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The evolution of biological membranes was essential to enable early life to separate ions and metabolites from the environment, establish gradients of these molecules and ultimately generate and store energy for metabolism and communication1. The necessity to exchange molecules across membranes while simultaneously leveraging their potential energy to drive import or export is the most likely evolutionary driving force for the emergence of SLC proteins in the cell. Unlike ABC transporters or P-type ATPases, which use the free energy of ATP hydrolysis to drive the conformational changes required for ligand transport, SLCs operate under the influence of either internal or externally directed ion and/or metabolite gradients and function through a mechanism referred to as the alternating access cycle2 (Fig. 1, right). In its simplest form, the alternating access cycle contains two states: an outward open state, in which a central binding site is accessible to the extracellular side of the membrane, and an inward-facing state, where the transporter has changed conformation and opened its binding site to the cytoplasm. Structurally, SLCs are predominantly α-helical integral membrane proteins, and, in many cases, the conformational changes are made by specific gating helices, which control access to the central binding site in response to ligand binding3. Over the last 20 years, starting in 2003 with the publication of the ground-breaking crystal structures of LacY, the proton-coupled lactose transporter4, and GlpT, the glycerol–phosphate antiporter5, there has been a steady but inexorable increase in the number of SLC transporter structures reported6. More than 200 crystal and cryo-electron microscopy (cryo-EM) structures of SLC transporters have been deposited in the Protein Data Bank7 to date, providing valuable insights into the evolution, biochemistry and pharmacology of this medically important protein family.

Fig. 1: The many roles of SLCs in the cell.
figure 1

SLCs transport ions, metabolites, toxins and drugs across membranes, with transport often driven by internal or external ion or metabolite gradients (right, pink). They have also been identified to function as receptors in metabolic and inflammatory signaling at the lysosome and endosomal membranes (middle, teal and pink). Furthermore, SLC-like proteins, such as the KDEL receptor, have emerged as essential regulators of protein trafficking by coupling ligand binding to pH changes in the secretory pathway and forming dynamic interactions with cytoplasmic coat protein complexes, COPI and COPII (left, orange).

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Membrane proteins continue to present unique challenges for structural and biochemical studies, such as membrane extraction, finding suitable overexpression systems and identifying constructs amenable for biochemical and biophysical characterization8. Crystallizing SLCs remains extremely challenging due to their inherent flexibility, which reduces long-range order in three-dimensional crystals. Several technological advances, combined with ingenious strategies to express and identify suitable targets, aided progress in the early years. These included the development of carbohydrate-based detergents for membrane extraction and using C-terminally fused green fluorescent protein (GFP) as a reporter for screening expression9,10, protein stability and homogeneity11. Advances in membrane protein-crystallization screens12, lipid-based crystallization methods13, microfocus beamlines and photon-counting detectors enabled fast data collection on micrometer-sized crystals that often exhibit extreme radiation sensitivity14. However, progress was still slow and hard won.

A step change in SLC structural biology emerged following the well-publicized advances in single-particle cryo-EM15. Advances in image analysis and detector sensitivity now result in structures being determined with only tens of micrograms of protein, substantially lowering technical barriers and opening up new opportunities to isolate and study endogenous proteins and SLC complexes16. Additionally, structures can now be generated for molecules with large regions of disorder, which would previously need careful trial-and-error construct design for X-ray crystallography. Nevertheless, it was only relatively recently, in 2018, that we started to see the first cryo-EM structures of SLCs reported in earnest. So, what have we learned, and what opportunities remain?

Therapeutic opportunities in the SLC space

The human genome has ~450 SLC genes, representing the second largest family of membrane proteins after G protein-coupled receptors (GPCRs)17. Historically, SLC drug-discovery programs have targeted the SLC6 family of monoamine and serotonin transporters as treatments for neurological and psychiatric disorders18. However, the central role of SLC systems as regulators of metabolite flux has led to a hunt for small molecules that inhibit or reprogram cell metabolism to treat metabolic diseases19. Key examples include inhibitors of the monocarboxylate transporter MCT1 (SLC16A1)20 to starve cancer cells of lactate or inhibiting the sodium–glucose cotransporter SGLT2 (SLC5A2)21,22 to treat patients with type 2 diabetes. Further opportunities exist in understanding and exploiting SLCs to optimize drug pharmacokinetics. In 2010, the first report by the International Transporter Consortium was published to help guide clinical studies on drug transporter interactions, highlighting the critical role of SLCs in drug uptake, clearance and breakdown23,24. By tailoring drugs to SLC members, specific sites of disease can be targeted. Alternatively, drug uptake into certain organs, such as the liver or kidneys, can be lowered to reduce toxicity or increase efficacy. For example, targeting the proton-coupled folate transporter PCFT (SLC46A1) is a promising route to increase the effectiveness of antifolates to treat non-small cell lung cancer, as solid tumors upregulate PCFT to take up folic acid for cell growth25.

