Voltage-gated Ca2+ channels, by mediating the entry of messenger Ca2+ into excitable cells, play a key role in neural, muscular and secretory activities. During excitation-contraction coupling in cardiac myocytes, Ca2+ influx through L-type Ca2+ channels (LCC) triggers abutting ryanodine receptors in the sarcoplasmic reticulum, resulting in """"""""Ca2+ sparks"""""""". Here, using combined confocal laser-scanning microscopy and patch clamp techniques, we visualize the fundamental trigger event, i.e., microdomain Ca2+ transient produced by unitary L-type Ca2+ current. In rat ventricular myocytes whose SR function is paralyzed by thapsigargin, caffeine and/or ryanodine, cell-attached patch-clamp is established with 10 uM FPL64176, an LCC agonist, and 20 mM [Ca2+] in the pipette. Upon depolarization from a holding potential of -50 mV to test potentials between -40 and 0 mV, confocal imaging visualizes tiny flashes of fluo-4 fluorescence, dubbed """"""""Ca2+ sparklets,"""""""" occurring just beneath the patch membrane. Ca2+ sparklets were resistant to bath application of ryanodine (10 uM), but were completely abolished by the LCC antagonist nifedipine (2 uM). These data indicate that Ca2+ sparklets originate from single LCC openings, and thus represent the first optical measurement of voltage-gated single-channel activity in intact cells. Unlike Ca2+ sparks of RyR origin, which typically last for ~30 ms, Ca2+ sparklets exhibited a highly irregular duration; the onset and termination of sparklets correlated tightly with the opening and closing of the channel, respectively. To characterise sparklet properties in relation to Ca2+ influx, we measured the """"""""signal mass"""""""" of sparklets (i.e., space-time integral of local fluo-4 fluorescence) and integrated the corresponding unitary Ca2+ influx (qCa). Over the voltage range from -30 mV to +10 mV, where LCC openings can be clearly identified, there was a nice linear correlation between qCa (~8,000 to 100,000 Ca2+ ions) and sparklet signal mass. Hence, Ca2+ sparklets provide a faithful alternative readout of LCC unitary current. Furthermore, under whole-cell voltage-clamp conditions, depolarization-evoked Ca2+sparklets localize to T-tubules, and their amplitudes range between 10~30 nM at 10 mM extracellular [Ca2+], whereas Ca2+ sparks are typically 150 nM. Since LCC open duration is prolonged by >10-fold in our experiments, these data suggest that microscopic """"""""gain"""""""" of EC coupling (spark:sparklet flux ratio) is ~100 under physiological conditions. The direct visualization of single-channel Ca2+ fluxes makes it possible to track single-channel activity in circumstances when electrophysiological single-channel analysis is precluded, and may have important impact on studies of EC coupling and Ca2+ signaling in general.
