Conformationally Sensitive Proximity of Extracellular Loops 2 and 4 of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Inferred from Paired Cysteine Mutagenesis

Hilwi, Maram; Dayan, Oshrat; Kanner, Baruch I.

doi:10.1074/jbc.m114.593061

Cited by 3 publications

(1 citation statement)

References 33 publications

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“…Cross-linking of introduced Cys pairs is an established approach to confirm the proximity of Cys-substituted residues in membrane transporters (45)(46)(47). Recently, this approach was applied to identify residues in transmembrane helices that form an extracellular or intracellular gate at external or internal interfaces of the aqueous translocation pathway based upon the inward-open or outward-open predicted structures, respectively.…”

Section: Discussionmentioning

confidence: 99%

Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking

Zhao

Najmi

Fiser

et al. 2016

Journal of Biological Chemistry

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Mammals meet their biosynthetic needs for one-carbon donors by absorption of dietary folates mediated by the protoncoupled folate transporter (PCFT) 2 (SLC46A1) (1, 2). Mutations in the PCFT gene that result in loss of the protein or its function are the basis for hereditary folate malabsorption (OMIM 229050), a rare autosomal recessive disorder (1, 3).PCFT transports protons along with folates as reflected in the current and acidification accompanying folate transport in oocytes that express this transporter (1, 4 -6). Structure-function studies, based on site-directed and random mutagenesis, have begun to define residues and domains that play a key role in PCFT function. Elucidation of the secondary structure of PCFT, along with the residues that contribute to the aqueous translocation pathway, have been documented by the substituted cysteine accessibility method (7). The protein consists of 12 transmembrane helices, divided in half by a large intracellular loop, with C and N termini oriented into cytoplasm (8 -10). Residues critical to folate/antifolate binding, proton coupling, and proton binding have been identified (5,(11)(12)(13). The roles of residues in the 2nd, 4th transmembrane domains (TMDs), and the loop connecting the 2nd and 3rd TMDs have been studied (14,15). Analyses of the functional defects that occur in subjects with hereditary folate malabsorption have provided insights into residues important to PCFT protein folding, stability, and/or trafficking to the cell membrane, substrate binding, and oscillation of the carrier between its conformational states (13, 16 -20).PCFT expression is limited to eukaryotes; there are no closely related proteins present in archaea or bacteria, so that a highresolution x-ray crystal structure with a high level of sequence identity is not available that would facilitate a structure/function analysis of the human or rodent proteins. Rather, homology modeling has identified the structure of the bacterial homolog, the glycerol-3-transporter, GlpT, as having a protein structure most like PCFT despite a low (14%) sequence identity (5,10,21). In this model, the transporter is in a conformation that is closed to the outside and open to the inside. According to the alternative access model, PCFT oscillates between this conformation and a conformation that is closed to the inside and open to the outside. This computational model has been used for correlation with functional data and for structural predictions as in the current study (5, 12-14, 21, 22).In this inward facing homology model of PCFT, TMD-1, -2, -7, and -11 are predicted to cluster together in proximity to the extracellular interface to form an extracellular gate. To confirm the presence of such a gate, Cys pairs predicted to be in proximity were introduced into these TMDs at locations near the extracellular surface. The possibility that the pairs are sufficiently close to be cross-linked and/or form a complex was evaluated by assessment of function, which should be decreased when the mobility of the gate is r...

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Section: Discussionmentioning

confidence: 99%

Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking

Zhao

Najmi

Fiser

et al. 2016

Journal of Biological Chemistry

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Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

Rannversson

Wilson

Kristensen

et al. 2015

Journal of Biological Chemistry

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Background:The role of extracellular loop regions during substrate translocation in the serotonin transporter (SERT) is not well understood. Results: A mutation in the extracellular loop 4 of SERT disrupts the conformational equilibrium by favoring an outward-facing conformation. Conclusion: Extracellular loop 4 is important for conformational transitions in SERT.Significance: New insights are gained into the coordinated conformational changes associated with substrate translocation in SERT.

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Metal bridges to probe membrane ion channel structure and function

Linsdell

2015

Biomolecular Concepts

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Ion channels are integral membrane proteins that undergo important conformational changes as they open and close to control transmembrane flux of different ions. The molecular underpinnings of these dynamic conformational rearrangements are difficult to ascertain using current structural methods. Several functional approaches have been used to understand two- and three-dimensional dynamic structures of ion channels, based on the reactivity of the cysteine side-chain. Two-dimensional structural rearrangements, such as changes in the accessibility of different parts of the channel protein to the bulk solution on either side of the membrane, are used to define movements within the permeation pathway, such as those that open and close ion channel gates. Three-dimensional rearrangements – in which two different parts of the channel protein change their proximity during conformational changes – are probed by cross-linking or bridging together two cysteine side-chains. Particularly useful in this regard are so-called metal bridges formed when two or more cysteine side-chains form a high-affinity binding site for metal ions such as Cd2+ or Zn2+. This review describes the use of these different techniques for the study of ion channel dynamic structure and function, including a comprehensive review of the different kinds of conformational rearrangements that have been studied in different channel types via the identification of intra-molecular metal bridges. Factors that influence the affinities and conformational sensitivities of these metal bridges, as well as the kinds of structural inferences that can be drawn from these studies, are also discussed.

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Conformationally Sensitive Proximity of Extracellular Loops 2 and 4 of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Inferred from Paired Cysteine Mutagenesis

Cited by 3 publications

References 33 publications

Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking

Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

Metal bridges to probe membrane ion channel structure and function

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