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

Abstract

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.

Identification and expression analysis of type II and type III Pi transporters in the opossum kidney cell line

“…A hydropathy analysis revealed an uncommon structure of 11 TMDs, with the amino end located intracellularly and the carboxy terminus located extracellularly. This contrasts with the established 12‐TMD model, in which both ends of the protein are located extracellularly (Bøttger & Pedersen, ; Ravera et al., ). The 11‐TMD proposal is attributable to the absence, in OK cell PiT1, of the first accepted TMD of the 12‐TMD model, which has the consensus sequence ILGFIIAFVLAFSVG in the other orthologue proteins (Figure b).…”

“…Common characteristics include a glycosylation site at asparagine N94 within the first extracellular loop (amino acids 83–96); two phosphorylation sites at S261 and S265 in the main intracellular loop; and two phosphate transporter family domains (pfam01384) at amino acids 43–140 and 561–659, respectively (PHO4; https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=pfam01384). The first loop (amino acids 83–96) is a region involved in P i affinity and, according to the results of a combination of the two‐electrode voltage clamp and substituted cysteine accessibility methods, this region seems to form part of a hydrophilic pore where the substrates interact with specific amino acids (Ravera, Murer, & Forster, ).…”

“…Consequently, the first TMD of the accepted model of PiT1 in other species is not predicted for OK cell PiT1, and therefore, the model changes from 12 TMDs (Bøttger & Pedersen, 2011;Ravera et al, 2013) to 11 TMDs.…”

“…The main difference was related to PiT1, because the first 39 amino acids were very different with respect to the remaining orthologues, resulting in high hydrophilicity and low hydrophobicity of the amino‐terminus, according to the hydropathy results (Figure c). Consequently, the first TMD of the accepted model of PiT1 in other species is not predicted for OK cell PiT1, and therefore, the model changes from 12 TMDs (Bøttger & Pedersen, ; Ravera et al., ) to 11 TMDs.…”

“…All the other characteristics present in PiT1 and PiT2 from OK cells have been described previously. This includes N-glycosylation and critical amino acids for P i transport in the external region between TMDs 1 and 2 in PiT1 and TMDs 2 and 3 in PiT2 (Ravera et al, 2013), in addition to the two pfam01384 domains, the PD1131 homology domains (Salaün, Gyan, Rodrigues, & Heard, 2002) and the PiT signature sequences (Bøttger & Pedersen, 2011).…”