Plasma membrane vesicles of Beta vulgaris storage root show high water channel activity that is regulated by cytoplasmic pH, and a dual range of calcium concentrations

KARINA ALLEVA; CHRISTA NIEMIETZ; MOIRA SUTKA; CHRISTOPHE MAUREL; MARIO PARISI; GABRIELA AMODEO

JOURNAL OF EXPERIMENTAL BOTANY

Oxford University Press

Lugar: Lancaster; Año: 2006 p. 609 - 621

0022-0957

Plasma membrane vesicles isolated by two-phase partitioning from Beta vulgaris storage root show atypically high water permeability that is equivalent to those reported only for active aquaporins in tonoplast or animal red cells (Pf= 542 mm s-1). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. In order to study short term regulation of water transport that could be the result of channel gating we tested the effects of pH, divalent cations, and protection against dephosphorylation. The observed high Pf at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility for changes in Ca2+ to finely up- or down-regulate water channel activity.  The calcium effect is predominantly on the cytoplasmic face and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.