NONPOLAR ENVIRONMENT OF TRYPTOPHANS IN ERYTHROCYTE WATER CHANNEL CHIP28 DETERMINED BY FLUORESCENCE QUENCHING

CHIP28 is an abundant water-transporting protein in erythrocytes, kidn ey proximal tubule, and other fluid-transporting tissues. To determine the environment of the four tryptophans in CHIP28, fluorescence spect ra and quenching by polar and nonpolar compounds were measured in stri pped human erythrocyte membranes containing CHIP28 and in proteoliposo mes reconstituted with purified CHIP28; comparative studies were perfo rmed in membranes containing MIP26. Functional analysis showed that CH IP28 water permeability was not affected by the polar quenchers iodide and acrylamide nor the nonpolar n-anthroyloxy fatty acids (n-AF). The emission maximum of CHIP28 tryptophan fluorescence was at 324 +/- 2 n m and did not change with the addition of quenchers; the maximum for M IP26 was at 335 +/- 5 nm. There was weak quenching of CHIP28 tryptopha n fluorescence by the polar compounds iodide and acrylamide, with Ster n-Volmer constants of 0.13 and 0.71 M-1, respectively. HgCl2 inhibited water permeability by >95% at 50 muM and quenched CHIP28 fluorescence reversibly by up to 70% with a biphasic concentration dependence; que nching by HgCl2 and acrylamide was not additive. The membrane-associat ed n-AF probes quenched CHIP28 fluorescence by up to 80% with the grea test quenching for n = 2 and 12; addition of HgCl2 or acrylamide after n-AF caused a small, anthroyloxy-position-dependent increase in quenc hing which was greatest at n = 6. These studies indicate that the tryp tophans in CHIP28 are in a nonpolar, membrane-associated environment. Mathematical modeling of then-AF results suggests that the tryptophans are clustered near the surface and center of the bilayer. Site-direct ed mutagenesis of the highly conserved tryptophan 210 to leucine had n o significant effect on the CHIP28 water-transport function, as assaye d in Xenopus oocytes. These results provide new information about CHIP 28 and MIP26 structure that was not anticipated from the analysis of h ydropathy.