Excitatory amino acid transporters (EAAT/SLC1) mediate Na+-dependent uptake of extracellular glutamate and are potential drug targets for neurological disorders. Conventional methods to assess... Show moreExcitatory amino acid transporters (EAAT/SLC1) mediate Na+-dependent uptake of extracellular glutamate and are potential drug targets for neurological disorders. Conventional methods to assess glutamate transport in vitro are based on radiolabels, fluorescent dyes or electrophysiology, which potentially compromise the cell's physiology and are generally less suited for primary drug screens. Here, we describe a novel label-free method to assess human EAAT function in living cells, i.e., without the use of chemical modifications to the substrate or cellular environment. In adherent HEK293 cells overexpressing EAAT1, stimulation with glutamate or aspartate induced cell spreading, which was detected in real-time using an impedance-based biosensor. This change in cell morphology was prevented in the presence of the Na+/K+-ATPase inhibitor ouabain and EAAT inhibitors, which suggests the substrate-induced response was ion-dependent and transporter-specific. A mechanistic explanation for the phenotypic response was substantiated by actin cytoskeleton remodeling and changes in the intracellular levels of the osmolyte taurine, which suggests that the response involves cell swelling. In addition, substrate-induced cellular responses were observed for cells expressing other EAAT subtypes, as well as in a breast cancer cell line (MDA-MB-468) with endogenous EAAT1 expression. These findings allowed the development of a label-free high-throughput screening assay, which could be beneficial in early drug discovery for EAATs and holds potential for the study of other transport proteins that modulate cell shape. Show less
Solute carrier transporters (SLCs) limit receptor activation via uptake of extracellular ligands. Novel concepts are emerging that describe the modulation of intracellular and plasma membrane... Show moreSolute carrier transporters (SLCs) limit receptor activation via uptake of extracellular ligands. Novel concepts are emerging that describe the modulation of intracellular and plasma membrane receptors by ligand influx and efflux via SLCs, respectively. Here, we evaluate recent insights and provide an outlook for developing potential therapeutic strategies. Show less
The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs... Show moreThe solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery. Show less
The Innovative Medicines Initiative Consortium RESOLUTE has started to develop tools and produce data sets to de-orphanize transporters in the solute carrier protein (SLC) superfamily, thereby... Show moreThe Innovative Medicines Initiative Consortium RESOLUTE has started to develop tools and produce data sets to de-orphanize transporters in the solute carrier protein (SLC) superfamily, thereby lowering the barrier for the scientific community to explore SLCs as an attractive drug target class Show less
Polymerase delta is essential for eukaryotic genome duplication and synthesizes DNA at both the leading and lagging strands. The polymerase delta complex is a heterotetramer comprising the... Show morePolymerase delta is essential for eukaryotic genome duplication and synthesizes DNA at both the leading and lagging strands. The polymerase delta complex is a heterotetramer comprising the catalytic subunit POLD1 and the accessory subunits POLD2, POLD3, and POLD4. Beyond DNA replication, the polymerase delta complex has emerged as a central element in genome maintenance. The essentiality of polymerase delta has constrained the generation of polymerase delta-knockout cell lines or model organisms and, therefore, the understanding of the complexity of its activity and the function of its accessory subunits. To our knowledge, no germline biallelic mutations affecting this complex have been reported in humans. In patients from 2 independent pedigrees, we have identified what we believe to be a novel syndrome with reduced functionality of the polymerase delta complex caused by germline biallelic mutations in POLD1 or POLD2 as the underlying etiology of a previously unknown autosomal-recessive syndrome that combines replicative stress, neurodevelopmental abnormalities, and immunodeficiency. Patients' cells showed impaired cell-cycle progression and replication-associated DNA lesions that were reversible upon overexpression of polymerase delta. The mutations affected the stability and interactions within the polymerase delta complex or its intrinsic polymerase activity. We believe our discovery of human polymerase delta deficiency identifies the central role of this complex in the prevention of replication-related DNA lesions, with particular relevance to adaptive immunity. Show less
