Ca2+ release from sarcoplasmic reticulem in cardiac cells during excitation-contraction coupling is inatiated by Ca2+ influx through L-type Ca2+ channels. We recently demonstrated the feasibility to visualize Ca2+ influxes of single L-type Ca2+ channel (LCC) openings- """"""""Ca2+ sparklets""""""""- using confocal linescan imaging combined with patch clamp techniques. Here we directly visualize the process that Ca2+ sparklets trigger Ca2+ sparks in rat ventricular myocytes. To preserve the delicate molecular architecture of E-C coupling, loose-patch clamp is established by gently pressing the patch pipette against cell surface without suction. In the presence of 10 microM FPL64176 and 20 mM [Ca2+]o, depolarization pulses (-40 to +50 mV) evoke local Ca2+ transients that fall into two distinct categories. The low-amplitude events with irregular duration were resistant to ryanodine (10 mM), but were abolished by 2 mM nifedipine, and thus represent Ca2+ sparklets. The high-amplitude events were identified as Ca2+ sparks by virtue of morphology and sensitivity to ryanodine. The blockade of Ca2+ sparklets by substitution of Ca2+ with Ba2+ in the pipette abolished the Ca2+ spark activity, indicating that the sparks are triggered by sparklets. Our data provide direct evidence that Ca2+ flux from single L-type channel is capable of triggering a Ca2+ spark. The simultaneous recording of sparklets and sparks make it possible to probe into kinetics of cardiac excitation-contraction coupling at the single-channel level. During 400-ms depolarising pulses, 71% of the first local Ca2+ events occurred as triggered sparks, suggesting that a Ca2+ sparklet does not always elicit a spark. When a sparklet did trigger a spark, the latency varied widely from case to case. Interestingly, the coupling fidelity was significantly reduced to 30% after the first triggered spark, reflecting a local refractoriness of SR Ca2+ release. These evidence suggest that the LCC-RyR coupling is probabilistic rather than deterministic by nature, and that the coupling efficiency varies in a use-dependent manner. Ca2+ sparklets of known iCa also offer a natural standard to gauge the SR Ca2+ efflux underlying the spark (jspark). Upon repolerization to -70 mV, tail current-evoked sparklets displayed a rising time constant of 9.3 ms and a steady-state amplitude of 57 nM. By comparison, an averaged Ca2+ spark exhibited a peak amplitude that was 3.1-fold greater than the sparklet at the same rising time. Assuming a linear relation between Ca2+ flux and local Ca2+ transient, we estimated that the average jspark mounts to 2.1 pA, suggesting that 4 ~ 6 RyRs are tipically involved in an evoked Ca2+ spark. Altogether, our work provide direct evidence that Ca2+ sparklets due to single LCCs constitute fundamental trigger events of SR Ca2+ release, which ignite elemental Ca2+ sparks originated from 4 ~ 6 RyRs. We found that cardiac E-C coupling at the molecular level is inherently stochastic. The optical analysis of single-channel physiology and intermolecular crosstalk affords a powerful means for elucidation of Ca2+ signalling mechanisms at the molecular level.
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