Related Experiment Videos
Selected cysteine point mutations confer mercurial sensitivity to the mercurial-insensitive water channel MIWC/AQP-4
1Department of Medicine, University of California, San Francisco 94143-0521, USA.
Abstract:
The mercurial-insensitive water channel (MIWC or AQP-4) is a 30-32 kDA integral membrane protein expressed widely in fluid-transporting epithelia [Hasegawa et al. (1994) J. Biol. Chem. 269, 5497-5500]. To investigate the mercurial insensitivity and key residues involved in MIWC-mediated water transport, amino acids just proximal to the conserved NPA motifs (residues 69-74 and 187-190) were mutated individually to cysteine. Complementary RNAs were expressed in Xenopus oocytes for assay of osmotic water permeability (Pf) and HgCl2 inhibition dose-response. Oocytes expressing the cysteine mutants were highly water permeable, with Pf values (24-33 x 10(-3) cm/s) not different from that of wild-type (WT) MIWC. Pf was reversibly inhibited by HgCl2 in mutants S70C, G71C, G72C, H73C, and S189C but insensitive to HgCl2 in the other mutants. K1/2 values for 50% inhibition of Pf by HgCl2 were as follows (in millimolar): 0.40 (S70C), 0.36 (G71C), 0.14 (G72C), 0.45 (H73C), 0.24 (S189C), and > 1 for WT MIWC and the other mutants. To test the hypothesis that these residues are near the MIWC aqueous pore, residues 72 and 188 were mutated individually to the larger amino acid tryptophan. Pf in oocytes expressing mutants G72W or A188W (1.3-1.4 x 10(-3) cm/s) was not greater than that in water-injected oocytes even though these proteins were expressed at the oocyte plasma membrane as shown by quantitative immunofluorescence. Coinjection of cRNAs encoding WT MIWC and G72W or A188W indicated a dominant negative effect; Pf (x 10(-3) cm/s) was 22 (0.25 ng of WT), 10 (0.25 ng of WT + 0.25 ng of G72W), and 12 (0.25 ng of WT + 0.25 ng of A188W). Taken together, these results suggest the MIWC is mercurial-insensitive because of absence of a cysteine residue near the NPA motifs and that residues 70-73 and 189 are located at or near the MIWC aqueous pore. In contrast to previous data for the channel-forming integral protein of 28kDa (CHIP28), the finding of a dominant negative phenotype for mutants G72W and A188W indicates that MIWC monomers interact at a functional level.
Insights
The mercurial-insensitive water channel (MIWC) is not inhibited by mercury due to a lack of cysteine near its NPA motifs. Specific residues (70-73 and 189) are crucial for water transport through the MIWC pore.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Biology
Background:
- The mercurial-insensitive water channel (MIWC), also known as AQP-4, is an integral membrane protein vital for fluid transport in epithelia.
- Understanding the structural basis for MIWC's insensitivity to mercury and its role in water transport is crucial for elucidating aquaporin function.
Purpose of the Study:
- To investigate the structural determinants of mercurial insensitivity in MIWC.
- To identify key amino acid residues involved in MIWC-mediated water transport.
- To explore the location of these residues within the MIWC aqueous pore.
Main Methods:
- Site-directed mutagenesis of amino acids proximal to the conserved NPA motifs in MIWC.
- Expression of complementary RNAs in Xenopus oocytes for functional assays.
- Measurement of osmotic water permeability (Pf) and inhibition by HgCl2.
- Quantitative immunofluorescence to confirm protein expression.
- Dominant-negative effect assays using co-injection of wild-type and mutant cRNAs.
Main Results:
- Mutating specific residues (S70C, G71C, G72C, H73C, S189C) to cysteine rendered MIWC's water permeability sensitive to HgCl2 inhibition.
- Mutations to larger amino acids (G72W, A188W) significantly reduced water permeability, suggesting these residues are critical for pore function.
- G72W and A188W mutants exhibited a dominant-negative effect when co-expressed with wild-type MIWC, indicating functional monomer interactions.
Conclusions:
- MIWC's mercurial insensitivity is attributed to the absence of cysteine residues near the NPA motifs.
- Residues 70-73 and 189 are located within or near the MIWC aqueous pore, playing a critical role in water translocation.
- MIWC monomers interact functionally, as evidenced by the dominant-negative effects observed in specific mutants.