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C. T. Leung, A. Li, K. Peterson-Yantorno, M. M. Civan; Regulatory Role of ClC-3 and Wide-Bore Channels in Degranulation of LAD2 Mast Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):698.
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Antigens bind IgE immunoglobulin on mast-cell surfaces, leading to Ca2+ influx through CRAC channels that critically depends on membrane potential (Vm) established by K+ and possibly Cl- channels. The Ca2+ influx triggers degranulation of histamine and other products that are likely crucial in certain aspects of allergic conjunctivitis. In large part, the perceived role of Cl- channels rests on results with blockers that also inhibit wide-bore channels, including pannexin and connexin hemichannels. This study aimed at testing the roles of the ClC-3 Cl- channels and hemichannels.
Channel expression in human LAD2 mast cells was probed by RT-PCR. Cells were pre-incubated for 30min with/without the following inhibitors: (1) nonspecific Cl--channel blockers [flufenamic acid (FFA, 500µM) and diphenylamine-2-carboxylic acid (DPC, 500 µM)]; (2) connexin (Cx) and pannexin (Px) hemichannel blockers [meclofenamic acid (MFA, 200 µM), carbenoxolone (CBX, 100µM, mefloquine (MFQ, 50µM), probenecid (Prob, 1mM) and octanol (OCT, 500µM)]; and (3) the P2X7 receptor blocker KN-62 (1 µM). Degranulation was triggered by C3a, the Ca2+ ionophore ionomycin or cross-linking FcεRI-anchored IgE, and monitored as percentage β-hexosaminidase release. To knock down ClC-3, cells were transfected with human CLCN-3 siRNA for 72 hours. Knockdown efficacy was tested by qPCR.
LAD2 cells expressed ClC-3, pannexin Px1, and connexins Cx26, Cx31 and Cx43. KN-62 had no effect, suggesting P2X7 does not contribute directly to LAD2-cell degranulation. The other blockers inhibited degranulation triggered by the 3 stimuli (N=4 -12 wells). At saturating concentration, the Px1 blocker Prob reduced degranulation by 60-80% (N=4-12, P<0.001), and submaximal 50-µM concentrations of CBX inhibited C3a- and FcεRI-triggered degranulation by ~50% (N=4 each, P<0.001). siRNA knocked down expression of ClC-3 by 75%±4% (N=23) and reduced C3a-triggered degranulation by ~25% (N=59, P<0.05). Hydrolyzing released ATP with apyrase had no effect (N=4).
This is the first direct evidence that ClC-3-dependent Cl- transfer regulates mast-cell degranulation, likely by modulating Vm. Both Cx and Px1 hemichannels likely regulate degranulation through their signaling, rather than conduit, role. The results suggest a novel approach for addressing allergic conjunctivitis by interrupting hemichannel- and ClC-3-dependent degranulation.
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