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Biomedical Imaging Group and Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655

Correspondence to Ronghua ZhuGe: ronghua.zhuge{at}umassmed.edu

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 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 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 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.

© 2008 Bao et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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