A complete set of rate constants for a transporter’s catalytic cycle

AdiC functions by the alternating access mechanism proposed by Jardetsky (1966) for the Na⁺, K⁺-ATPase (Fig. 1). The mechanism requires that the transport protein “must contain a slit or cavity in the interior of the molecule” and “be able to assume two different configurations, such that the molecular cavity is open to one side of the membrane in one configuration and to the opposite side in the other.” The mechanism was originally proposed for an active pump but also applies to uniporters, exchangers, and cotransporters.

Early support for alternating access in coupled transporters came from studies of the erythrocyte anion exchanger band 3. The kinetics of tracer anion fluxes showed that the K_1/2 for extracellular Cl⁻ depends on the intracellular concentration as predicted by an alternating access model (Gunn and Fröhlich, 1979). Biochemical studies showed that anion gradients and various inhibitors can alter the proportions of band 3 in inward-facing and outward-facing conformations (Grinstein et al., 1979; Rothstein et al., 1979). The very high number of copies of band 3 per cell made it possible to determine the stoichiometry of a half turnover of the catalytic cycle (Jennings, 1982), showing that a half cycle of Cl⁻ efflux can take place without influx, as expected for an alternating access mechanism.

Studies on erythrocyte uniporter GLUT1 also provided early support for alternating access, including measurement of a half turnover (Appleman and Lienhard, 1985; Lowe and Walmsley, 1987) and estimates of inward and outward translocation rate constants of loaded and unloaded transporter (Appleman and Lienhard, 1989; Mueckler and Thorens, 2013). Some characteristics of red cell GLUT1 were not consistent with alternating access but can be explained by an oligomeric model that retains the basic feature of inward-facing and outward-facing conformations (Lloyd et al., 2017).

Although these early studies and much subsequent work strongly suggested an alternating access mechanism, the most convincing evidence was the determination of distinct structures of inward-facing and outward-facing conformations of individual transporters (Forrest et al., 2008; Krishnamurthy et al., 2009; Drew and Boudker, 2016). Recent advances in cryo-EM and computational protein chemistry have resulted in structures of inward-open, outward-open, and occluded states for many transporters. In a recent review of alternating access transporters, Drew and Boudker (2024) point out that “the wealth of structural information on these proteins provides an illusionary sense that the ion-coupling mechanisms are well understood.” Accordingly, a full understanding of the mechanism of a given transporter will require knowledge not only of structures but also the numerical values of the unimolecular rate constants for each transition between protein conformations in the catalytic cycle.

Steady-state fluxes are determined by multiple elementary steps (e.g., Schicker et al., 2021), and it is difficult to use steady-state fluxes to estimate rate constants of elementary events. For transporters with charge-translocating events, electrophysiological measurement of transient currents offers a way to dissect catalytic cycles and measure rates of some of the elementary events (e.g., Wadiche et al., 1995). Bazzone et al. (2023) recently compiled an extensive set of rate constants for the catalytic cycle of SGLT1, using new data from solid-supported membrane-based electrophysiology combined with earlier data from Wright and coworkers (see Wright et al. [2011]). A model was developed using these rate constants but did not include voltage-dependent events in the reorientation of the empty transporter.

Single-molecule techniques have been very useful for investigating elementary events in transporter catalytic cycles. The most widely used single-molecule method is FRET, which was first applied to the bacterial oxalate–formate exchanger OxlT by Knauf and coworkers (Lesoine et al., 2006) and to LacY by Kaback and coworkers (Majumbdar et al., 2007). Zhao et al. (2010) used single-molecule FRET to study the dynamics of substrate-induced conformational changes in the neurotransmitter transporter homolog LeuT. As noted in commentary on that article, the single-molecule FRET technique made it possible “to visualize several steps in the cycle of a transporter as it transfers a substrate across a membrane, an important breakthrough for the field” (Karpowich and Wang, 2010). Subsequent single-molecule FRET studies on LeuT revealed new partially open intermediate states (Terry et al., 2018).

A single-molecule FRET method based on detection of substrate, without modification of the transport protein (Fitzgerald et al., 2019), demonstrated that the LeuT-fold transporter MhsT has a catalytic cycle in which the rate-limiting step, the return of empty transporter, is, unexpectedly, dependent on the substrate that was transported inward in the previous half cycle. These and other applications of single-molecule FRET to transporters (Bartels et al., 2021) have revealed important information about individual events in transporters.

