Spatial organization of RYRs and BK channels underlying the activation of STOCs by Ca2+ sparks in airway myocytes

摘要

Short-lived, localized Ca²⁺ events mediate Ca²⁺ signaling with high efficiency and great fidelity largely as a result of the close proximity between Ca²⁺-permeable ion channels and their molecular targets. However, in most cases, direct evidence of the spatial relationship between these two types of molecules is lacking, and, thus, mechanistic understanding of local Ca²⁺ signaling is incomplete. In this study, we use an integrated approach to tackling this issue on a prototypical local Ca²⁺ signaling system composed of Ca²⁺ sparks resulting from the opening of ryanodine receptors (RYRs) and spontaneous transient outward currents (STOCs) caused by the opening of Ca²⁺-activated K⁺ (BK) channels in airway smooth muscle. Biophysical analyses of STOCs and Ca²⁺ sparks acquired at 333 Hz demonstrate that these two events are associated closely in time, and approximately eight RYRs open to give rise to a Ca²⁺ spark, which activates ∼15 BK channels to generate a STOC at 0 mV. Dual immunocytochemistry and 3-D deconvolution at high spatial resolution reveal that both RYRs and BK channels form clusters and RYR1 and RYR2 (but not RYR3) localize near the membrane. Using the spatial relationship between RYRs and BK channels, the spatial-temporal profile of [Ca²⁺] resulting from Ca²⁺ sparks, and the kinetic model of BK channels, we estimate that an average Ca²⁺ spark caused by the opening of a cluster of RYR1 or RYR2 acts on BK channels from two to three clusters that are randomly distributed within an ∼600-nm radius of RYRs. With this spatial organization of RYRs and BK channels, we are able to model BK channel currents with the same salient features as those observed in STOCs across a range of physiological membrane potentials. Thus, this study provides a mechanistic understanding of the activation of STOCs by Ca²⁺ sparks using explicit knowledge of the spatial relationship between RYRs (the Ca²⁺ source) and BK channels (the Ca²⁺ target).