Functional independence of monomeric CHIP28 water channels revealed by expression of wild-type mutant heterodimers.
Functional independence of monomeric CHIP28 water channels revealed by expression of wild-type mutant heterodimers.
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发表时间：
1994-04
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通讯作者：
Lan-bo Shi;W. Skach;A. Verkman
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作者：
Lan-bo Shi;W. Skach;A. Verkman
CHIP28 是红细胞和肾脏中的主要水转运蛋白，其在膜中形成四聚体（Verbavatz, J. M.、Brown, D.、Sabolic, I.、Valenti, G.、Ausiello, D. A.、Van Hoek, A. N.、Ma, T. 和 Verkman, A. S. (1993) J. Cell Biol. 123, 605-618)。为了确定 CHIP28 单体是否独立发挥作用，构建了嵌合 cDNA 二聚体，其中包含与野生型 CHIP28、非水转运 CHIP28 突变体 (C189W) 或有功能但对汞不敏感的 CHIP28 突变体 (C189S) 串联的野生型 CHIP28。将转录的 cRNA 注射到非洲爪蟾卵母细胞中，并通过定量免疫荧光测定质膜表达。通过渗透诱导的膨胀来测量水通道功能。 CHIP28 同二聚体和异二聚体靶向卵母细胞质膜并充当水通道。相对渗透水渗透率（Pf）值（针对单体亚基的质膜表达标准化）为：1.0（CHIP28单体）、0.0（C189W）、1.07（C189S）、1.10（CHIP28-CHIP28二聚体）和0.52（CHIP28-C189W）。卵母细胞 Pf 的增加与野生型 CHIP28 和 C189S 亚基的质膜表达线性相关。 HgCl2 (0.3 mM) 可抑制表达野生型 CHIP28 单体和二聚体的卵母细胞中通道介导的 Pf 85-90%，但不抑制表达 C189S 的卵母细胞中的 Pf。 HgCl2 将表达 CHIP28-C189S 二聚体的卵母细胞中的 Pf 抑制了 44 +/- 7%，这与异二聚体中的一种汞敏感亚基和一种不敏感亚基一致。这些结果表明，尽管单体 CHIP28 亚基以四聚体形式组装，但其独立地发挥水通道的功能。
CHIP28 is a major water transporting protein in erythrocytes and kidney which forms tetramers in membranes (Verbavatz, J. M., Brown, D., Sabolic, I., Valenti, G., Ausiello, D. A., Van Hoek, A. N., Ma, T., and Verkman, A. S. (1993) J. Cell Biol. 123, 605-618). To determine whether CHIP28 monomers function independently, chimeric cDNA dimers were constructed which contained wild-type CHIP28 in series with either wild-type CHIP28, a non-water transporting CHIP28 mutant (C189W), or a functional but mercurial-insensitive CHIP28 mutant (C189S). Transcribed cRNAs were injected in Xenopus oocytes and plasma membrane expression was assayed by quantitative immunofluorescence. Water channel function was measured by osmotically induced swelling. CHIP28 homo- and heterodimers were targeted to the oocyte plasma membrane and functioned as water channels. Relative osmotic water permeability (Pf) values (normalized for plasma membrane expression of monomeric subunits) were: 1.0 (CHIP28 monomer), 0.0 (C189W), 1.07 (C189S), 1.10 (CHIP28-CHIP28 dimer) and 0.52 (CHIP28-C189W). The increase in oocyte Pf was linearly related to plasma membrane expression of wild-type CHIP28 and C189S subunits. HgCl2 (0.3 mM) inhibited channel-mediated Pf in oocytes expressing wild-type CHIP28 monomers and dimers by 85-90%, but did not inhibit Pf in oocytes expressing C189S. HgCl2 inhibited Pf in oocytes expressing CHIP28-C189S dimers by 44 +/- 7%, consistent with one mercurial-sensitive and one insensitive subunit in the heterodimer. These results indicate that despite their assembly in tetramers, monomeric CHIP28 subunits function independently as water channels.