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

• Main Authors: Silvia Ravera, Heini Murer, Ian C. Forster
• Format: Article
• Language: English
• Published: Cell Physiol Biochem Press GmbH & Co KG 2013-07-01
• Series: Cellular Physiology and Biochemistry
• Subjects: Na+/Pi cotransport; Two-electrode voltage clamp; SLC20; Cysteine scanning
• Online Access: http://www.karger.com/Article/FullText/350135

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 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 2nd and 3rd 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 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.