Identification of functionally important sites in the1st intracellular loop of the NaPi-IIa cotransporter

doi:10.1152/ajprenal.00282.2001

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Am J Physiol Renal Physiol (November 13, 2001). doi:10.1152/ajprenal.00282.2001

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Articles in PresS, published online ahead of print November 13, 2001
Am J Physiol Renal Physiol, 10.1152/ajprenal.00282.2001
Submitted on September 13, 2001
Accepted on October 30, 2001

Identification of functionally important sites in the1^st intracellular loop of the NaPi-IIa cotransporter

Katja Kohler¹, Ian C Forster¹^*, Gerti Stange¹, Jurg Biber¹, and Heini Murer¹

¹ Physiology, University of Zurich, Zurich, Switzerland

^* To whom correspondence should be addressed. E-mail: IForster{at}access.unizh.ch.

Intrasequence comparison of the NaPi-IIa cotransporter revealed two regions with high similarity in the 1^st intracellular (ICL-1) and the 3^rd extracellular (ECL-3) loops. As ECL-3 contains functionally important sites identified by cysteine scanning, we applied this method to corresponding sites in ICL-1. Accessibility of novel cysteines by methanethiosulfonate reagents was assayed electrophysiologically. N199C and V202C were fully inhibited after 2-aminoethyl methanethiosulfonate hydrobromide exposure, whereas other mutants showed marginal reductions in cotransport function. None showed significant functional loss after exposure to impermeant 2-(trimethylammonium)ethyl methanethiosulfonate bromide. This suggested a sidedness of Cys-modification. Compared to the WT, A203C showed altered Na⁺-leak kinetics, whereas N199C exhibited decreased apparent substrate affinities. To delineate the role of residue N199 in conferring substrate affinity, other mutations at this site were made. Only two gave significant ³²P_i uptake and inward P_i-induced currents with decreased P_i affinity, whereas for the others, P_i application suppressed the Na⁺-leak only. We suggest that ICL-1 and ECL-3 contribute to the transport pathway and site N199 is implicated in defining the transport mode.

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