The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel

Water channels provide the plasma membranes of red cells and renal proximal tubules with high permeability to water, thereby permitting water to move in the direction of an osmotic gradient. Molecular identification of CHIP28 protein as the membrane water channel was first accomplished by measurement of osmotic swelling of Xenopus oocytes injected with CHIP28 RNA (Preston, G. M., Carroll, T. P., Guggino, W. B., and Agre, P. (1992) Science 256, 385-387). Since water channels are pharmacologically inhibited by submillimolar concentrations of Hg²⁺, site-directed mutagenesis was undertaken to demonstrate which of the 4 cysteines (87, 102, 152, or 189) is the Hg²⁺-sensitive residue in the CHIP28 molecule. Each cysteine was individually replaced by serine, and oocytes expressing each of the four mutants exhibited osmotic water permeability (P_f) equivalent to wild-type CHIP28. After incubation in HgCl₂, all were significantly inhibited, except C189S which was not inhibited even at 3 DIM HgCl₂. CHIP28 exists as a multisubunit complex in the native membrane; however, although oocytes injected with mixed CHIP28 and C189S RNAs exhibited P, corresponding to the sum of their individual activities, exposure to Hg²⁺ only reduced the P_f to the level of the C189S mutant. Of the six substitutions at residue 189, only the serine and alanine mutants exhibited increased P_f and had glycosylation patterns resembling wild-type CHIP28 on immunoblots. These studies demonstrated: (i) CHIP28 water channel activity is retained despite substitution of individual cysteines with serine; (ii) cysteine 189 is the Hg²⁺-sensitive residue; (iii) the subunits of the CHIP28 complex are individually active water pores; (iv) residue 189 is critical to proper processing of the CHIP28 protein.