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

Cellular Physiology and Biochemistry

关键词: Na+/Pi cotransport;    Two-electrode voltage clamp;    SLC20;    Cysteine scanning;   

DOI  :  10.1159/000350135

学科分类：分子生物学，细胞生物学和基因

来源: S Karger AG

【 摘 要 】

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.

【 授权许可】

CC BY-NC-ND