An amino acid channel activated by hypotonically induced swelling of Leishmania major promastigotes

Abstract

Leishmania promastigotes accumulate amino acids (AAs) by an uphill transport mechanism that is dependent on membrane potential. The accumulated AAs provide the cell with an osmotic reservoir that can be utilized for osmoregulation. Exposure of Leishmania promastigotes to hypotonic media induced a rapid release of AAs that was proportional to the imposed osmotic gradients and independent of the ionic strength or the presence of Cl-, K+, Na+ or Ca2+ in the medium. The hypotonically activated AA release pathway was of relatively low chemical specificity. The solutes released included most of the zwitterionic and anionic AAs, predominantly alanine, hydroxyproline, glycine and glutamic acid, whereas cationic AAs were virtually excluded. AA release was markedly blocked by classical anion transport inhibitors such as the disulphonic stilbene 4,4´-di-isothiocyanostilbene-2,2´-disulphonate (DIDS) and its dihydro derivative H2DIDS and others, by restoration of isotonicity or by lowering the temperature (4 °C). The temperature profile of AA release showed a low energy of activation (Ea 46±1.3 (S.E.M.) kJ/mol) in the range 15-30 °C and a very high Ea (147±8 kJ/mol) in the range 4–15 °C. Parasites exposed to hypotonic media containing AAs also showed a hypotonically stimulated AA uptake under favourable solute concentration gradients. This uptake was analogous for L- and D-isomers of threonine. After hypotonic exposure, cells underwent a depolarization that was largely prevented by anion transport blockers. On the basis of all these results we propose that after hypotonic stress Leishmania promastigotes restore their internal volume by a regulated release of AAs, which involves activation of channels that allow the passage of both neutral and anionic AAs and possibly other anionic substances.