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Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-48247

Ghezzi, C; Meinild, A K; Murer, H; Forster, I C (2011). Voltage- and substrate-dependent interactions between sites in putative re-entrant domains of a Na(+)-coupled phosphate cotransporter. Pflügers Archiv: European Journal of Physiology (Pflugers Archiv), 461(6):645-663.

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Abstract

A common structural feature characterises sodium-coupled inorganic phosphate cotransporters of the SLC34 family (NaPi-IIa/b/c): a pair of inverted regions in the N- and C-terminal halves of the protein. These regions are hypothesised to contain re-entrant domains that associate to allow alternating access of the substrates from either side of the membrane. To investigate if these domains interact during the NaPi-II transport cycle, we introduced novel cysteines at three functionally important sites associated with the predicted re-entrant domains of the flounder NaPi-IIb for the purpose of fluorescent labelling and cross-linking. Single and double mutants were expressed in Xenopus oocytes and their function analysed using electrophysiological and real-time fluorometric assays. The substitution at the cytosolic end of the first re-entrant domain induced a large hyperpolarizing shift in the voltage dependence of steady-state and presteady-state kinetics, whereas the two substitutions at the external face were less critical. By using Cu-phenanthroline to induce disulfide bridge formation, we observed a loss of transport activity that depended on the presence of sodium in the incubation medium. This suggested that external sodium increased the probability of NaPi-IIb occupying a conformation that favours interaction between sites in the re-entrant domains. Furthermore, voltage-dependent fluorescence data supported the hypothesis that a localised interaction between the two domains occurs that depends on the membrane potential and substrate present: we found that the fluorescence intensity reported by a labelled cysteine in one domain was dependent on the side chain substituted at a functionally critical site in the opposed domain.

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
DDC:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2011
Deposited On:06 Jun 2011 12:21
Last Modified:27 Nov 2013 16:37
Publisher:Springer
ISSN:0031-6768
Additional Information:The original publication is available at www.springerlink.com
Publisher DOI:10.1007/s00424-011-0948-z
PubMed ID:21384128
Citations:Web of Science®. Times Cited: 8
Google Scholar™
Scopus®. Citation Count: 9

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