Dynamics of signaling between Ca2+ sparks and Ca2+-activated K+ channels studied with a novel image-based method for direct intracellular measurementof ryanodine receptor Ca2+ current
R. Zhuge et al., Dynamics of signaling between Ca2+ sparks and Ca2+-activated K+ channels studied with a novel image-based method for direct intracellular measurementof ryanodine receptor Ca2+ current, J GEN PHYSL, 116(6), 2000, pp. 845-864
Ca2+ sparks are highly localized cytosolic Ca2+ transients caused by a rele
ase of Ca2+ front the sarcoplasmic reticulum via ryanodine receptors (RyRs)
; they are the elementary events underlying global changes in Ca2+ in skele
tal and cardiac muscle, In smooth muscle and some neurons, Ca2+ sparks acti
vate large conductance Ca2+-activated K+ channels (BK channels) in the spar
k microdomain, causing spontaneous transient outward currents (STOCs) that
regulate membrane potential and, hence, voltage-gated channels. Using the f
luorescent 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 fir
st 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, a
nd 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 magnit
ude of I-Ca(spark) in different sparks varies more than fivefold, Ca2+ spar
ks 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 m
ore than threefold, a finding that is best explained by variability in coup
ling 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 th
at is independent of the magnitude of signal mass and has a time constant c
lose 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 [C
a2+] decline at the membrane. Computer simulations were carried out to dete
rmine the spa tiotemporal distribution of the Ca2+ concentration resulting
from the measured range of I-Ca(spark). At the onset of a spark, the Ca2+ c
oncentration 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 BR channels lie
close to the release site and are exposed to a saturating [Ca2+] with the r
ise and fall of the STOCs determined by BK channel kinetics. The mechanism
of signaling between RyRs and BR channels may provide a model for Ca2+ acti
on on a variety of molecular targets within cellular microdomains.