Twelve Transmembrane Helices Form the Functional Core of Mammalian MATE1 (Multidrug and Toxin Extruder 1) Protein

The x-ray structure of the prototypic MATE family member, NorM from Vibrio cholerae, reveals a protein fold composed of 12 transmembrane helices (TMHs), confirming hydropathy analyses of the majority of (prokaryotic and plant) MATE transporters. However, the mammalian MATEs are generally predicted to have a 13(th) TMH and an extracellular C terminus. Here we affirm this prediction, showing that the C termini of epitope-tagged, full-length human, rabbit, and mouse MATE1 were accessible to antibodies from the extracellular face of the membrane. Truncation of these proteins at or near the predicted junction between the 13(th) TMH and the long cytoplasmic loop that precedes it resulted in proteins that (i) trafficked to the membrane and (ii) interacted with antibodies only after permeabilization of the plasma membrane. CHO cells expressing rbMate1 truncated at residue Gly-545 supported levels of pH-sensitive transport similar to that of cells expressing the full-length protein. Although the high transport rate of the Gly-545 truncation mutant was associated with higher levels of membrane expression (than full-length MATE1), suggesting the 13(th) TMH may influence substrate translocation, the selectivity profile of the mutant indicated that TMH13 has little impact on ligand binding. We conclude that the functional core of MATE1 consists of 12 (not 13) TMHs. Therefore, we used the x-ray structure of NorM to develop a homology model of the first 12 TMHs of MATE1. The model proved to be stable in molecular dynamic simulations and agreed with topology evident from preliminary cysteine scanning of intracellular versus extracellular loops.     

Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp²³⁸, Pro²³⁹, and Glu²⁴⁷. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe²³⁰, Gly²³¹, Ala²³⁶, Val²³⁷, Ala²⁴⁴, Val²⁴⁵, and Glu²⁴⁸ were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn²²⁷, Ile²³³, and Leu²⁴³ lining the translocation pore.     

Structural and Functional Analysis of Transmembrane Segment IV of the Salt Tolerance Protein Sod2

Sod2 is the plasma membrane Na⁺/H⁺ exchanger of the fission yeast Schizosaccharomyces pombe. It provides salt tolerance by removing excess intracellular sodium (or lithium) in exchange for protons. We examined the role of amino acid residues of transmembrane segment IV (TM IV) (¹²⁶FPQINFLGSLLIAGCITSTDPVLSALI¹⁵²) in activity by using alanine scanning mutagenesis and examining salt tolerance in sod2-deficient S. pombe. Two amino acids were critical for function. Mutations T144A and V147A resulted in defective proteins that did not confer salt tolerance when reintroduced into S. pombe. Sod2 protein with other alanine mutations in TM IV had little or no effect. T144D and T144K mutant proteins were inactive; however, a T144S protein was functional and provided lithium, but not sodium, tolerance and transport. Analysis of sensitivity to trypsin indicated that the mutations caused a conformational change in the Sod2 protein. We expressed and purified TM IV (amino acids 125–154). NMR analysis yielded a model with two helical regions (amino acids 128–142 and 147–154) separated by an unwound region (amino acids 143–146). Molecular modeling of the entire Sod2 protein suggested that TM IV has a structure similar to that deduced by NMR analysis and an overall structure similar to that of Escherichia coli NhaA. TM IV of Sod2 has similarities to TM V of the Zygosaccharomyces rouxii Na⁺/H⁺ exchanger and TM VI of isoform 1 of mammalian Na⁺/H⁺ exchanger. TM IV of Sod2 is critical to transport and may be involved in cation binding or conformational changes of the protein.     

Regulatory activation is accompanied by movement in the C terminus of the Na-K-Cl cotransporter (NKCC1)

The Na-K-Cl cotransporter (NKCC1) is expressed in most vertebrate cells and is crucial in the regulation of cell volume and intracellular chloride concentration. To study the structure and function of NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins at two sites within the C terminus and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Both singly and doubly tagged NKCC1s were appropriately produced, trafficked to the plasma membrane, and exhibited (86)Rb transport activity. When both fluorescent probes were placed within the same C terminus of an NKCC1 transporter, we recorded an 11% FRET decrease upon activation of the transporter. This result clearly demonstrates movement of the C terminus during the regulatory response to phosphorylation of the N terminus. When we introduced CFP and YFP separately in different NKCC1 constructs and cotransfected these in HEK cells, we observed FRET between dimer pairs, and the fractional FRET decrease upon transporter activation was 46%. Quantitatively, this indicates that the largest FRET-signaled movement is between dimer pairs, an observation supported by further experiments in which the doubly tagged construct was cotransfectionally diluted with untagged NKCC1. Our results demonstrate that regulation of NKCC1 is accompanied by a large movement between two positions in the C termini of a dimeric cotransporter. We suggest that the NKCC1 C terminus is involved in transport regulation and that dimerization may play a key structural role in the regulatory process. It is anticipated that when combined with structural information, our findings will provide a model for understanding the conformational changes that bring about NKCC1 regulation.     

