩ transients in HEK293 cells grown on coverslips were monitored with the fluorescence Ca 2ϩ indicator Fluo-4. Cellular Ca 2ϩ release was induced by the addition of ϳ8 mM caffeine and monitored in individual cells using the EasyRatioPro algorithm (Photon Technology International, Lawrenceville, NJ).

[ 3 H]Ryanodine binding
Ryanodine binds with high specificity to RyR1 and is widely used to probe RyR activity and content (20).  Shown are the predicted interactions of Ca 2ϩ with RyR1-WT and RyR1-E3893Q/E3967Q, E3893V/E3967V, and T5001A variants under nominally Ca 2ϩ -free (PDB code 5TB0) and Ca 2ϩ /ATP/caffeine (PDB code 5TAQ and 5TAL) conditions. Residues displaying electrostatic interactions with Ca 2ϩ in the Ca 2ϩ /ATP/caffeine state are depicted in a stick representation, and the backbone of RyR1 is shown in a ribbon representation. Ca 2ϩ is shown as a green sphere in Ca 2ϩ -bound states and as a hollow sphere in Ca 2ϩ -free states. Strong electrostatic interactions (d Ͻ 3.5 Å) are shown as blue dashed lines, and weak electrostatic interactions (3.5 Ͻ d Ͻ 4 Å) are shown as red dashed lines between Ca 2ϩ and RyR1 residues. Distances less than 3.5 Å formed interresidue contacts and are shown as blue dashed lines.

Activation of RyR1 by Ca 2؉
the presence of 10 M unlabeled ryanodine. Amounts of bound [ 3 H]ryanodine were determined using a filtration assay (30).

Single-channel recordings
Membrane isolates were added to the cis (cytosolic) chamber of the bilayer apparatus (35). Single-channel recordings took advantage of the impermeability of RyRs to Cl Ϫ and high conductance of K ϩ relative to Ca 2ϩ . Channel activities were recorded using 0.25 M KCl, 20 mM KHEPES, pH 7.4, on both sides of the bilayer, 2 M trans (SR luminal), and the indicated cis (cytosolic) Ca 2ϩ concentrations. The trans side of the bilayer was defined as ground. Electrical signals were filtered at 2 kHz, digitized at 10 kHz, and analyzed at 50% threshold setting (35). Data acquisition and analysis of 2-min recordings were performed using commercially available software (pClamp, Axon Instruments). Channel activities were also recorded in symmetrical 0.25 M KCl solution with 10 mM Ca 2ϩ in the trans bilayer chamber. The reversal potential was measured to determine Ca 2ϩ /K ϩ permeability ratios using a modified form of the Goldman-Hodgkin-Katz equation (35).

Computational methods
The effects of RyR1 mutations on Ca 2ϩ binding were modeled by performing in silico amino acid substitutions at chosen positions in three RyR1 cryo-EM structures solved under different physiological conditions (PDB entries 5TB0, 5TAQ, and 5TAL) (9). The position of Ca 2ϩ in the Ca 2ϩ -free RyR1 struc-ture (PDB code 5TB0) was simulated by superposing the closed RyR1 structure (PDB code 5TAQ). In silico mutations at chosen residue positions in RyR1 structures were performed using the Mutagenesis tool in the PyMOL molecular visualization suite (36). We chose side-chain rotamers for substituted amino acids at selected positions based on backbone dependence and minimum clash score. For further refinement, side-chain optimizations were executed on mutated RyR1 structures using GROMACS version 4 (37). Optimized structures were visualized and analyzed for loss or gain of Ca 2ϩ interactions with surrounding residues using PyMOL (36).

Biochemical assays and data analysis
Free Ca 2ϩ concentrations were obtained by including in the solutions the appropriate amounts of Ca 2ϩ and EGTA using a Ca 2ϩ -selective electrode. Free Ca 2ϩ concentrations following the addition of 2 mM ATP (Fig. 6) were calculated using Max-Chelator and constants from Theo Schoenmakers' Chelator. Differences between samples were analyzed using SigmaPlot 11 Statistics. Comparison of two groups was determined by Student's t test or Mann-Whitney rank sum test (when data failed the normality test). Three sample groups or more were determined by one-way ANOVA with Tukey's test, Kruskal-Wallis one-way ANOVA on ranks followed by Dunn's method (when the normality test failed in one-way ANOVA), or twoway ANOVA using the Holm-Sidak method, where p Ͻ 0.05 was considered significant.