Ion specificity and ionic strength dependence of the osmoregulatory ABC transporter OpuA.

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.