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Abstract

CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2;4 that is highly homologous to HvPIP2;3 did not. The isoleucine residue at position 254 of HvPIP2;3 was conserved in PIP2 aquaporins of barley, except HvPIP2;4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2;3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity.

Isoleucine 254 (I-254) is an important factor for CO2 permeability of HvPIP2;3

Simultaneous substitution of four amino acids of HvPIP2;3, A-268, L-281, R-289 and S-290, to L-268, F-281, S-289 and R-290 [HvPIP2;3(LFSR)] did not affect the acidification of the cytosol of HvPIP2;3(LFSR) cRNA-injected oocytes compared with HvPIP2;3-injected oocytes (). The osmotic water permeability (Pf) of HvPIP2;3 and HvPIP2;3(LFSR) was not significantly different (P > 0.05, ). This indicates that the four amino acids in the C-terminal stretch are not involved in the CO2 permeability of HvPIP2;3. Substitution of I-254 of HvPIP2;3, which is localized at the edge of the E-loop, by methionine (C) resulted in substantial repression of the PCO2 (D). Substitution of methionine 254 (M-254) of HvPIP2;4 by isoleucine, HvPIP2;4(M254I) (B) caused activation of PCO2 (D). The Pf of HvPIP2;3(I254M) and HvPIP2;4(M254I) was examined to test whether the mutated aquaporins were functional (E). The substitution of I-254 of HvPIP2;3 and M-254 of HvPIP2;4 did not show any apparent effect on Pf. This indicates that I-254 of HvPIP2;3 is crucial to the CO2 permeability.