Despite these advances, it has not yet been possible to measure the rate constant for every elementary event in the catalytic cycle of a transporter until the article by Lewis et al. (2025) on E. coli AdiC. The function of AdiC is to mediate the influx of arginine and efflux of agmatine (the product of arginine decarboxylation in the bacterial cytosol). The result is acid consumption and protection of the bacterium against excessive acidification in the stomach lumen (Iyer et al., 2003; Fang et al., 2007).

Lewis et al. (2025) covalently labeled AdiC with bifunctional rhodamine at introduced cysteine residues in α-helix 6A, which has distinct orientations in four identified conformational states of the protein, as shown in an earlier publication from the same investigators (Zhou et al., 2023): outward open (E_o), inward open (I_o), outward-occluded (E_x), and inward-occluded (I_x). The structures of E_o and E_x are known for AdiC, and the structure of I_o and I_x are inferred from the structures of homologous proteins. Improved resolution of the polarization microscopy technique made it possible to detect relatively small changes in orientation and thereby determine which conformation a single-protein molecule assumes at a given moment and identify events resulting in changes in conformation.

The method was applied to labeled AdiC in lipid nanodiscs. The time courses of changes in orientation of the 6A helix were measured as a function of the arginine or agmatine concentration. Each of the four conformations can exist in one of three binding states: without substrate, with bound arginine, or with bound agmatine. The time course of exit from a given conformation was found to be a double exponential, indicative of two separate states within each conformation, implying a total of 24 states (4 conformations with 2 states each, 3 possible occupancies). Connectivity constraints among states reduce the number of possible transition events, but the system is still quite complex, with 60 rate constants for transitions among states and 4 dissociation constants for arginine and agmatine binding to E_o and I_o conformations.

The key to assigning numerical values to this many parameters was the ability to determine dwell times for all intermediate states at various concentrations of arginine and agmatine. From the time course of occupancy of each state, both time constants for exiting the state as well as the fraction of the population associated with each time constant could be measured. From these parameters (measured at many different substrate concentrations) and connectivity constraints among the states, it was possible to derive numerical values for 60 rate constants and 4 dissociation constants that are consistent with the single-molecule fluorescence polarization data and also can account for steady-state AdiC-mediated tracer arginine flux data in the literature.

Uniporters and 1:1 exchangers have very similar catalytic cycles (Fig. 1, right), the only difference being the rate of translocation of unloaded transporter. The rate constants for AdiC determined by Lewis et al. (2025) showed that, contrary to expectation for an obligatory 1:1 exchanger, the translocation events in the absence of ligand are not much slower than those of the loaded transporter. New transport data in the article show that the time course of tracer influx and retention in vesicles is also consistent with AdiC being a uniporter rather than a tightly coupled 1:1 exchanger, in agreement with the rate constant data. The rate constants measured for AdiC by fluorescence polarization microscopy were in an unsided preparation (nanodiscs) at pH 5 (to allow comparison with existing transport data at this pH) and no membrane potential. Membrane potential is known to affect AdiC-mediated transport (Tsai et al., 2012), and a challenge for future work will be to determine how membrane potential affects individual events in the catalytic cycle.

In summary, the work of Lewis et al. (2025) is significant because it reports the most complete set of rate constants for the elementary events of a transporter catalytic cycle that has been published to date. Each of the four identified conformations is associated with a known structure of either AdiC itself or a homolog, providing a framework for future work toward understanding why the rate constants have the observed values and why the translocation rate for unloaded transporter is so close to that for the loaded transporter. In addition, the article demonstrates the power of single-molecule fluorescence polarization microscopy as applied to transporters. Unlike single-molecule FRET, single-molecule fluorescence polarization microscopy makes it possible to track transitions among all the major conformations of a transporter if the conformations have sufficiently distinct orientations of a labeled site.

Olaf S. Andersen served as an editor.

The author acknowledges helpful input from Michael Romero, Mark Parker, and Rossana Occhipinti about the literature on transporter rate constants.

Author contributions: M.L. Jennings: conceptualization, visualization, and writing—original draft, review, and editing.