Molecular mechanisms of band 3 inhibitors. 3. Translocation inhibitors

During the translocation of the band 3 transport site between the inward- and outward-facing orientations, the Cl- transport site complex passes through a transition state lying on the reaction pathway between the two extreme orientations. Niflumic acid, 2-[(7-nitrobenzofurazan-4-yl)amino]ethanesulfonate, and 2,4,6-trichlorobenzenesulfonate each are translocation blockers that can bind to both the inward- and outward-facing conformations of band 3. The principal mechanism of these inhibitors is a reduction in the translocation rate, since they have essentially no effect on the apparent KD for Cl- binding to the transport site and the migration of Cl- between the transport site and solution. Instead, these inhibitors raise the free energy of formation of the transition state during translocation and thereby can lock the transport site into either the inward- or outward-facing orientation. In contrast, 2,4-dinitrofluorobenzene (DNFB) appears to restrict the accessibility of the transport site to solution Cl-; also, the DNFB reaction rate is increased by Cl-, suggesting that DNFB modification may occur during translocation. Thus DNFB is proposed to trap the Cl--transport site complex site during translocation to yield a conformation intermediate to the inward- and outward-facing orientations. A model is presented for the molecular mechanism of transport across biological membranes. The transport machinery is proposed to contain greater than or equal to 6 transmembrane helices that surround a central channel containing a sliding hydrophobic barrier. The transport site lies between two of the channel-forming helices and remains stationary while the hydrophobic barrier slides from one end of the channel to the other, thereby exposing the transport site to the opposite solution compartment.