Phosphate Transport Kinetics and Structure-Function Relationships of SLC34 and SLC20 Proteins

Forster, Ian C.; Hernando, Nati; Biber, Jürg; Murer, Heini

doi:10.1016/b978-0-12-394316-3.00010-7

Cited by 63 publications

(80 citation statements)

References 145 publications

(242 reference statements)

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“…Voltage-dependent transport proteins, including Na + cotransporters and pumps, exhibit these capacitive currents (7,(10)(11)(12). Transient charge movements may be fit with a Boltzmann relation to estimate the voltage at half-maximal charge transfer (V 0.5 ), where the protein is equally distributed between the two extreme states, the apparent valence of the moveable charge (z) and the number of transporters in the plasma membrane (Q max ) (10, 13).…”

mentioning

confidence: 99%

Functional identification and characterization of sodium binding sites in Na symporters

Loo

Jiang

Gorraitz

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na + in binding sites is beyond the resolution of the structures, two Na + binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (K d ) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na + binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two -OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium K d by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na + and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na + binding, and that Na + binding to Na2 promotes binding to Na1 and also sugar binding.I on coupled symporters, or cotransporters, such as hSGLT1 use electrochemical potential gradients to drive solutes into cells. A common finding for these transporters is that external Na + binds before the substrate. Na + binding induces a conformational change of the protein, resulting in the substrate vestibule becoming open to the external membrane surface. After substrate binding, the two ligands are transported across the membrane and are released into the cytoplasm. The atomic structures of several sodium dependent transporters have been solved [leucine transporter (LeuT), vibrio parahaemolyticus sodium glucose (vSGLT), sodium hydantoin (Mhp1), and sodium betaine (BetP)] (1-6). They share a common structural fold with a five-helix inverted repeat, the "LeuT fold". The substrate binding sites are located in the middle of the protein, isolated from the external and membrane surfaces by hydrophobic gates, and putative Na + sites have been identified. Testing these binding sites is a problem due to the fact that phenomenological kinetic constants for ligand transport (K 0.5 , half-saturation values) are interdependent on each other (7). Here we have developed a method to estimate the intrinsic Na + dissociation constants (K d ) for human sodium glucose cotransport (hSGLT1) that may be broadly applicable to other symporters. We then use this to investigate the importance of hSGLT1 residues predicted to be at or near the two Na + binding sites, Na1 and Na2.The method is based on the fact that voltage-dependent membrane proteins exhibit transient charge movements (capacitive transients) i...

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mentioning

confidence: 99%

Functional identification and characterization of sodium binding sites in Na symporters

Loo

Jiang

Gorraitz

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…SCAM has previously proven useful when applied to members of the type II Na + /P i cotransporter (SLC34 family) (for review see [3]). An important caveat for its successful application is that only the substituted Cys should be the target of the thiol reaction.…”

Section: Discussionmentioning

confidence: 99%

“…The gene products of the SLC34 family (NaPi-IIa, NaPi-IIb and NaPi-IIc) are responsible for P i absorption and reabsorption across the apical membranes of many epithelia, including kidney and small intestine, where they play essential roles in P i homeostasis (for review see [1,2,3,4]). Their transport properties have been extensively characterized by heterologous expression in Xenopus oocytes and a number of structure-function studies based on cysteine scanning and voltage-clamp fluorometry have been described (reviewed in [3]). The gene products of the SLC20 family (SLC20A1 or PiT-1and SLC20A2 or PiT-2, also referred to as type III Na + /P i transporters) were initially identified as retroviral receptors and later shown to be Na + -coupled P i cotransporters [5,6,7].…”

Section: Introductionmentioning

confidence: 99%

An Externally Accessible Linker Region in the Sodium-Coupled Phosphate Transporter PiT-1 (SLC20A1) is Important for Transport Function

