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1 Biomedical Imaging Group, University of Massachusetts Medical School, Worcester, MA, USA; Physiology, University of Massachusetts Medical School, Worcester, MA, USA
2 Information Services, University of Massachusetts Medical School, Worcester, MA, USA
3 Neuroscience & Biomedical Systems, University of Glasgow, Glasgow, United Kingdom
4 Physiology, University of Massachusetts Medical School, Worcester, MA, USA
* To whom correspondence should be addressed. E-mail: john.walsh{at}umassmed.edu.
Ca2+ sparks are highly localized Ca2+ transients due to Ca2+ release from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca2+ sparks activate nearby large conductance K+ (BK) channels to generate spontaneous transient outward currents (STOCs). The properties of individual sites that give rise to Ca2+ sparks have not been systematically examined. Here we characterize individual sites in amphibian gastric smooth muscle cells with simultaneous high-speed imaging of Ca2+ sparks using widefield digital microscopy and patch-clamp recording of STOCs using whole-cell mode. A signal mass approach was used to measure the total Ca2+ released at a site and to estimate the Ca2+ current flowing through RyRs (ICa(spark)). The variance between spark sites was significantly greater than the intrasite variance for the following parameters: Ca2+ signal mass; ICa(spark); STOC amplitude; and 5 msec isochronic STOC amplitude. Sites that failed to generate STOCs did so consistently while those at the remaining sites generated STOCs without failure, allowing the sites to be divided into STOC-generating and STOCless sites. We also determined the average number of spark sites which was 42 per cell at a minimum and more likely on the order of at least 400 per cell. We conclude that (1) spark sites differ in the number of RyRs, BK channels and the coupling ratio of RyRs/BK channels; (2) there are numerous Ca2+ spark-generating sites in smooth muscle cells. The implications of these findings for the organization of the spark microdomain are explored.
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