title: An externally accessible linker region in the sodium-coupled phosphate transporter PiT-1 (SLC20A1) is important for transport function.
abstract: OBJECTIVE
  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 Pi homeostasis as well as being implicated in vascular calcification and renal Pi 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 (32)P 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 Pi 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.
authors.affiliation: Institute of Physiology and Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland.
authors.forename: Silvia
authors.initials: S
authors.lastname: Ravera
authors.forename: Heini
authors.initials: H
authors.lastname: Murer
authors.forename: Ian C
authors.initials: IC
authors.lastname: Forster
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D003545:Cysteine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050611:Sodium-Phosphate Cotransporter Proteins, Type III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014982:Xenopus laevis / Q000254:growth & development / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D003545:Cysteine