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

摘要

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.