In humans, more than 50 transporters are responsible for the traffic and balance of amino acids within and between cells and tissues, and half of them have been associated with disease 1. Covering ...all common amino acids, Heteromeric Amino acid Transporters (HATs) are one class of such transporters. This review first highlights structural and functional studies that solved the atomic structure of HATs and revealed molecular clues on substrate interaction. Moreover, this review focuses on HATs that have a role in the central nervous system (CNS) and that are related to neurological diseases, including: (i) LAT1/CD98hc and its role in the uptake of branched chain amino acids trough the blood brain barrier and autism. (ii) LAT2/CD98hc and its potential role in the transport of glutamine between plasma and cerebrospinal fluid. (iii) y
+
LAT2/CD98hc that is emerging as a key player in hepatic encephalopathy. xCT/CD98hc as a potential therapeutic target in glioblastoma, and (iv) Asc-1/CD98hc as a potential therapeutic target in pathologies with alterations in NMDA glutamate receptors.
L-amino acid transporters (LATs) play key roles in human physiology and are implicated in several human pathologies. LATs are asymmetric amino acid exchangers where the low apparent affinity ...cytoplasmic side controls the exchange of substrates with high apparent affinity on the extracellular side. Here, we report the crystal structures of an LAT, the bacterial alanine-serine-cysteine exchanger (BasC), in a non-occluded inward-facing conformation in both apo and substrate-bound states. We crystallized BasC in complex with a nanobody, which blocks the transporter from the intracellular side, thus unveiling the sidedness of the substrate interaction of BasC. Two conserved residues in human LATs, Tyr 236 and Lys 154, are located in equivalent positions to the Na1 and Na2 sites of sodium-dependent APC superfamily transporters. Functional studies and molecular dynamics (MD) calculations reveal that these residues are key for the asymmetric substrate interaction of BasC and in the homologous human transporter Asc-1.
Abstract The function of the chloride channel ClC‐1 is crucial for the control of muscle excitability. Thus, reduction of ClC‐1 functions by CLCN1 mutations leads to myotonia congenita. Many ...different animal models have contributed to understanding the myotonia pathophysiology. However, these models do not allow in vivo screening of potentially therapeutic drugs, as the zebrafish model does. In this work, we identified and characterized the two zebrafish orthologues ( clc‐1a and clc‐1b ) of the ClC‐1 channel. Both channels are mostly expressed in the skeletal muscle as revealed by RT‐PCR, western blot, and electrophysiological recordings of myotubes, and clc‐1a is predominantly expressed in adult stages. Characterization in Xenopus oocytes shows that the zebrafish channels display similar anion selectivity and voltage dependence to their human counterparts. However, they show reduced sensitivity to the inhibitor 9‐anthracenecarboxylic acid (9‐AC), and acidic pH inverts the voltage dependence of activation. Reduction of clc‐1a/b expression hampers spontaneous and mechanically stimulated movement, which could be reverted by expression of human ClC‐1 but not by some ClC‐1 containing myotonia mutations. Treatment of clc‐1 ‐depleted zebrafish with mexiletine, a typical drug used in human myotonia, improves the motor behaviour. Our work extends the repertoire of ClC channels to evolutionary structure–function studies and proposes the zebrafish clcn1 crispant model as a simple tool to find novel therapies for myotonia. image Key points We have identified two orthologues of ClC‐1 in zebrafish (clc‐1a and clc‐1b) which are mostly expressed in skeletal muscle at different developmental stages. Functional characterization of the activity of these channels reveals many similitudes with their mammalian counterparts, although they are less sensitive to 9‐AC and acidic pH inverts their voltage dependence of gating. Reduction of clc‐1a/b expression hampers spontaneous and mechanically stimulated movement which could be reverted by expression of human ClC‐1. Myotonia‐like symptoms caused by clc‐1a/b depletion can be reverted by mexiletine, suggesting that this model could be used to find novel therapies for myotonia.
