Winters C J, Reeves W B, Andreoli T E
Department of Internal Medicine, University of Arkansas College of Medicine, Little Rock.
Kidney Int. 1994 Mar;45(3):803-10. doi: 10.1038/ki.1994.106.
Cl- channels fused from basolateral mTAL membranes into planar bilayers have distinctive functional characteristics which, when taken together, are unique among Cl- channels. The properties of these 50 to 60 pS channels can account for the characteristics of basolateral Cl- conductances in microperfused mTAL segments and thus may mediate net basolateral Cl- absorption in the intact mTAL. In the present studies, we solubilized basolateral membranes from rabbit mTAL. Since basolateral mTAL Cl- channels contain arginine- and lysine-rich domains, we exposed these solubilized membranes to sequential cation- and anion-exchange chromatography. The bound and unbound eluates from cation- and anion-exchange chromatography were reconstituted into proteoliposomes which, when fused into bilayers, yielded Cl- channels whose properties were virtually identical to those described above for native basolateral mTAL channels fused into bilayers. As judged by valinomycin-sensitive conductive 36Cl- uptake, proteoliposomes reconstituted from the unbound eluates after anion-exchange chromatography were enriched at least 30-fold in Cl- channel activity and had about 30% of the total Cl- channel activity solubilized in native vesicles.
从基底外侧髓袢升支粗段（mTAL）膜融合到平面双层膜中的氯离子通道具有独特的功能特性，综合起来，这些特性在氯离子通道中是独一无二的。这些50到60皮西门子（pS）通道的特性可以解释微灌注mTAL节段中基底外侧氯离子电导的特征，因此可能介导完整mTAL中基底外侧氯离子的净吸收。在本研究中，我们溶解了兔mTAL的基底外侧膜。由于基底外侧mTAL氯离子通道含有富含精氨酸和赖氨酸的结构域，我们将这些溶解的膜依次进行阳离子和阴离子交换层析。阳离子和阴离子交换层析的结合和未结合洗脱液被重构成蛋白脂质体，当它们融合到双层膜中时，产生的氯离子通道的特性与上述融合到双层膜中的天然基底外侧mTAL通道的特性几乎相同。通过缬氨霉素敏感的导电36Cl-摄取判断，阴离子交换层析后从未结合洗脱液中重构的蛋白脂质体的氯离子通道活性至少富集了30倍，并且具有溶解在天然囊泡中的总氯离子通道活性的约30%。
