Functional analysis of conserved polar residues in Vc-NhaD, Na+/H+ antiporter of Vibrio cholerae

Vc-NhaD is a Na(+)/H(+) antiporter from Vibrio cholerae with a sharp maximum of activity at pH approximately 8.0. NhaD homologues are present in many bacteria as well as in higher plants. However, very little is known about structure-function relations in NhaD-type antiporters. In this work 14 conserved polar residues associated with putative transmembrane segments of Vc-NhaD have been screened for their possible role in the ion translocation and pH regulation of Vc-NhaD. Substitutions S150A, D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, and S431A completely abolished the Vc-NhaD-mediated Na(+)-dependent H(+) transfer in inside-out membrane vesicles. Substitutions T157A and S428A caused a significant increase of apparent K(m) values for alkali cations, with the K(m) for Li(+) elevated more than that for Na(+), indicating that Thr-157 and Ser-428 are involved in alkali cation binding/translocation. Of six conserved His residues, mutation of only His-93 and His-210 affected the Na(+)(Li(+))/H(+) antiport, resulting in an acidic shift of its pH profile, whereas H93A also caused a 7-fold increase of apparent K(m) for Na(+) without affecting the K(m) for Li(+). These data suggest that side chains of His-93 and His-210 are involved in proton binding and that His-93 also contributes to the binding of Na ions during the catalytic cycle. These 15 residues are clustered in three distinct groups, two located at opposite sides of the membrane, presumably facilitating the access of substrate ions to the third group, a putative catalytic site in the middle of lipid bilayer. The distribution of these key residues in Vc-NhaD molecule also suggests that transmembrane segments IV, V, VI, X, XI, and XII are situated close to one another, creating a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported cations.