Sodium-glucose transporters (SGLTs) facilitate the movement of water across the cell membrane, playing a central role in cellular homeostasis. Here, we present a detailed analysis of the mechanism of water permeation through the inward-facing state of vSGLT based on nearly 10 μs of molecular dynamics simulations. These simulations reveal the transient formation of a continuous water channel through the transporter that permits water to permeate the protein. Trajectories in which spontaneous release of galactose is observed, as well as those in which galactose remains in the binding site, show that the permeation rate, although modulated by substrate occupancy, is not tightly coupled to substrate release. Using a, to our knowledge, novel channel-detection algorithm, we identify the key residues that control water flow through the transporter and show that solvent gating is regulated by side-chain motions in a small number of residues on the extracellular face. A sequence alignment reveals the presence of two insertion sites in mammalian SGLTs that flank these outer-gate residues. We hypothesize that the absence of these sites in vSGLT may account for the high water permeability values for vSGLT determined via simulation compared to the lower experimental estimates for mammalian SGLT1.

Background and purpose: Amphetamines bind to the plasmalemmal transporters for the monoamines dopamine (DAT), norepinephrine (NET) and serotonin (SERT); influx of amphetamine leads to efflux of substrates. Various models have been put forth to account for this amphetamine-induced reverse transport in mechanistic terms. A most notable example is the molecular stent hypothesis, which posits a special amphetamine-induced conformation that is not foreseen in alternate access models of transport. The current study was designed to evaluate the explanatory power of these models and the molecular stent hypothesis. Experimental approach: Xenopus laevis oocytes and HEK293 cells expressing human (h)SERT were voltage clamped and exposed to serotonin (5-HT), p-chloroamphetamine (pCA) or methylenedioxyamphetamine (MDMA). Key results: In contrast to currents induced by 5-HT, pCA-triggered currents through SERT decayed slowly (i.e., with a half-life of 20 s at 3 micromolar) in Xenopus laevis oocytes once the agonist was removed (consistent with the molecular stent hypothesis). However, when SERT was expressed in HEK293 cells, currents induced by 3 micromolar or 100 micromolar pCA decayed 10 or 100 times faster, respectively, after pCA removal. Conclusions and implications: This discrepancy in decay rates is inconsistent with the molecular stent hypothesis. In contrast, a multi-state version of the alternate access model accounts for all the observations and reproduces the kinetic parameters extracted from the electrophysiological recordings. A crucial feature that explains the action of amphetamines is their lipophilic nature, which allows for rapid diffusion through the membrane.