Abstract
In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73–80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg2+, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg2+ (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5–2.0 μM Hg2+, concentrations that activate AQP6. The Hg lysis was completely blocked by β-mercaptoethanol which disrupts Hg2+-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO3 − > Br− > I− > Cl− and was facilitated by acidic pH. The anion selectivity for NO3 − and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg2+-sensitive anion channel in rat parotid secretory granules.
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Acknowledgments
Supported in part by a Nihon University Multidisciplinary research grant for 2009, a Grant-in-Aid for Young Researcher Scientists, Nihon University School of Dentistry at Matsudo (2010-018 and 2011-001), and a JSPS KAKENHI Grant Number (24791986).
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Matsuki-Fukushima, M., Fujita-Yoshigaki, J., Murakami, M. et al. Involvement of AQP6 in the Mercury-Sensitive Osmotic Lysis of Rat Parotid Secretory Granules. J Membrane Biol 246, 209–214 (2013). https://doi.org/10.1007/s00232-012-9522-7
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DOI: https://doi.org/10.1007/s00232-012-9522-7