Skeletal muscle uses voltage sensors in the transverse tubular membrane that are linked by protein-protein interactions to intracellular ryanodine receptors, which gate the release of calcium from the sarcoplasmic reticulum. Here we show, by using voltage-clamped single fibres and confocal imaging, that stochastic calcium-release events, visualized as Ca2+ sparks, occur in skeletal muscle and originate at the triad. Unitary triadic Ca(2+)-release events are initiated by the voltage sensor in a steeply voltage-dependent manner, or occur spontaneously by a mechanism independent of the voltage sensor. Large-amplitude events also occur during depolarization and consist of two or more unitary events. We propose a 'dual-control' model for discrete Ca2+ release events from the sacroplasmic reticulum that unifies diverse observations about Ca(2+)-signalling in frog skeletal muscle, and that may be applicable to other excitable cells.

A three dimensional (3D) model of Ca(2+) diffusion and binding within a sarcomere of a myofibril, including Ca(2+) binding sites troponin, parvalbumin, sarcoplasmic reticulum Ca(2+) pump, and fluorescent Ca(2+)-indicator dye (fluo-3), was developed to numerically simulate laser scanning confocal microscope images of Ca(2+) "sparks" in skeletal muscle. Diffusion of free dye (D), calcium dye (CaD), and Ca(2+) were included in the model. The Ca(2+) release current was assumed to last 8 ms, to arise within 4 x 10(-5) microm(3) at the triad and to be constant during release. Line scan confocal fluorescence images of Ca(2+) sparks were simulated by 3D convolution of the calculated distribution of CaD with a Gaussian kernel approximating the point spread function of the microscope. Our results indicate that the amplitude of the simulated spark is proportional to the Ca(2+) release current if all other model parameters are constant. For a given release current, the kinetic properties and concentrations of the binding sites and the diffusion parameters of D, CaD, and Ca(2+) all have significant effects on the simulated Ca(2+) sparks. The simulated sparks exhibited similar amplitudes and temporal properties, but less spatial spread than experimentally observed sparks.

Halocyclization of alkenes is a powerful bond-forming tool in synthetic organic chemistry and a key step in natural product biosynthesis, but catalyzing halocyclization with high enantioselectivity remains a challenging task. Identifying suitable enzymes that catalyze enantioselective halocyclization of simple olefins would therefore have significant synthetic value. Flavin-dependent halogenases (FDHs) catalyze halogenation of arene and enol(ate) substrates. Herein, we reveal that FDHs engineered to catalyze site-selective aromatic halogenation also catalyze non-native bromolactonization of olefins with high enantioselectivity and near-native catalytic proficiency. Highly selective halocyclization is achieved by characterizing and mitigating the release of HOBr from the FDH active site using a combination of reaction optimization and protein engineering. The structural origins of improvements imparted by mutations responsible for the emergence of halocyclase activity are discussed. This expansion of FDH catalytic activity presages the development of a wide range of biocatalytic halogenation reactions.