Transporters Through the Looking Glass: An Insight into the Mechanisms of Ion-Coupled Transport and Methods That Help Reveal Them

Abstract

Cell membranes, despite providing a barrier to protect intracellular constituents, require selective gating for influx of important metabolites including ions, sugars, amino acids, neurotransmitters and efflux of toxins and metabolic end-products. The machinery involved in carrying out this gating process comprises of integral membrane proteins that use ionic electrochemical gradients or ATP hydrolysis, to drive concentrative uptake or efflux. The mechanism through which ion-coupled transporters function is referred to as alternating-access. In the recent past, discrete modes of alternating-access have been described with the elucidation of new transporter structures and their snapshots in altered conformational states. Despite X-ray structures being the primary sources of mechanistic information, other biophysical methods provide information related to the structural dynamics of these transporters. Methods including EPR and smFRET, have extensively helped validate or clarify ion-coupled transport mechanisms, in a near-native environment. This review seeks to highlight the mechanistic details of ion-coupled transport and delve into the biophysical tools and methods that help in understanding these fascinating molecules.

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

“…The TableS1: List of maleimide fluorophores used in this study including their extinction coefficients. Sources were company web-portals of Thermo-Fischer for AlexaFluor dyes [12], ATTOTEC [13], Lumiprobe [14] and ref. [15] for Cy3B.…”

“…Over the past years [13], various labs have introduced single-molecule tools to investigate the structural dynamics of active membrane transporters [14][15][16][17][18][19][20][21][22][23][24][25]. Förster resonance energy transfer in combination with single-molecule detection (smFRET [26][27][28]) has proven to be a particularly useful tool for the validation of structural models [29][30][31], and for revealing functional features of transporters which are mechanistically important, such as conformational heterogeneity [28,[32][33][34].…”

Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor

“…Porters mediate the passage of small molecules and ions like glucose and sodium and chloride ions [ 50 ]. The binding of substrates induces a conformational change in the porter protein, allowing the movement of these substrates across the cell plasma membrane [ 50 ]. Porters can be further classified based upon the number of types of molecules they convey [ 50 , 51 ].…”

“…The binding of substrates induces a conformational change in the porter protein, allowing the movement of these substrates across the cell plasma membrane [ 50 ]. Porters can be further classified based upon the number of types of molecules they convey [ 50 , 51 ]. Whereas uniporters transport one type of molecule, symporters and antiporters are classified as cotransporters that organize the exchange of two different substrates ( Figure 1 E) [ 50 , 51 ].…”

Structure and Gating Dynamics of Na+/Cl– Coupled Neurotransmitter Transporters

“…The structural and mechanistic similarities with LeuT have typified the underlying mechanisms of substrate transport in eukaryotic neurotransmitter transporters (Focke et al, 2013; Penmatsa and Gouaux, 2014; Navratna and Gouaux, 2019). The architecture of Na + /Cl − coupled neurotransmitter transporters reveal 12 TM helices of which helical bundles 1–5 and 6–10 display a pseudo 2-fold symmetry that allows, “alternating-access” within this family of transporters (Figure 3A) (Jardetzky, 1966; Drew and Boudker, 2016; Majumder et al, 2018). Within the symmetric helices, TMs 1 and 6 serve as gating helices with a non-helical junction in the center that allows movement of extracellular and intracellular halves independent of each other to regulate the exposure and closure of the ligand-binding pocket to solvent access.…”