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Essential cysteine residues of the type IIa Na+/Pi cotransporter.

Authors: Kohler, K  Forster, IC  Stange, G  Biber, J  Murer, H 
Citation: Kohler K, etal., Pflugers Arch. 2003 May;446(2):203-10. Epub 2003 Mar 26.
Pubmed: (View Article at PubMed) PMID:12739158
DOI: Full-text: DOI:10.1007/s00424-003-1039-6

The rat renal Na(+)/P(i) cotransporter (NaP(i)-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 NaP(i)-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 NaP(i)-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 NaP(i)-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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RGD ID: 7243150
Created: 2013-05-08
Species: All species
Last Modified: 2013-05-08
Status: ACTIVE



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