Abstract
The rat renal Na+/Pi cotransporter (NaPi-IIa) contains 12 native cysteines. When individually replaced by a serine, none appears essential for proper expression and function. Nevertheless, the formation of one essential cysteine bridge (C5/C6), together with a postulated second bridge, is necessary. To determine the minimum cysteine residues required for functional NaPi-IIa, with the goal of generating a Cys-less backbone for structure–function studies, mutants were constructed in which multiple endogenous cysteines were replaced by serines in different combinations. In Xenopus oocytes, most mutants were functional, except those where cysteine pairs C4/C9, C4/C12 or C9/C12 were simultaneously deleted. This suggested that one of these pairs could form the second cysteine bridge essential for expression and/or protein function. Up to eight cysteines could therefore be removed to give a functional Cys-reduced NaPi-IIa with activity and kinetics comparable to the wild-type (WT). This construct, like all intermediate mutants and the WT, was insensitive to cysteine-modifying methanethiosulfonate (MTS) reagents. Moreover, by introducing a novel cysteine into the Cys-reduced NaPi-IIa at a site functionally important in the WT (Ser-460), the loss of transport function reported for mutant S460C, after exposure to MTS reagents, was recapitulated. This confirmed that the MTS reagent site of action was Cys-460 and that modification of native cysteines does not contribute to S460C behavior.
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Köhler, K., Forster, I.C., Stange, G. et al. Essential cysteine residues of the type IIa Na+/Pi cotransporter. Pflugers Arch - Eur J Physiol 446, 203–210 (2003). https://doi.org/10.1007/s00424-003-1039-6
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DOI: https://doi.org/10.1007/s00424-003-1039-6