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I. Cysteine Scanning Mutagenesis
Address correspondence to Ian C. Forster, Physiologisches Institut, Universität Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Fax: 41-1-635 5715; email: IForster{at}access.unizh.ch
The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V 
–80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.
Key Words: mutagenesis site directed • electrophysiology • phosphate transport proteins • electrogenic • Xenopus laevis
K. Kohler's present address is Laboratory of Morphogenesis and Cell Signaling, UMR144, Institut Curie, Paris, France.
Abbreviations used in this paper: DTT, dithiothreitol; MTS, methanethiosulfonate; MTSEA, 2-aminoethyl MTS hydrobromide; MTSES, sodium (2-sulfonatoethyl) MTS; MTSET, 2-(trimethylammonium)ethyl MTS bromide; NaPi-IIa, type IIa Na+/Pi cotransporter; PFA, phosphonoformic acid; SCAM, substituted cysteine accessibility method; WT, wild type.
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