A Novel Ste20-related Proline/Alanine-rich Kinase (SPAK)-independent Pathway Involving Calcium-binding Protein 39 (Cab39) and Serine Threonine Kinase with No Lysine Member 4 (WNK4) in the Activation of Na-K-Cl Cotransporters

Na⁺-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na⁺-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.     

Transmembrane Segment 11 Appears to Line the Purine Permeation Pathway of the Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1)

Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate K_m. We investigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methanethiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an α-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60° arc suggesting that TM11 is largely α helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure.     

Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies

The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (K_d = 0.18 μm) and an ATPase activity with a K_m of 0.99 mm. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.     

Mutation of Glu521 or Glu535 in cytoplasmic loop 5 causes differential misfolding in multiple domains of multidrug and organic anion transporter MRP1 (ABCC1)

The polytopic 5-domain multidrug resistance protein 1 (MRP1/ABCC1) extrudes a variety of drugs and organic anions across the plasma membrane. Four charged residues in the fifth cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 are critical for stable expression of MRP1 at the plasma membrane. Thus Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) all cause misfolding of MRP1 and target the protein for proteasome-mediated degradation. Of four chemical chaperones tested, 4-phenylbutyric acid (4-PBA) was the most effective at restoring expression of MRP1 mutants K513A, K516A, E521A, and E535A. However, although 4-PBA treatment of K513A resulted in wild-type protein levels (and activity), the same treatment had little or no effect on the expression of K516A. On the other hand, 4-PBA treatment allowed both E521A and E535A to exit the endoplasmic reticulum and be stably expressed at the plasma membrane. However, the 4-PBA-rescued E535A mutant exhibited decreased transport activity associated with reduced substrate affinity and conformational changes in both halves of the transporter. By contrast, E521A exhibited reduced transport activity associated with alterations in the mutant interactions with ATP as well as a distinct conformational change in the COOH-proximal half of MRP1. These findings illustrate the critical and complex role of CL5 for stable expression of MRP1 at the plasma membrane and more specifically show the differential importance of Glu(521) and Glu(535) in interdomain interactions required for proper folding and assembly of MRP1 into a fully transport competent native structure.     

A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin

Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocin^P118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2^P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin^P118L and MEC-2^P134S did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.     

Expression,Folding, and Proton Transport Activity of Human Uncoupling Protein-1 (UCP1) in Lipid Membranes: EVIDENCE FOR ASSOCIATED FUNCTIONAL FORMS*

Uncoupling protein-1 (UCP1) is abundantly expressed in the mitochondrial inner membrane of brown adipose tissues and has an important role in heat generation, mediated by its proton transport function. The structure and function of UCP1 are not fully understood, partially due to the difficulty in obtaining native-like folded proteins in vitro. In this study, using the auto-induction method, we have successfully expressed UCP1 in Escherichia coli membranes in high yield. Overexpressed UCP1 in bacterial membranes was extracted using mild detergents and reconstituted into phospholipid bilayers for biochemical studies. UCP1 was folded in octyl glucoside, as indicated by its high helical content and binding to ATP, a known UCP1 proton transport inhibitor. Reconstituted UCP1 in phospholipid vesicles also exhibited highly helical structures and proton transport that is activated by fatty acids and inhibited by purine nucleotides. Self-associated functional forms of UCP1 in lipid membranes were observed for the first time. The self-assembly of UCP1 into tetramers was unambiguously characterized by circular dichroism and fluorescence spectroscopy, analytical ultracentrifugation, and semi-native gel electrophoresis. In addition, the mitochondrial lipid cardiolipin stabilized the structure of associated UCP1 and enhanced the proton transport activity of the protein. The existence of the functional oligomeric states of UCP1 in the lipid membranes has important implications for understanding the structure and proton transport mechanism of this protein in brown adipose tissues as well as structure-function relationships of other mammalian UCPs in other tissues.     

Rate and Regulation of Copper Transport by Human Copper Transporter 1 (hCTR1)

Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using ⁶⁴Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher K_m value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and K_m values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu⁺ first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry.     

Conserved intramolecular disulfide bond is critical to trafficking and fate of ATP-binding cassette (ABC) transporters ABCB6 and sulfonylurea receptor 1 (SUR1)/ABCC8

The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.     

