A point mutation at cysteine 189 blocks the water permeability of rat kidney water channel CHIP28k.
CHIP28k is an important water-transporting protein in the kidney proximal tubule and the thin descending limb of Henle [Zhang, Skach, Hasegawa, Van Hoek, & Verkman (1993) J. Cell Biol. 120, 359-369] that is homologous to human erythrocyte CHIP28 [Preston & Agre (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11110-11114]. Oligonucleotide-directed mutagenesis was used to identify the cysteine(s) involved in inhibition of the water-transporting function of CHIP28k by the mercurial HgCl2. Each of the four cysteines (at positions 87, 102, 152, and 189) were mutated to serine individually, or in combinations. In vitro transcribed cRNA was expressed in Xenopus oocytes for measurement of osmotic water permeability (Pf) in the absence or presence of 0.3 mM HgCl2. Pf (in cm/s x 10(-4) measured at 10 degrees C) was 7 +/- 1 in water-injected oocytes. In wild-type CHIP28k, Pf was 58 +/- 7 (-HgCl2) and 12 +/- 1 (+HgCl2). Mutation of cysteine 87, 102, or 152, individually or in combinations, had little effect on oocyte Pf or on the inhibition by HgCl2. Mutation of cysteine 189 to serine or glycine gave similar Pf values of 49-56 (-HgCl2); however, Pf was not inhibited up to 1 mM HgCl2. Mutation of cysteine 189 to the larger amino acid tryptophan gave a low Pf of 9 +/- 1; coexpression with wild-type CHIP28k indicated that the tryptophan mutation was not dominant negative. Mutation of the asparagine 42 and 205 glycosylation sites to threonine had little effect on Pf.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhang
R
R
Department of Medicine, University of California, San Francisco 94143-0532.
van Hoek
A N
AN
Biwersi
J
J
Verkman
A S
AS
D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D001483:Base Sequence; D002463:Cell Membrane Permeability; D003545:Cysteine / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D006031:Glycosylation; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008627:Mercuric Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014867:Water / Q000378:metabolism*; D014982:Xenopus laevis
C496022:Aqp1 protein, rat; D020346:Aquaporins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014867:Water; D051398:Aquaporin 1; D008627:Mercuric Chloride; D003545:Cysteine