The evolution of a mechanistic model: from 1990 to 2025. For simplicity, only six TMs are shown. The cycle is assumed to involve six steps (numbered). In the absence of a proton gradient, the dominant population is of the lumen-facing conformation, as indicated by the transporter’s ability to bind TBZ but not reserpine. Upon acidification of the lumen, binding of protons enables the conformational switch to the cytoplasm-facing conformation (step 1), whereas binding of substrate enables the change to the lumen-facing conformation (step 4). Binding of TBZ locks the transporter in a conformation that appears incompatible with substrate binding and is therefore presumably not cytoplasm facing (shown as an off-cycle state connected to step 6). Binding of reserpine also locks the transporter in a dead-end conformation, but reserpine binding competes with substrate binding, and therefore the reserpine-bound conformation is presumably cytoplasm facing (shown as an off-cycle state connected to step 2). We propose that the second proton is needed for release of the substrate in the lumen (step 5). Residues contributing to the cytoplasmic gate are shown as purple circles. Adapted from Yaffe et al. (2016).