Abstract-Ventricular tachycardia (VT) is the leading cause of sudden death, and the cardiac ryanodine receptor (RyR2) is emerging as an important focus in its pathogenesis. RyR2 mutations have been linked to VT and sudden death, but their precise impacts on channel function remain largely undefined and controversial. We have previously shown that several disease-linked RyR2 mutations in the C-terminal region enhance the sensitivity of the channel to activation by luminal Ca 2ϩ . Cells expressing these RyR2 mutants display an increased propensity for spontaneous Ca 2ϩ release under conditions of store Ca 2ϩ overload, a process we referred to as store overload-induced Ca 2ϩ release (SOICR). To determine whether common defects exist in disease-linked RyR2 mutations, we characterized 6 more RyR2 mutations from different regions of the channel. Stable inducible HEK293 cell lines expressing Q4201R and I4867M from the C-terminal region, S2246L and R2474S from the central region, and R176Q(T2504M) and L433P from the N-terminal region were generated. All of these cell lines display an enhanced propensity for SOICR. HL-1 cardiac cells transfected with disease-linked RyR2 mutations also exhibit increased SOICR activity. Single channel analyses reveal that disease-linked RyR2 mutations primarily increase the channel sensitivity to luminal, but not to cytosolic, Ca

SUMMARY Within dendritic spines, actin is presumed to anchor receptors in the postsynaptic density and play numerous roles regulating synaptic transmission. However, the submicron dimensions of spines have limited examination of actin dynamics within spines, and prevented live-cell discrimination of perisynaptic actin filaments. Using photoactivated localization microscopy, we measured movement of individual actin molecules within living spines. Velocity of single actin molecules along filaments, an index of filament polymerization rate, was highly heterogeneous within individual spines. Most strikingly, molecular velocity was elevated in discrete, well-separated foci occurring not principally at the spine tip, but in subdomains throughout the spine, including the neck. Whereas actin velocity on filaments at the synapse was substantially elevated, those at the endocytic zone showed no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.

Spontaneous Ca2+ release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca2+ release (SOICR) can result in Ca2+ waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here, we show that a point mutation E4872A in the helix bundle crossing (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, RyR2 Ca2+ activation. Introducing metal-binding histidines at this site converts RyR2 into a luminal Ni2+ gated channel. Mouse hearts harboring an RyR2 mutation at this site (E4872Q+/−) are resistant to store overload-induced Ca2+ waves and completely protected against Ca2+-triggered VTs. These data show that the RyR2 gate directly senses store Ca2+, explaining RyR2 store Ca2+ regulation, Ca2+ wave initiation, and Ca2+-triggered arrhythmias. This novel store-sensing gate structure is conserved in all RyRs and inositol 1,4,5-trisphosphate receptors.

Through the interplay of high-resolution scanning tunneling microscopy (STM) imaging/manipulation and density functional theory (DFT) calculations, we have demonstrated that an unprecedented selective aryl-aryl coupling via direct C-H bond activation can be successfully achieved on Cu(110). These findings present a simple and generalized route for preparing low dimensional carbon nanomaterials.

On-surface fabrication of covalently interlinked conjugated nanostructures has attracted significant attention, mainly because of the high stability and efficient electron transport ability of these structures. Here, from the interplay of scanning tunneling microscopy imaging and density functional theory calculations, we report for the first time on-surface formation of one-dimensional polyphenylene chains through Bergman cyclization followed by radical polymerization on Cu(110). The formed surface nanostructures were further corroborated by the results for the ex situ-synthesized molecular product after Bergman cyclization. These findings are of particular interest and importance for the construction of molecular electronic nanodevices on surfaces.

Caffeine has long been used as a pharmacological probe for studying ryanodine receptor (RyR)-mediated Ca2+ release and cardiac arrhythmias. However, the precise mechanism by which caffeine activates RyRs is elusive. Here we investigated the effects of caffeine on spontaneous Ca2+ release and on the response of single cardiac RyR (RyR2) channels to luminal or cytosolic Ca2+. We found that HEK293 cells expressing RyR2 displayed partial or “quantal” Ca2+ release in response to repetitive additions of submaximal concentrations of caffeine. This quantal Ca2+ release was abolished by ryanodine. Monitoring of endoplasmic reticulum luminal Ca2+ revealed that caffeine reduced the luminal Ca2+ threshold at which spontaneous Ca2+ release occurs. Interestingly, spontaneous Ca2+ release in the form of Ca2+ oscillations persisted in the presence of 10 mM caffeine, and was diminished by ryanodine, demonstrating that unlike ryanodine, caffeine, even at high concentrations, does not hold the channel open. At the single channel level, caffeine markedly reduced the threshold for luminal Ca2+ activation, but had little effect on the threshold for cytosolic Ca2+ activation, indicating that the major action of caffeine is to reduce the luminal, but not the cytosolic, Ca2+ activation threshold. Furthermore, as with caffeine, the clinically relevant, pro-arrhythmic methylxanthines aminophylline and theophylline potentiated luminal Ca2+ activation of RyR2, and increased the propensity for spontaneous Ca2+ release, mimicking the effects of diseased-linked RyR2 mutations. Collectively, our results demonstrate that caffeine triggers Ca2+ release by reducing the threshold for luminal Ca2+ activation of RyR2, and suggest that disease-linked RyR2 mutations and RyR2-interacting pro-arrhythmic agents may share the same arrhythmogenic mechanism.

