Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.

译文

成年牛或小牛晶状体的去污剂溶解的质膜蛋白和高效液相色谱纯化的晶状体的主要内在蛋白 (MIP) 重构为单层囊泡和平面脂质双层。冷冻断裂研究表明,囊泡中膜内颗粒的密度与蛋白质/脂质比率成正比。在高比率下,这些颗粒像透镜纤维中的MIP一样结晶成四方阵列。由纯化的MIP或去污剂溶解的蛋白质诱导的通道具有基本相同的特性。多通道膜的电导在0 mV附近最大,并且在大于80 mV的电压下降至最大值的0.49 +/- 0.08。两态玻尔兹曼分布很好地拟合了电导对电压的依赖性。大于30 mV的电压阶跃引起欧姆电流阶跃,随后缓慢 (秒) 双指数下降。振幅和时间常数取决于幅度,而不取决于电压的符号。从100 mV到电压小于30 mV的阶跃导致通道以毫秒时间常数呈指数打开。对电压阶跃后首次闭合的潜伏期进行分析,得出的时间常数与多通道动力学几乎相同。单通道电导与KCl中0.1至1.0 M的盐浓度成正比。在0.1M KCl中,通道具有两个优选的电导状态,其幅度为380和160 pS,以及三个额外的子状态。多通道和单通道数据表明该通道具有两个在动力学上重要的开放状态。通道具有轻微的阴离子选择性。通道的性质在pH 7.4至5.8或pca7至2之间没有明显变化。我们建议具有这些特性的通道可以有助于维持镜片的透明度和流体平衡。

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