Ravera¹,

Murer²,

Forster³

2013

Cell Physiol Biochem

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Background/Aims: Members of the SLC20 cotransporter family (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform housekeeping functions for intracellular P_i homeostasis as well as being implicated in vascular calcification and renal P_i reabsorption. The aims of this study were to investigate the topology of a linker region in PiT-1 between the predicted 2^nd and 3^rd transmembrane domains and to investigate the functional consequences of cysteine substitutions in this region. Methods: Cysteines were substituted at 18 sites in the Xenopus PiT-1 isoform and the mutants were expressed in Xenopus laevis oocytes. Transport function of the mutants was investigated by ³²P tracer or two electrode voltage clamp before and after thiol modification of the novel Cys. Results: Exposure to the thiol reactive reagent resulted in diminished transport function for 7 mutants. The apparent accessibility of 5 of the mutated sites, estimated from the rate of functional thiol modification, was site-dependent. Cysteine substitution at some sites also altered the apparent affinity for P_i and cation (Na⁺/Li⁺) and substrate (phosphate/arsenate) selectivity, further underscoring the importance of this linker in defining PiT-1 transport characteristics. Conclusions: The external accessibility of a linker in PiT-1 was confirmed and sites were identified that determine substrate selectivity and transport function.

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“…(15). We made Cys-substitutions in AAD-IIc and investigated the constructs by means of conventional TEVC (steady-state kinetics) and voltage clamp fluorometry (VCF) (7,19,20,34,35).…”

Section: Cysteine Substitutions In Aad-iicmentioning

confidence: 99%

Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step

Patti

Ghezzi

Forster

2013

Pflugers Arch - Eur J Physiol

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The SLC34 family of Na(+)-dependent inorganic phosphate cotransporters comprises two electrogenic isoforms (NaPi-IIa, NaPi-IIb) and an electroneutral isoform (NaPi-IIc). Both fulfill essential physiological roles in mammalian phosphate homeostasis. By substitution of three conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc, electrogenicity was re-established and the Na(+)/P i stoichiometry increased from 2:1 to 3:1. However, this engineered electrogenic construct (AAD-IIc) had a reduced apparent P i affinity and different presteady-state kinetics from the wildtype NaPi-IIa/b. We investigated AAD-IIc using electrophysiology and voltage clamp fluorometry to elucidate the compromised behavior. The activation energy for cotransport was threefold higher than for NaPi-IIc and 1.5-fold higher than for NaPi-IIa and the temperature dependence of presteady-state charge displacements suggested that the large activation energy was associated with the empty carrier reorientation. AAD-IIc shows a weak interaction of external Na(+) ions with the electric field, and thus retains the electroneutral cooperative interaction of two Na(+) ions preceding external P i binding of NaPi-IIc. Most of the presteady-state charge movement was accounted for by the empty carrier (in the absence of external P i ), and the cytosolic release of one Na(+) ion (in the presence of P i ). Simulations using a kinetic model recapitulated the presteady-state and steady-state behavior and allowed identification of two critical partial reactions: the final release of Na(+) to the cytosol and external P i binding. Fluorometric recordings from AAD-IIc mutants with Cys substituted at functionally important sites established that AAD-IIc undergoes substrate-and voltage-dependent conformational changes that correlated qualitatively with its presteady-state kinetics. Manuscript-revised Click here to download Manuscript: AAD-EJP 2nd sub-final.docx Click here to view linked References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 AbstractThe SLC34 family of Na + -dependent inorganic phosphate cotransporters comprises two electrogenic isoforms (NaPi-IIa, NaPi-IIb) and an electroneutral isoform (NaPi-IIc). Both fulfil essential physiological roles in mammalian phosphate homeostasis. By substitution of 3 conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc, electrogenicity was re-established and the Na + :P i stoichiometry increased from 2:1 to 3:1. However, this engineered electrogenic construct (AAD-IIc) had a reduced apparent P i affinity and different presteady-state kinetics from the wild-type NaPi-IIa/b. We investigated AAD-IIc using electrophysiology and voltage clamp fluorometry to elucidate the compromised behavior. The activation energy for cotransport was 3-f...

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Phosphate Transport Kinetics and Structure-Function Relationships of SLC34 and SLC20 Proteins

Cited by 63 publications

References 145 publications

Functional identification and characterization of sodium binding sites in Na symporters

Functional identification and characterization of sodium binding sites in Na symporters

An Externally Accessible Linker Region in the Sodium-Coupled Phosphate Transporter PiT-1 (SLC20A1) is Important for Transport Function

Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step

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