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Original Article

^a Biomedical Imaging Group, University of Massachusetts Medical School, Worcester, Massachusetts 01655
^b Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
Department of Physiology, University of Massachusetts Medical Center, Worcester, MA 01655-027.(508) 856-5997

john.walsh{at}umassmed.edu

Ca²⁺ sparks are highly localized cytosolic Ca²⁺ transients caused by a release of Ca²⁺ from the sarcoplasmic reticulum via ryanodine receptors (RyRs); they are the elementary events underlying global changes in Ca²⁺ in skeletal and cardiac muscle. In smooth muscle and some neurons, Ca²⁺ sparks activate large conductance Ca²⁺-activated K⁺ channels (BK channels) in the spark microdomain, causing spontaneous transient outward currents (STOCs) that regulate membrane potential and, hence, voltage-gated channels. Using the fluorescent Ca²⁺ indicator fluo-3 and a high speed widefield digital imaging system, it was possible to capture the total increase in fluorescence (i.e., the signal mass) during a spark in smooth muscle cells, which is the first time such a direct approach has been used in any system. The signal mass is proportional to the total quantity of Ca²⁺ released into the cytosol, and its rate of rise is proportional to the Ca²⁺ current flowing through the RyRs during a spark (I_Ca(spark)). Thus, Ca²⁺ currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of I_Ca(spark) in different sparks varies more than fivefold, Ca²⁺ sparks appear to be caused by the concerted opening of a number of RyRs. Sparks with the same underlying Ca²⁺ current cause STOCs, whose amplitudes vary more than threefold, a finding that is best explained by variability in coupling ratio (i.e., the ratio of RyRs to BK channels in the spark microdomain). The time course of STOC decay is approximated by a single exponential that is independent of the magnitude of signal mass and has a time constant close to the value of the mean open time of the BK channels, suggesting that STOC decay reflects BK channel kinetics, rather than the time course of [Ca²⁺] decline at the membrane. Computer simulations were carried out to determine the spatiotemporal distribution of the Ca²⁺ concentration resulting from the measured range of I_Ca(spark). At the onset of a spark, the Ca²⁺ concentration within 200 nm of the release site reaches a plateau or exceeds the [Ca²⁺]_EC50 for the BK channels rapidly in comparison to the rate of rise of STOCs. These findings suggest a model in which the BK channels lie close to the release site and are exposed to a saturating [Ca²⁺] with the rise and fall of the STOCs determined by BK channel kinetics. The mechanism of signaling between RyRs and BK channels may provide a model for Ca²⁺ action on a variety of molecular targets within cellular microdomains.

Key Words: widefield digital microscope • sarcoplasmic reticulum • microdomain • STOC • smooth muscle release

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