Dynamics of Signaling between Ca2+ Sparks and Ca2+- Activated K+ Channels Studied with a Novel Image-Based Method for Direct Intracellular Measurement of Ryanodine Receptor Ca2+ Current

Abstract

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 (ICa(spark)). Thus, Ca2+ currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of ICa(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 ICa(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.

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes

“…1 and 2) because this would be expected to increase the total Ca 2ϩ release during each spark. If this Ca 2ϩ is mainly cleared from the cytoplasm by diffusion in smooth muscle cells (38), then the increased Ca 2ϩ load should also increase the spark width. Thus, although we cannot rule out the possibility that increased spark duration and spread may reflect recruitment of additional RyRs after an increase in their Ca 2ϩ sensitivity (7), it seems that an increase in open probability and mean time to closure could adequately explain most of the changes seen.…”

“…It is well established that Ca 2ϩ sparks can activate Ca 2ϩ -sensitive conductances in the adjacent plasma membrane of smooth muscle cells. In the case of Ca 2ϩ -activated K ϩ channels, the high [Ca 2ϩ ] generated by localized Ca 2ϩ release into the microdomain adjacent to the membrane increases K ϩ channel open probability, resulting in a STOC (38,39). It follows, therefore, that interventions that increase spark activity are also likely to promote STOCs.…”

Depletion “skraps” and dynamic buffering inside the cellular calcium store

“…The rate of change dM͞dt of signal mass (Fig. 2E) is used to approximate time course of release flux during the average spark (13,14).…”

“…The flux of Ca 2ϩ release underlying a spark (green trace in Fig. 2E) estimated by the rate of production of signal mass (14) was essentially over by the time the spark peaked. Surprisingly, [Ca 2ϩ ] SR continued to decrease afterward, reaching nadir 50 ms later.…”

Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle

“…In addition, the signal mass of sparklets was determined. Details of the nP s and signal mass analyses can be found elsewhere (ZhuGe et al, 2000;Zou et al, 2002;Navedo et al, 2005Navedo et al, , 2006 …”