A Close Association of RyRs with Highly Dense Clusters of Ca2+-activated Cl− Channels Underlies the Activation of STICs by Ca2+ Sparks in Mouse Airway Smooth Muscle

Ca2+ sparks are highly localized, transient releases of Ca2+ from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca2+ sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca2+-activated K+ channels, and also gate Ca2+-activated Cl− (Cl(Ca)) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca2+ sparks is well understood, little information is available on how Ca2+ sparks activate STICs. In the present study, we investigated the spatial organization of RyRs and Cl(Ca) channels in spark sites in airway myocytes from mouse. Ca2+ sparks and STICs were simultaneously recorded, respectively, with high-speed, widefield digital microscopy and whole-cell patch-clamp. An image-based approach was applied to measure the Ca2+ current underlying a Ca2+ spark (ICa(spark)), with an appropriate correction for endogenous fixed Ca2+ buffer, which was characterized by flash photolysis of NPEGTA. We found that ICa(spark) rises to a peak in 9 ms and decays with a single exponential with a time constant of 12 ms, suggesting that Ca2+ sparks result from the nonsimultaneous opening and closure of multiple RyRs. The onset of the STIC lags the onset of the ICa(spark) by less than 3 ms, and its rising phase matches the duration of the ICa(spark). We further determined that Cl(Ca) channels on average are exposed to a [Ca2+] of 2.4 μM or greater during Ca2+ sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca2+] produced by a reaction-diffusion simulation with measured ICa(spark). Finally we estimated that the number of Cl(Ca) channels localized in Ca2+ spark sites could account for all the Cl(Ca) channels in the entire cell. Taken together these results lead us to propose a model in which RyRs and Cl(Ca) channels in Ca2+ spark sites localize near to each other, and, moreover, Cl(Ca) channels concentrate in an area with a radius of ∼600 nm, where their density reaches as high as 300 channels/μm2. This model reveals that Cl(Ca) channels are tightly controlled by Ca2+ sparks via local Ca2+ signaling

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

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)