Abstract ClC‐K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or ...BSND , encoding ClC‐Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline‐tyrosine motif in the C‐terminus of barttin stimulates ClC‐K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co‐expressing ClC‐K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC‐K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock‐in mouse. Comparing wild‐type (WT) and knock‐in mice under a standard diet, we could not observe any difference in ClC‐K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high‐sodium low‐potassium diet, known to induce hyperplasia of distal convoluted tubules, knock‐in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. image Key points It is revealed by mutagenesis and functional experiments that a previously identified proline‐tyrosine motif regulating ClC‐K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC‐K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo . Knock‐in mice with a mutation in this motif, under conditions of a high‐sodium low‐potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild‐type animals, indicating a gain of function of the channel in vivo .
Age-related hearing loss (ARHL) is the most common sensory deficit in the elderly. The disease has a multifactorial etiology with both environmental and genetic factors involved being largely ...unknown. SLC7A8/SLC3A2 heterodimer is a neutral amino acid exchanger. Here, we demonstrated that SLC7A8 is expressed in the mouse inner ear and that its ablation resulted in ARHL, due to the damage of different cochlear structures. These findings make SLC7A8 transporter a strong candidate for ARHL in humans. Thus, a screening of a cohort of ARHL patients and controls was carried out revealing several variants in
, whose role was further investigated by in vitro functional studies. Significant decreases in SLC7A8 transport activity was detected for patient's variants (p.Val302Ile, p.Arg418His, p.Thr402Met and p.Val460Glu) further supporting a causative role for SLC7A8 in ARHL. Moreover, our preliminary data suggest that a relevant proportion of ARHL cases could be explained by SLC7A8 mutations.
Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of ...pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
We examined the role of the concentrative nucleoside transporter CNT3 in the establishment of a transepithelial flux of natural
nucleosides and their pharmacologically active derivatives in renal ...epithelial cell lines. Murine PCT cells grown on a transwell
dish showed endogenous CNT3 activity at their apical membrane that was responsible for the sodium-dependent transepithelial
flux of both purine and pyrimidine nucleosides. hCNT3 was also identified in human kidney and its role in the transport of
nucleosides was tested. To this end, MDCK cells, lacking endogenous CNT3 activity, were genetically engineered to express
the human orthologue of CNT3 (hCNT3-MDCK cells). In these cells, hCNT3 was inserted into the apical membrane, thus generating,
as for PCT cells, a transepithelial flux of both nucleosides and nucleoside-derived drugs. Apical-to-basolateral transepithelial
flux was present in all cells expressing a functional CNT3 transporter and was significantly higher than that found either
in PCT cells in absence of sodium or in mock-transfected MDCK cells. Nevertheless in all cases a significant amount of the
transported nucleoside was retained and transformed inside cells. However release to the opposite compartment was CNT3 dependent,
not only in terms of absolute flux (much higher when an apical CNT3 transporter was active) but also regarding metabolic transformations
of the apically absorbed nucleosides. These results underline a critical role of CNT3 in the renal reabsorption of nucleosides
and their derivatives as well as in their intracellular metabolism.
Heteromeric amino acid transporters (HATs) are relevant targets for structural studies. On the one hand, HATs are involved in inherited and acquired human pathologies. On the other hand, these ...molecules are the only known examples of solute transporters composed of two subunits (heavy and light) linked by a disulfide bridge. Unfortunately, structural knowledge of HATs is scarce and limited to the atomic structure of the ectodomain of a heavy subunit (human 4F2hc-ED) and distant prokaryotic homologues of the light subunits that share a LeuT-fold. Recent data on human 4F2hc/LAT2 at nanometer resolution revealed 4F2hc-ED positioned on top of the external loops of the light subunit LAT2. Improved resolution of the structure of HATs, combined with conformational studies, is essential to establish the structural bases for light subunit recognition and to evaluate the functional relevance of heavy and light subunit interactions for the amino acid transport cycle.
Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral ...heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4-3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1.
Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, ...AdiC, is the main APC structural paradigm and shares the "5 + 5 inverted repeat" fold found in other families like the Na⁺-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg⁺-bound occluded. However, the role of Arg⁺ during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg⁺-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg⁺-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.
PDF
