title: 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.
abstract: Ca(2+) sparks are highly localized, transient releases of Ca(2+) from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca(2+) sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca(2+)-activated K(+) channels, and also gate Ca(2+)-activated Cl(-) (Cl((Ca))) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca(2+) sparks is well understood, little information is available on how Ca(2+) 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. Ca(2+) 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 Ca(2+) current underlying a Ca(2+) spark (I(Ca(spark))), with an appropriate correction for endogenous fixed Ca(2+) 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 Ca(2+) 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 [Ca(2+)] of 2.4 microM or greater during Ca(2+) sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca(2+)] produced by a reaction-diffusion simulation with measured I(Ca(spark)). Finally we estimated that the number of Cl((Ca)) channels localized in Ca(2+) 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 Ca(2+) spark sites localize near to each other, and, moreover, Cl((Ca)) channels concentrate in an area with a radius of approximately 600 nm, where their density reaches as high as 300 channels/microm(2). This model reveals that Cl((Ca)) channels are tightly controlled by Ca(2+) sparks via local Ca(2+) signaling.
authors.affiliation: Biomedical Imaging Group and Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Rongfeng
authors.initials: R
authors.lastname: Bao
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Lifshitz
authors.forename: Richard A
authors.initials: RA
authors.lastname: Tuft
authors.forename: Karl
authors.initials: K
authors.lastname: Bellvé
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Fogarty
authors.forename: Ronghua
authors.initials: R
authors.lastname: ZhuGe
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000502:physiology*; D003198:Computer Simulation; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D004594:Electrophysiology; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008954:Models, Biological; D009130:Muscle, Smooth / Q000166:cytology / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D010782:Photolysis; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology*; D014132:Trachea / Q000166:cytology; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; D018118:Chloride Channels; D019837:Ryanodine Receptor Calcium Release Channel; D014966:Xanthenes; C085112:2-nitrophenyl-EGTA; C059715:Fluo-3; D004533:Egtazic Acid; D002118:Calcium