The 12.6-kDa FK506-binding protein (FKBP12.6) is considered to be a key regulator of the cardiac ryanodine receptor (RyR2), but its precise role in RyR2 function is complex and controversial. In the present study we investigated the impact of FKBP12.6 removal on the properties of the RyR2 channel and the propensity for release or store overload-induced Ca 2؉ release (SOICR). FK506 increased the amplitude and decreased the frequency of SOICR in HEK293 cells expressing RyR2 with or without FKBP12.6, indicating that the action of FK506 on SOICR is independent of FKBP12.6. As with recombinant RyR2, the conductance and ligand-gating properties of single RyR2 channels from FKBP12.6-null mice were indistinguishable from those of single wild type channels. Moreover, FKBP12.6-null mice did not exhibit enhanced susceptibility to stress-induced ventricular arrhythmias, in contrast to previous reports. Collectively, our results demonstrate that the loss of FKBP12.6 has no significant effect on the conduction and activation of RyR2 or the propensity for spontaneous Ca 2؉ release and stress-induced ventricular arrhythmias.

The phosphorylation of the cardiac Ca 2؉ -release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) has been extensively characterized, but its functional consequence remains poorly defined and controversial. We have previously shown that RyR2 is phosphorylated by PKA at two major sites, serine 2030 and serine 2808, of which Ser-2030 is the major PKA site responding to ␤-adrenergic stimulation. Here we investigated the effect of the phosphorylation of RyR2 by PKA on the properties of single channels and on spontaneous Ca 2؉ release during sarcoplasmic reticulum Ca 2؉ overload, a process we have referred to as store overload-induced Ca 2؉ release (SOICR). We found that PKA activated single RyR2 channels in the presence, but not in the absence, of luminal Ca 2؉ . On the other hand, PKA had no marked effect on the sensitivity of the RyR2 channel to activation by cytosolic Ca 2؉ . Importantly, the S2030A mutation, but not mutations of Ser-2808, diminished the effect of PKA on RyR2. Furthermore, a phosphomimetic mutation, S2030D, potentiated the response of RyR2 to luminal Ca 2؉ and enhanced the propensity for SOICR in HEK293 cells. In intact rat ventricular myocytes, the activation of PKA by isoproterenol reduced the amplitude and increased the frequency of SOICR. Confocal line-scanning fluorescence microscopy further revealed that the activation of PKA by isoproterenol increased the rate of Ca 2؉ release and the propagation velocity of spontaneous Ca 2؉ waves, despite reduced wave amplitude and resting cytosolic Ca 2؉ . Collectively, our data indicate that PKA-dependent phosphorylation enhances the response of RyR2 to luminal Ca 2؉ and reduces the threshold for SOICR and that this effect of PKA is largely mediated by phosphorylation at Ser-2030. Ventricular tachycardia (VT)4 is the leading cause of sudden death, particularly in patients with heart failure (HF), but the molecular mechanisms underlying the high incidence of VT in HF are not completely understood (1). A major cause of VT is believed to be delayed afterdepolarizations, which are produced by spontaneous Ca 2ϩ release from the sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) during SR Ca 2ϩ overload (2-5), a process we referred to as store overload-induced Ca 2ϩ release (SOICR) (6, 7). Physical or emotional stresses, which activate the ␤-adrenergic receptor (AR)/protein kinase A (PKA) signaling pathway, are common triggers for SOICR. Marks' group has shown that RyR2 is phosphorylated by PKA at a single residue, Ser-2808 (10, 11), which was originally identified as a unique Ca 2ϩ -and calmodulin-dependent protein kinase II phosphorylation site (12,13 (23, 24). These results are seemingly inconsistent with those of in vitro studies. The reasons for this apparent discrepancy are unknown.Moderate modulation of RyR2 activity has been shown to have no sustained effect on stimulated SR Ca 2ϩ release due to the regulation of RyR2 by luminal Ca 2ϩ , a phenomenon often referred to as "SR auto-regulation" (25), but it does exert a ...