Abstract-Hyperphosphorylation of the cardiac Ca 2ϩ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca 2ϩ dependence of [ 3 H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute ␤-adrenergic stimulation, and that

As an inhibitor of Ca 2؉ release through ryanodine receptor (RYR) channels, the skeletal muscle relaxant dantrolene has proven to be both a valuable experimental probe of intracellular Ca 2؉ signaling and a lifesaving treatment for the pharmacogenetic disorder malignant hyperthermia. However, the molecular basis and specificity of the actions of dantrolene on RYR channels have remained in question. Here we utilize 2؉ . In contrast to the RYR1 isoform, the cardiac RYR2 isoform was unaffected by dantrolene, both in native cardiac SR vesicles and when heterologously expressed in HEK-293 cells. By comparison, the RYR3 isoform expressed in HEK-293 cells was significantly inhibited by dantrolene, and the extent of RYR3 inhibition was similar to that displayed by the RYR1 in native SR vesicles. Our results thus indicate that both the RYR1 and the RYR3, but not the RYR2, may be targets for dantrolene inhibition in vivo.

To investigate the channel properties of the mammalian type 3 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed the cDNA in HEK293 cells. Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca 2؉ release measurements using the fluorescence Ca 2؉ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine-and ryanodine-sensitive Ca 2؉ Ryanodine receptors are a family of intracellular Ca 2ϩ release channels that were originally identified in the sarcoplasmic reticulum (SR) 1 of striated muscles. To date, three members of this family have been identified in mammalian tissues, namely the skeletal muscle (RyR1), the cardiac muscle (RyR2), and the brain (RyR3) ryanodine receptor. These proteins are the products of different genes and share 66 -70% amino acid sequence identity (1-5). Earlier studies using RNA blot analysis revealed that the expression patterns of these isoforms were very different (6). RyR1 was predominantly expressed in skeletal muscle, whereas RyR2 was mainly expressed in heart and brain. The expression of RyR3 was detected in smooth muscle tissues and certain regions of the brain. However, results of recent ribonuclease protection assay demonstrate that all three RyR isoforms are widely and differentially expressed (7). These studies also indicate that most tissues express more than one RyR isoform. For example, skeletal muscles express both RyR1 and RyR3, although RyR3 is expressed at a much lower level than that of RyR1.

The 12.6-kDa FK506-binding protein (FKBP12.6) interacts with the cardiac ryanodine receptor (RyR2) and modulates its channel function. However, the molecular basis of FKBP12.6-RyR2 interaction is poorly understood. To investigate the significance of the isoleucineproline (residues 2427-2428) dipeptide epitope, which is thought to form an essential part of the FKBP12.6 binding site in RyR2, we generated single and double mutants, P2428Q, I2427E/P2428A, and P2428A/L2429E, expressed them in HEK293 cells, and assessed their ability to bind GST-FKBP12.6. None of these mutations abolished GST-FKBP12.6 binding, indicating that this isoleucine-proline motif is unlikely to form the core of the FKBP12.6 binding site in RyR2. To systematically define the molecular determinants of FKBP12.6 binding, we constructed a series of internal and NH 2 -and COOHterminal deletion mutants of RyR2 and examined the effect of these deletions on GST-FKBP12.6 binding. These deletion analyses revealed that the first 305 NH 2 -terminal residues and COOH-terminal residues 1937-4967 are not essential for GST-FKBP12.6 binding, whereas multiple sequences within a large region between residues 305 and 1937 are required for GST-FKBP12.6 interaction. Furthermore, an NH 2 -terminal fragment containing the first 1937 residues is sufficient for GST-FKBP12.6 binding. Co-expression of overlapping NH 2 and COOH-terminal fragments covering the entire sequence of RyR2 produced functional channels but did not restore GST-FKBP12.6 binding. These data suggest that FKBP12.6 binding is likely to be conformationdependent. Binding of FKBP12.6 to the NH 2 -terminal domain may play a role in stabilizing the conformation of this region.

