Oxidative gating of water channels (aquaporins) in Chara by hydroxyl radicals

Hydroxyl radicals (*OH) as produced in the Fenton reaction (Fe2+ + H2O2 = Fe3+ + OH- + *OH) have been used to reversibly inhibit aquaporins in the plasma membrane of internodes of Chara corallina. Compared to conventional agents such as HgCl2, *OH proved to be more effective in blocking water channels and was less toxic to the cell. When internodes were treated for 30 min, cell hydraulic conductivity (Lp) decreased by 90% or even more. This effect was reversed within a few minutes after removing the radicals from the medium. In contrast to HgCl2, radical treatment reduced membrane permeability of small lipophilic organic solutes (ethanol, acetone, 1-propanol, and 2-propanol) by only 24 to 52%, indicating some continued limited movement of these solutes across aquaporins. The biggest effect of *OH treatment on solute permeability was found for isotopic water (HDO), which largely used water channels to cross the membrane. Inhibition of aquaporins reduced the diffusional water permeability (P-d) by about 70%. For the organic test solutes, which mainly use the bilayer to cross the membrane, channel closure caused anomalous (negative) osmosis; that is, cells had negative reflection coefficients (sigma(s)) and were transiently swelling in a hypertonic medium. From the ratio of bulk (Lp or osmotic permeability coefficient, P-f) to diffusional (P-d) permeability of water, the number (N) of water molecules that align in water channels was estimated to be N = P-f/P-d = 46 (on average). Radical treatment decreased N from 46 to 11, a value still larger than unity, which would be expected for a membrane lacking pores. The gating of aquaporins by *OH radicals is discussed in terms of a direct action of the radicals when passing the pores or by an indirect action via the bilayer. The rapid recovery of inhibited channels may indicate an easy access of cytoplasmic antioxidants to closed water channels. As hydrogen peroxide is a major signalling substance during different biotic and abiotic stresses, the reversible closure of water channels by *OH (as produced from H2O2 in the apoplast in the presence of transition metals such as Fe2+ or Cu+) may be downstream of the H2O2 signalling. This may provide appropriate adjustments in water relations (hydraulic conductivity), and a common response to different kinds of stresses.