Ca2+-induced Ca2+ release is a general mechanism that most cells use to amplify Ca2+ signals. In heart cells, this mechanism is operated between voltage-gated L-type Ca2+ channels (LCCs) in the plasma membrane and Ca2+ release channels, commonly known as ryanodine receptors, in the sarcoplasmic reticulum. The Ca2+ influx through LCCs traverses a cleft of roughly 12 nm formed by the cell surface and the sarcoplasmic reticulum membrane, and activates adjacent ryanodine receptors to release Ca2+ in the form of Ca2+ sparks. Here we determine the kinetics, fidelity and stoichiometry of coupling between LCCs and ryanodine receptors. We show that the local Ca2+ signal produced by a single opening of an LCC, named a 'Ca2+ sparklet', can trigger about 4-6 ryanodine receptors to generate a Ca2+ spark. The coupling between LCCs and ryanodine receptors is stochastic, as judged by the exponential distribution of the coupling latency. The fraction of sparklets that successfully triggers a spark is less than unity and declines in a use-dependent manner. This optical analysis of single-channel communication affords a powerful means for elucidating Ca2+-signalling mechanisms at the molecular level.

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.

Intracellular Ca 2؉ release in many types of cells is mediated by ryanodine receptor Ca 2؉ release channels (RyRCs) that are assembled into two-dimensional paracrystalline arrays in the endoplasmic͞sarcoplasmic reticulum. However, the in situ operating mechanism of the RyRC array is unknown. Here T he ryanodine receptor Ca 2ϩ release channel (RyRC) is a prototypical member of the Ca 2ϩ release channel superfamily located in the endoplasmic reticulum͞sarcoplasmic reticulum (SR) of eukaryotic cells and plays a pivotal role in intracellular Ca 2ϩ signaling (1-5). Instances of local Ca 2ϩ release, in the form of ''Ca 2ϩ sparks'' or their equivalents, constitute the elementary Ca 2ϩ signaling events in heart, brain, and muscle cells (6-12). Intriguingly, RyRCs in intact cells are almost exclusively found at discrete spark-generating sites, where Ϸ100 channels are assembled into two-dimensional paracrystalline arrays (13)(14)(15). This pattern of RyRC organization seems to be highly conserved from crustaceans to vertebrates (13,15,16), suggesting that array formation is critical to RyRC-mediated Ca 2ϩ signaling in vivo. Thus, understanding array-based RyRC behavior is of fundamental importance for elucidation of intracellular Ca 2ϩ signaling mechanism.Despite a wealth of information on behavior of RyRCs in planar lipid bilayers and other cell-free systems (5,(17)(18)(19)(20), little is known on how RyRCs operate in situ; many fundamental issues regarding the genesis and termination of Ca 2ϩ sparks remain unanswered (5,21,22). Based on in vitro properties of RyRCs, the classic Ca 2ϩ -induced Ca 2ϩ release (CICR) mechanism (23) would predict an all-or-none activation of the RyRC array and an everlasting local Ca 2ϩ release, which cannot explain the prompt termination of Ca 2ϩ sparks and the microstability of intracellular signaling (24, 25). The relatively constant Ca 2ϩ spark rise time (Ϸ10 ms in the heart) indicates a stereotypical open time of RyRCs (25) whereas RyRCs in vitro always follow exponential open-time distributions (5,17,20). The brief spark duration also contrasts sharply with the coupled gating (19) kinetics of RyRCs, in which the open time of physically linked channels acting in unison is prolonged by orders of magnitude (up to 2,500 ms) (20). These paradoxical observations underscore that in vivo operation of RyRCs may involve gating mechanisms that are apparently absent in cell-free systems. Alternatively, the formation of RyRC array may endow the channels with new regulatory mechanisms that are unshared by a corresponding set of solitary RyRCs.In the present study, we sought to investigate RyRC operation in their native arrays. Because intracellular location of RyRC arrays makes them inaccessible to electrophysiological means, we devised an optical approach to analyze the Ca 2ϩ release flux underlying the spark (I spark ) in intact cardiac myocytes. By splitting I spark from individual RyRC arrays into single-channel components, we provided a view of the in situ gating of a single RyRC and demonstrat...