We have investigated the molecular basis for ryanodine receptor (RyR) activation by Ca 2؉ by using sitedirected mutagenesis together with functional assays consisting of Ca 2؉ release measurements and single channel recordings in planar lipid bilayers. We report here that a single substitution of alanine for glutamate at position 3885 (located in the putative transmembrane sequence M2 of the type 3 RyR) reduces the Ca 2؉ sensitivity, as measured by single channel activation, by more than 10,000-fold, without apparent changes in channel conductance and in modulation by other ligands (e.g. ATP and ryanodine). Co-expression of the wild type and mutant RyR proteins results in the synthesis of single channels that have intermediate Ca 2؉sensitivities. These results suggest that the glutamates at position 3885 of each monomer may act in a coordinated way to form the Ca 2؉ sensor in the tetrameric structure corresponding to RyR.Ryanodine receptors (RyRs) 1 are a family of Ca 2ϩ channels which mediate intracellular Ca 2ϩ release that is essential for a variety of cellular functions including muscle contraction, egg fertilization, and synaptic transmission (1, 2). Three RyR isoforms (RyR1, RyR2, and RyR3) have been identified in mammalian tissues; all three are activated by Ca 2ϩ (3)(4)(5)(6)(7)(8). Activation of RyR by Ca 2ϩ is the mechanism underlying Ca 2ϩ -induced Ca 2ϩ release from the sarco(endo)plasmic reticulum (9 -11).Of the many ligands known to modulate the activity of RyR, Ca 2ϩ is the essential regulator. Most other ligands exert their effect on RyR activity by influencing the Ca 2ϩ sensitivity of 12). Alterations in the Ca 2ϩ sensitivity of RyR have been implicated in at least one disease, malignant hyperthermia (13). Thus, understanding the molecular mechanism that controls the Ca 2ϩ sensitivity is fundamental to the understanding of RyR regulation and intracellular Ca 2ϩ signaling. RyR activation by Ca 2ϩ is thought to be mediated by high affinity Ca 2ϩ binding sites in the protein (14), but the molecular identity of these Ca 2ϩ activation sites, the Ca 2ϩ sensor, has yet to be defined. It has been shown that negatively charged residues within a transmembrane sequence are often involved in binding and translocation of cations across the membrane (15-17). Analysis of the amino acid sequences of RyRs reveals that of the 12 predicted transmembrane sequences of RyR (18), four (M1, M2, M7, and M10) contain negatively charged amino acid residues that are conserved in all known RyR isoforms ( Fig. 1A) (19 -26). To investigate their roles in RyR function, we have mutated these negatively charged residues in the rabbit type 3 RyR. The functional consequence of one of these point mutations, a glutamate-to-alanine mutation at position 3885 (E3885A) located in the M2 transmembrane sequence (Fig. 1B), was assessed. Our results demonstrate that glutamate 3885 plays an essential role in determining the Ca 2ϩ sensitivity and provide important new insights into the Ca 2ϩ -sensing mechanism of RyR. EXPERIMENTAL PROCEDUR...

Abstract-We recently showed that phosphoinositide-3-kinase-␥-deficient (PI3K␥ Ϫ/Ϫ ) mice have enhanced cardiac contractility attributable to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca 2ϩ content and release but not L-type Ca 2ϩ current (I Ca,L ), demonstrating PI3K␥ locally regulates cAMP levels in cardiomyocytes. Because phosphodiesterases (PDEs) can contribute to cAMP compartmentation, we examined whether the PDE activity was altered by PI3K␥ ablation. Selective inhibition of PDE3 or PDE4 in wild-type (WT) cardiomyocytes elevated Ca 2ϩ transients, SR Ca 2ϩ content, and phospholamban phosphorylation (PLN-PO 4 ) by similar amounts to levels observed in untreated PI3K␥

Isoform 2 of the ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. In the present study, two kinds of RyR2 cDNA were constructed, one encoding the wild type mouse RyR2 (RyR2 wt ) and the other encoding modified RyR2, into which was inserted a cDNA encoding green fluorescent protein (GFP). GFP was inserted into the divergent region 1 (DR1) of RyR2, after the Asp-4365 (RyR2 D4365-GFP ). HEK293 cells expressing both RyR2 wt and RyR2 D4365-GFP cDNAs showed caffeine-and ryanodine-sensitive calcium release, demonstrating that both wild type and modified RyR2s form functional calcium release channels. Cells expressing the fusion protein, RyR2 D4365-GFP , were readily identified by their fluorescence due to the presence of GFP, indicating that the inserted GFP folded properly. Both expressed RyR2s were purified from cell lysates in a single step by affinity chromatography using a GST-FKBP12.6 as the affinity ligand. Cryoelectron microscopy of purified RyR2s showed structurally intact receptors, and three-dimensional reconstructions were obtained by single particle image processing. The three-dimensional reconstruction of RyR2 wt appeared very similar to that of the native RyR2 purified from dog heart. The location of the inserted GFP, and consequently of DR1, was mapped on the three-dimensional structure of RyR2 to one of the subunit's characteristic domains, domain 3, also known as the "handle" domain. This study describes the first internal fusion of a protein into a ryanodine receptor, and it demonstrates the potential of this technology for localizing functional and structural domains on the threedimensional structure of RyR.

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 threedimensional 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.