However, where will the next developments in SLC drug discovery come from? Two areas stand out to benefit from advances in structural biology. The first concerns exploiting our knowledge of the conformational landscape of the alternating access cycle. Designing small molecules that trap SLCs in defined states can overcome challenges in drug design18. For example, small molecules that trap an SLC in a cytoplasmic or outward-facing state can shift the inhibition mode between competitive and noncompetitive, which can have dramatic consequences for inhibitor efficacy in the context of competing substrates. Here, advances in in situ structural biology, such as determining structures in different membrane environments, will facilitate studies on the role of ion gradients, lipids, binding partners and voltage on the conformations accessible to SLCs under physiological, disease or drug-bound states. Computational and nuclear magnetic resonance (NMR) methods will also be essential to project our current static structures onto dynamic energy landscapes, enabling the complete transport cycle to be both visualized and analyzed in real time. The second area of progress concerns lipids, which have emerged as important regulators of SLC proteins by controlling oligomeric state and directly impacting transport function2. Cholesterol- and phospholipid-binding sites are being actively studied in neurotransmitter transporters, such as the serotonin transporter (SERT; SLC6A4)26, the dopamine transporter (DAT; SLC6A3)27, the glycine transporter (GlyT; SLC6A5)28 and the l-type amino acid transporter (LAT1; SLC7A5)29 for their potential to reveal new allosteric sites that can be targeted for drug discovery.

The third area where I foresee substantial advances will be the use of nanobodies to modulate SLC function. Nanobodies have proved important tools in trapping these dynamic proteins in states amenable to structure determination through either X-ray crystallography or cryo-EM. However, these and other protein scaffolds provide many opportunities to develop new ways to modulate SLC function and overcome the disadvantages of small-molecule therapeutics and chemical tools. For example, unlike small molecules, which have difficulty discriminating between closely related SLC family members, antibody-based reagents can exhibit exquisite selectivity30. As discussed below, many SLCs function as part of intracellular signaling complexes. Nanobody-based reagents have the potential to disrupt these pathways without resulting in off-target effects. Synthetic pipelines also allow easier identification of nanobodies that can bind SLCs in defined states, helping to map and trap the conformational landscape for structural analysis and therapeutic drug development31. Combined with advances in super-resolution imaging and mass spectrometry, these tools will be invaluable in decoding the role of SLCs in the broader context of cell homeostasis and disease32.

Atypical and orphan SLCs: roles beyond transport

An important advance in SLC biology has been the discovery that, within the traditional role of transporting ligands across biological membranes, SLCs can function as intracellular signaling and trafficking receptors33 (Fig. 1). It is perhaps unsurprising that evolution adapted the SLC architecture for signaling, as these proteins contain many of the same features we observe in specialized systems, such as GPCRs, Toll-like receptors and receptor tyrosine kinases. For example, SLCs undergo large conformational changes across the membrane in response to binding metabolites and rapidly respond to changes in ion gradients, which are key hallmarks for the metabolic health of the cell. They also function as scaffolding platforms to recruit and localize enzymes and trafficking complexes to membrane surfaces. These are all fundamental properties needed for membrane signaling systems. Indeed, SLCs are likely to represent ancient signaling systems focused on metabolic sensing with therapeutic potential.

To date, the most notable examples of such metabolic transceptors have been identified in the yeast Saccharomyces cerevisiae, where SLCs responsible for phosphate, amino acids and sulfates control the activity of protein kinase A and direct cells to enter high- or low-growth states in response to changes in nutrient availability34. A similar system exists in mammalian cells, where SLC36 and SLC38 families of amino acid transporters regulate the mammalian target of rapamycin (mTORC1) kinase35 (Fig. 1, middle). SLC38A9 has emerged as a crucial regulator of cell metabolism by sensing lysosomal amino acids, triggering the recruitment of the Ragulator complex, and activating mTORC1 on the lysosomal membrane36. Members of the SLC36 family of proton-coupled amino acid transporters function similarly as recruitment scaffolds to activate mTORC1 on the trans-Golgi network37. The emerging consensus is that SLCs function as necessary decoders of cellular metabolism through their ability to recognize and respond to both metabolite levels and changes in ion gradients in response to fluctuating metabolic activity. However, the molecular mechanisms linking SLC transport and receptor functions are still unknown and represent an important area of future studies.

Protein trafficking is another area where SLCs have emerged as essential regulators in the cell (Fig. 1, left). The KDEL receptor, for example, which is responsible for the pH-dependent retrieval of endoplasmic reticulum located folding chaperones in the early secretory pathway, is structurally and evolutionarily linked to the PQ-loop family of SLC transporters38. On the other hand, the chaperone UNC93B1, which is responsible for trafficking Toll-like receptors to the endolysosome, belongs to the largest family of SLCs, the major facilitator superfamily39. Another recent example linking SLC function and lysosomal biology is SLC15A4, an atypical SLC in the lysosome, which regulates activation of the innate immune adaptor protein TASL40. As we uncover the many layers of SLC behavior in the cell, new therapeutic opportunities will emerge and new biology will be revealed.

Outlook

SLCs are remarkable proteins that have evolved to respond to various cellular stimuli, including metabolite concentrations, ion gradients, voltage and lipids. Structural and biochemical studies remain fundamental in uncovering the mechanisms through which these proteins recognize and respond to such stimuli. However, extending our understanding of SLC biology will require integrating studies on single proteins and expanding these into the larger membrane environment and cellular context. Progress toward this goal has already started, with the establishment of the RESOLUTE consortium in 2020, which took a cell-based approach to deorphanize SLCs using transcriptomics, proteomics and metabolomics41. Other exciting advances are being made with solution NMR42, high-speed atomic force microscopy to understand membrane protein dynamics43, native mass spectrometry44 and also super-resolution imaging to track and trace SLCs under different cell and disease states. Ultimately, understanding how SLC proteins function at the molecular, cellular and organism levels will be essential to fully uncover the biological links between metabolism, the membrane, cellular homeostasis and disease.