Propofol Binding to the Resting State of the Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Induces Structural Changes in the Inter- and Intrasubunit Transmembrane Domain (TMD) Cavities

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428–431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.     

The Sodium Bicarbonate Cotransporter (NBCe1) Is Essential for Normal Development of Mouse Dentition

Proximal renal tubular acidosis (pRTA) is a syndrome caused by abnormal proximal tubule reabsorption of bicarbonate resulting in metabolic acidosis. Patients with mutations to the SLC4A4 gene (coding for the sodium bicarbonate cotransporter NBCe1), have pRTA, growth delay, ocular defects, and enamel abnormalities. In an earlier report, we provided the first evidence that enamel cells, the ameloblasts, express NBCe1 in a polarized fashion, thereby contributing to trans-cellular bicarbonate transport. To determine whether NBCe1 plays a critical role in enamel development, we studied the expression of NBCe1 at various stages of enamel formation in wild-type mice and characterized the biophysical properties of enamel in NBCe1^−/− animals. The enamel of NBCe1^−/− animals was extremely hypomineralized and weak with an abnormal prismatic architecture. The expression profile of amelogenin, a known enamel-specific gene, was not altered in NBCe1^−/− animals. Our results show for the first time that NBCe1 expression is required for the development of normal enamel. This study provides a mechanistic model to account for enamel abnormalities in certain patients with pRTA.     

The Calsequestrin Mutation CASQ2D307H Does Not Affect Protein Stability and Targeting to the Junctional Sarcoplasmic Reticulum but Compromises Its Dynamic Regulation of Calcium Buffering

Mutations in cardiac ryanodine receptor (RYR2) and cardiac calsequestrin (CASQ2) genes are linked to catecholaminergic polymorphic ventricular tachycardia, a life-threatening genetic disease. They predispose young individuals to cardiac arrhythmia in the absence of structural abnormalities. One such mutation that changes an aspartic residue to histidine at position 307 in CASQ2 has been linked to catecholaminergic polymorphic ventricular tachycardia. In this study we made a transgenic mouse model expressing the mutant CASQ2^D307H protein in a CASQ2 null background and investigated if the disease is caused by accelerated degradation of the mutant protein. Our data suggest that the mutant protein can be expressed, is relatively stable, and targets appropriately to the junctional sarcoplasmic reticulum. Moreover, it partially normalizes the ultrastructure of the sarcoplasmic reticulum, which was altered in the CASQ2 null background. In addition, overexpression of the mutant protein does not cause any pathology and/or structural changes in the myocardium. We further demonstrate, using purified protein, that the mutant protein is very stable under chemical and thermal denaturation but shows abnormal Ca²⁺ buffering characteristics at high calcium concentrations. In addition, trypsin digestion studies reveal that the mutant protein is more susceptible to protease activity only in the presence of high Ca²⁺. These studies collectively suggest that the D307H mutation can compromise the dynamic behavior of CASQ2 including supramolecular rearrangement upon Ca²⁺ activation.     

A conserved asparagine residue in transmembrane segment 1 (TM1) of serotonin transporter dictates chloride-coupled neurotransmitter transport

Na(+)- and Cl(-)-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl(-) coordination of human serotonin transport have been identified, the role of Cl(-) in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl(-) binding to concentrative neurotransmitter uptake.     

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. Here, we report crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu⁵²⁰, which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, our structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.     

Severe neurologic impairment in mice with targeted disruption of the electrogenic sodium bicarbonate cotransporter NBCe2 (Slc4a5 gene)

The choroid plexus lining the four ventricles in the brain is where the majority of cerebrospinal fluid (CSF) is produced. The secretory function of the choroid plexus is mediated by specific transport systems that allow the directional flux of nutrients and ions into the CSF and the removal of toxins. Normal CSF dynamics and chemistry ensure that the environment for neural function is optimal. Here, we report that targeted disruption of the Slc4a5 gene encoding the electrogenic sodium bicarbonate cotransporter NBCe2 results in significant remodeling of choroid plexus epithelial cells, including abnormal mitochondrial distribution, cytoskeletal protein expression, and ion transporter polarity. These changes are accompanied by very significant abnormalities in intracerebral ventricle volume, intracranial pressure, and CSF electrolyte levels. The Slc4a5(-/-) mice are significantly more resistant to induction of seizure behavior than wild-type controls. In the retina of Slc4a5(-/-) mice, loss of photoreceptors, ganglion cells, and retinal detachment results in visual impairment assessed by abnormal electroretinogram waveforms. Our findings are the first demonstration of the fundamental importance of NBCe2 in the biology of the nervous system.     

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT,a Member of the l-Amino Acid Transporter (LAT) Family

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC₅₀ similar to the apparent K_M of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT.