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

作者：Bao Rongfeng; Lifshitz Lawrence M; Tuft Richard A; Bellve Karl; Fogarty Kevin E; ZhuGe Ronghua

来源：Journal of General Physiology, 2008, 132(1): 145-160.

DOI：10.1085/jgp.200709933

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 (I-Ca(spark)), with an appropriate correction for endogenous fixed Ca2+ buffer, which was characterized by flash photolysis of NPEGTA. We found that I-Ca(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 I-Ca(spark) by less than 3 ms, and its rising phase matches the duration of the I (Ca(spark)). We further determined that Cl-(Ca) channels on average are exposed to a [Ca2+] of 2.4 mu M 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 are action-diffusion simulation with measured I-Ca(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 similar to 600 nm, where their density reaches as high as 300 channels/mu m(2). This model reveals that Cl-(Ca) channels are tightly controlled by Ca2+ sparks via local Ca2+ signaling.

• 出版日期2008-7