Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a β-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.

A variety of N-tert-butanesulfinyl imines were reduced with NaBH4 in THF containing 2% water to provide the corresponding secondary sulfinamides in high yield and diastereoselectivity. By using the same sulfinyl imine starting materials and changing the reductant to L-Selectride, the stereoselectivity could be efficiently reversed to afford the opposite product diastereomer in high yield and selectivity.

Abstract-It is suggested that protein kinase A (PKA)-dependent phosphorylation of cardiac ryanodine receptors (RyR2) is linked to the development of heart failure and the generation of fatal cardiac arrhythmias. It is also suggested that RyR2 is phosphorylated to 75% of maximum levels in heart failure resulting in leaky, unregulated channels gating in subconductance states. We now demonstrate that this is unlikely, as RyR2 isolated from nonfailing cardiac muscle is phosphorylated to 75% of maximum at serine-2809, and in this situation, RyR2 displays low open probability (P o ) (0.059Ϯ0.010 [SEM]; nϭ30) and normal regulation of gating by Ca 2ϩ and other ligands. However, when serine-2809 is PKA phosphorylated to maximum levels on RyR2, unique changes in channel behavior are observed. The channel displays enhanced single-channel conductance, very long open states causing large increases in P o , and no evidence of subconductance states. Dephosphorylation of channels by protein phosphatase 1 (from 75% to near 0% at serine-2809) also enhances RyR2 channel activity through abbreviation of closed lifetimes. We propose that channels phosphorylated to 75% of maximum at serine-2809 occupy a natural low point in the RyR2 activity landscape. This optimizes channel control, which can be accomplished either by enhanced or decreased phosphorylation, making the channel particularly sensitive to the kinase:phosphatase balance. Pathological situations such as heart failure might upset this balance and thereby permit prolonged stoichiometric phosphorylation of serine-2809, which would be required for dysregulation of SR Ca 2ϩ release. 7 One model of RyR2 regulation by phosphorylation of serine-2809 has dominated discussions recently and has become something of a paradigm. This model suggests that hyperphosphorylation of RyR2 on serine-2809 (defined as 3 subunits per tetramer phosphorylated at serine-2809 1 ) occurs in heart failure and is a likely cause of the cardiac dysfunction through the pathological phenotype of hyperphosphorylated RyR2 channels. This phenotype is characterized by an enhanced P o and deranged control of channel function (subconductance states and loss of coupled gating 9 ). These results led to the suggestion that hyperphosphorylation of RyR2 at PKA sites can lead to severe defects in excitation contraction (EC) coupling and the generation of fatal arrhythmias. 1,2 Other investigators, however, have failed to support this model, observing no alteration in serine-2809 phosphorylation in heart failure. 4,5 In view of the controversy surrounding the molecular basis of heart failure, we have investigated the effects of PKAdependent phosphorylation on the gating and conduction of RyR2 and related the changes in single-channel function to serine-2809 phosphorylation levels. This study has defined clear functional consequences of RyR2 phosphorylation and evaluated whether serine-2809 phosphorylation is a worthwhile marker for these functional states. We show that PKA-dependent phosphorylation of serine-2809 on RyR...

The ryanodine receptor of cardiac muscle performs a central role in excitation-contraction coupling. Phosphorylation of the channel on serine 2809 (in rabbit or the corresponding serine 2808 in man) alters function in vitro, although the impact of this in vivo has not been established. We have produced a pair of antisera to the serine 2809 phosphorylation site to aid description of the incidence and consequence of phosphorylation of this receptor. One of these antisera is specific for the serine 2809 phosphorylated form of the cardiac ryanodine receptor; the other antiserum is specific for the serine 2809 dephosphorylated receptor. These antibodies have been used to demonstrate that both protein kinase A and calmodulin-dependent kinase II are capable of phosphorylating serine 2809 in vitro. Both kinases phosphorylate serine 2809 to full stoichiometry, but this is accompanied by the incorporation of more (radioactive) phosphate into the receptor by calmodulin-dependent kinase II than by protein kinase A. These data suggest that calmodulin-dependent kinase II phosphorylates at least four sites in addition to serine 2809 in vitro.The ryanodine receptor (RYR2) of cardiac muscle plays a central role in the coupling of electrical excitation of the muscle to mechanical contraction. It is a Ca 2ϩ channel, which resides primarily in the sarcoplasmic reticulum (SR) 1 at junctions between this organelle and the t-tubular system (a specialized invaginated domain of the plasma membrane). Upon depolarization of the plasma membrane, Ca 2ϩ enters the cell across the t-tubule membrane and interacts with the RYR2. Ca 2ϩ binding to RYR2 opens the channel, and Ca 2ϩ stored in the SR moves through the channel into the cytosol to initiate contraction (1).A variety of strategies are used by the cell to regulate RYR2 channel activity. It is anticipated that these regulatory strategies facilitate the fine control of E-C coupling, although evidence for this in live cells is rather limited. In vitro studies have shown that the binding of Ca 2ϩ (2), Mg 2ϩ (3), ATP (4), cADP ribose (5), calmodulin (6), and FKBP12.6 (7) affect channel activity, as does the binding of pharmacological agents such as the plant alkaloid ryanodine (which was originally used to identify the channel protein; Ref. 8). In addition the channel is phosphorylated on at least a single residue, and this phosphorylation alters channel behavior in vitro (7,9).In an effort to understand the regulatory role of RYR2 phosphorylation, research has focused on three aspects of the process. First, the identity of sites of phosphorylation in the receptor and kinases capable of using these sites; second, the functional consequence of site-specific phosphorylation in vitro; and third, the incidence and functional consequence of sitespecific phosphorylation of the receptor in living cells. To date Ser-2809 (in the rabbit sequence (10) or the corresponding Ser-2808 in man (11)) has been identified as a site of phosphorylation on RYR2, which is used in vitro (7,9). It has a counterpart in...

Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-, but not β2-, adrenoceptors (ARs). Compartmentation of β2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of β2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca2+ current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser16. Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during β2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed β2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of β2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit β2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled “Local Signaling in Myocytes”.

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Many cellular functions are regulated by activation of cell-surface receptors that mobilize calcium from internal stores sensitive to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The nature of these internal calcium stores and their localization in cells is not clear and has been a subject of debate. It was originally suggested that the Ins(1,4,5)P3-sensitive store is the endoplasmic reticulum, but a new organelle, the calciosome, identified by its possession of the calcium-binding protein, calsequestrin, and a Ca2+-ATPase-like protein of relative molecular mass 100,000 (100K), has been described as a potential Ins(1,4,5)P3-sensitive calcium store. Direct evidence on whether the calciosome is the Ins(1,4,5)P3-sensitive store is lacking. Using monoclonal antibodies raised against the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, we show that bovine adrenal chromaffin cells contain two Ca2+-ATPase-like proteins with distinct subcellular distributions. A 100K Ca2+-ATPase-like protein is diffusely distributed, whereas a 140K Ca2+-ATPase-like protein is restricted to a region in close proximity to the nucleus. In addition, Ins(1,4,5)P3-generating agonists result in a highly localized rise in cytosolic calcium concentration ([Ca2+]i) initiated in a region close to the nucleus, whereas caffeine results in a rise in [Ca2+]i throughout the cytoplasm. Our results indicate that chromaffin cells possess two calcium stores with distinct Ca2+-ATPases and that the organelle with the 100K Ca2+-ATPase is not the Ins(1,4,5)P3-sensitive store.

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