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Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-20249

Ghezzi, C; Murer, H; Forster, I C (2009). Substrate Interactions of the Electroneutral Na+-Coupled Inorganic Phosphate Cotransporter (NaPi-IIc). Journal of Physiology, 587(17):4293-4307.

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Abstract

The SLC34 solute carrier family comprises the electrogenic NaPi-IIa/b and the electroneutral NaPi-IIc, which display Na(+):P(i) cotransport stoichiometries of 3:1 and 2:1, respectively. We previously proposed that NaPi-IIc lacks one of the 3 Na(+) interaction sites hypothesised for the electrogenic isoforms, however, unlike NaPi-IIa/b, its substrate binding order is undetermined. By expressing NaPi-IIc in Xenopus oocytes, isotope influx and efflux assays gave results consistent with Na(+) being the first and last substrate to bind. To further investigate substrate interactions, we applied a fluorometry-based technique that uses site-specific labelling with a fluorophore to characterize substrate-induced conformational changes. A novel Cys was introduced in the 3rd extracellular loop of NaPi-IIc that could be labelled with a reporter fluorophore (MTS-TAMRA). Although labelling resulted in suppression of cotransport as previously reported for the electrogenic isoforms, changes in fluorescence were induced by changes in extracellular Na(+) concentration in the absence of P(i) and by changes in extracellular Pi concentration in presence of Na(+). These data, combined with (32)P uptake data, also support a binding scheme in which Na(+) is the 1(st) substrate to interact. Moreover, the apparent P(i) affinity from fluorometry agreed with that from (32)P uptake, confirming the applicability of the fluorometric technique for kinetic studies of electroneutral carriers. Analysis of the fluorescence data showed that like the electrogenic NaPi-IIb, 2 Na(+) ions interact cooperatively with NaPi-IIc before P(i) binding, which implies that only one of these is translocated. This result provides compelling evidence that SLC34 proteins share common motifs for substrate interaction and that cotransport and substrate binding stoichiometries are not necessarily equivalent.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Center for Integrative Human Physiology
04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
DDC:570 Life sciences; biology
610 Medicine & health
Language:English
Date:September 2009
Deposited On:18 Aug 2009 08:35
Last Modified:27 Nov 2013 22:36
Publisher:Wiley-Blackwell
ISSN:0022-3751
Additional Information:The definitive version is available at www.blackwell-synergy.com
Publisher DOI:10.1113/jphysiol.2009.175596
PubMed ID:19596895

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