Alternating Access as a Mechanism for Transmembrane Solute Transport

For the last 40 years, our understanding of the mechanistic basis for solute transport across membranes has been shaped by the alternating access mechanism (22, 37, 56). In this model, transported solutes bind to a site in the transporter that can be exposed by conformational changes to one side of the membrane or the other. Early mechanistic interpretations of this model suggested that the transporter (carrier) bound its substrate on one side of the membrane and then migrated to the other side, where substrate was released (8). Although this moving carrier model is probably accurate for ionophores such as valinomycin, nigericin, and 2,4-dinitrophenol, our current understanding of membrane protein structure suggests that the energetic barrier for such movement is prohibitive. Peter Mitchell suggested that, rather than a moving carrier, we should think about the transport process in terms of a moving barrier (34). The binding site for substrate could be at a fixed point within the transporter structure, but the permeability barrier between that site and the aqueous phases on either side of the membrane could move so that the site was accessible alternately from only one side of the membrane at a time. Although the moving carrier and moving barrier models for transport are quite different in their mechanistic details, they both predict similar “carrier kinetics,” which explain such phenomena as counterflow and accelerative exchange diffusion (52). Until recently, when the first crystal structures of transporters were solved, evaluation of specific mechanisms has not been possible. Now that such structures are appearing with regularity, the issue of specific transporter mechanism is at the forefront of the field. Conformational mechanisms of transport have been proposed for P-type ATPases, based on the numerous crystal structures of the SERCA Ca2+ pump (36, 53). Another mechanism has been proposed for lac permease, a H+coupled transporter, based on abundant biochemical and biophysical evidence (24, 32, 50). A compelling feature of the alternating access model is that it can account for many types of transport. In the simplest of these, uniport (also facilitated or mediated diffusion), the transporter allows passage of a single solute across the membrane. An example of this process is the transport of glucose into mammalian cells catalyzed by the GLUT family of transporters (5). For alternating access to account for uniport, the substrate would bind to the transporter from one side of the membrane, and a conformational change would close access to the binding site from that side and open access to the opposite side. The solute would then dissociate, and another conformational change would bring the transporter back to its original conformation (FIGURE 1). The alternating access mechanism also accounts for coupling the flux of two or more solutes. This process is sometimes referred to as secondary active transport to distinguish it from primary active transport, which is driven by metabolic energy such as ATP hydrolysis. In symport (co-transport) the solutes cross the membrane in the same direction, and in antiport (countertransport, exchange) they cross in opposite directions. For symport, two or more solutes bind and are released to the opposite side of the membrane in the same way as in uniport, followed by reorientation of the transporter as with uniport (FIGURE 1). In antiport, one or more solutes is transported across the membrane (like uniport or symport), but then before reorientation of the transporter, a different solute binds and is subsequently transported in the opposite direction (FIGURE 1). In both symport and antiport, the energy of a solute transmembrane concentration gradient can thus be used to concentrate another solute on one side of the membrane. If different transporters use this same general mechanism to catalyze uniport, symport, or antiport, there must be additional parameters that determine which kind of transport occurs in each case. Jencks (23) postulated that there must be “rules” followed by different coupled vectorial processes to account for different