Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Full Text Full Text (PDF, 214K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Lambert, G. Articles by Murer, H. Search for Related Content PubMed PubMed Citation Articles by Lambert, G. Articles by Murer, H. Social Bookmarking                            What's this?

Original Article

^a From the Institute for Physiology, University of Zürich, CH-8057 Zürich, Switzerland
Physiologisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.Fax: 41 1 636 6814;

forster{at}physiol.unizh.ch

The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na⁺/P_i cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 µM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P_i transport. Alkylation in 100 mM Na⁺ by either cationic {[2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA} or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P_i response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na⁺ resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P_i suppressed the slippage current equally, which indicated that P_i binding could occur without cotransport. Pre–steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na⁺ dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na⁺/P_i cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.

Key Words: mutagenesis • phosphate transport • electrophysiology • Xenopus laevis oocyte

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents