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An externally accessible linker region in the sodium-coupled phosphate transporter PiT-1 (SLC20A1) is important for transport function


Ravera, Silvia; Murer, Heini; Forster, Ian C (2013). An externally accessible linker region in the sodium-coupled phosphate transporter PiT-1 (SLC20A1) is important for transport function. Cellular Physiology and Biochemistry, 32(1):187-199.

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 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.

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 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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology

04 Faculty of Medicine > Center for Integrative Human Physiology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2013
Deposited On:12 Sep 2013 11:15
Last Modified:07 Dec 2017 22:19
Publisher:Karger
ISSN:1015-8987
Additional Information:The final, published version of this article is available at http://www.karger.com/?doi=10.1159/000350135
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1159/000350135
PubMed ID:23899881

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