Channel forming integral protein of 28 kD (CHIP28) functions as a water channel in erythrocytes, kidney proximal tubule and thin descending limb of Henle. CHIP28 morphology was examined by freeze-fracture EM in proteoliposomes reconstituted with purified CHIP28, CHO cells stably transfected with CHIP28k cDNA, and rat kidney tubules. Liposomes reconstituted with HPLC-purified CHIP28 from human erythrocytes had a high osmotic water permeability (Pf0.04 cm/s) that was inhibited by HgCl2. Freeze-fracture replicas showed a fairly uniform set of intramembrane particles (IMPs); no IMPs were observed in liposomes without incorporated protein. By rotary shadowing, the IMPs had a diameter of 8.5 +/- 1.3 nm (mean +/- SD); many IMPs consisted of a distinct arrangement of four smaller subunits surrounding a central depression. IMPs of similar size and appearance were seen on the P-face of plasma membranes from CHIP28k-transfected (but not mock-transfected) CHO cells, rat thin descending limb (TDL) of Henle, and S3 segment of proximal straight tubules. A distinctive network of complementary IMP imprints was observed on the E-face of CHIP28-containing plasma membranes. The densities of IMPs in the size range of CHIP28 IMPs, determined by non-linear regression, were (in IMPs/microns 2): 2,494 in CHO cells, 5,785 in TDL, and 1,928 in proximal straight tubules; predicted Pf, based on the CHIP28 single channel water permeability of 3.6 x 10(-14) cm3/S (10 degrees C), was in good agreement with measured Pf of 0.027 cm/S, 0.075 cm/S, and 0.031 cm/S, respectively, in these cell types. Assuming that each CHIP28 monomer is a right cylindrical pore of length 5 nm and density 1.3 g/cm3, the monomer diameter would be 3.2 nm; a symmetrical arrangement of four cylinders would have a greatest diameter of 7.2 nm, which after correction for the thickness of platinum deposit, is similar to the measured IMP diameter of approximately 8.5 nm. These results provide a morphological signature for CHIP28 water channels and evidence for a tetrameric assembly of CHIP28 monomers in reconstituted proteoliposomes and cell membranes.
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机译：通道形成的28 kD整合蛋白（CHIP28）在红细胞，肾近端小管和Henle的薄降肢中用作水通道。通过冷冻-断裂EM在用纯化的CHIP28重构的蛋白脂质体，用CHIP28k cDNA稳定转染的CHO细胞和大鼠肾小管中检查了CHIP28的形态。用HPLC纯化的CHIP28从人红细胞重构的脂质体具有高渗透水渗透性（Pf0.04 cm / s），受HgCl2抑制。冷冻断裂复制品显示出相当均匀的膜内颗粒（IMPs）集合。没有掺入蛋白质的脂质体中未观察到IMP。通过旋转遮蔽，IMP的直径为8.5 +/- 1.3 nm（平均+/- SD）；许多IMPs由围绕中央凹陷的四个较小的亚单元的独特排列组成。在CHIP28k转染（但未模拟转染）的CHO细胞，Henle的大鼠瘦降肢（TDL）和近端直小管S3段的质膜P面上看到相似大小和外观的IMP。在包含CHIP28的质膜的E面上观察到了互补的IMP印记的独特网络。通过非线性回归确定的CHIP28 IMPs大小范围内的IMPs密度（以IMPs /微米2为单位）：CHO细胞为2,494，TDL为5,785，近端直小管为1,928；根据CHIP28单通道水渗透率3.6 x 10（-14）cm3 / S（10摄氏度）预测的Pf与测得的Pf为0.027 cm / S，0.075 cm / S和0.031 cm / s高度吻合在这些细胞类型中分别为S。假设每个CHIP28单体是长度为5 nm，密度为1.3 g / cm3的右圆柱孔，则单体直径为3.2 nm；四个圆柱体的对称排列的最大直径为7.2 nm，在校正铂沉积物的厚度后，该直径类似于测得的IMP直径约8.5 nm。这些结果为CHIP28水通道提供了形态特征，并为CHIP28单体在重组蛋白脂质体和细胞膜中的四聚体组装提供了证据。
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