Klaerke D A
Biomembrane Research Centre, August Krogh Institute, University of Copenhagen, Denmark.
Kidney Int. 1995 Oct;48(4):1047-56. doi: 10.1038/ki.1995.388.
Reabsorption of NaCl in the thick ascending limb of Henle's loop in the kidney and in the surface cells in the distal colon involves the integrated function of several membrane transport systems including ion channels, the Na,K,Cl-cotransport system and the Na,K-pump. To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca(2+)-activated K+ channels from the luminal membrane of the TAL cells and from the basolateral membrane of the distal colon cells have been characterized by flux studies in plasma membrane vesicle preparations and by single channel measurements in lipid bilayers. The channels are found to be activated by Ca2+ in the physiological range of concentration with a strong dependence on intracellular pH and the membrane potential. The Ca(2+)-sensitivity of the K+ channels is modulated by phosphorylation and dephosphorylation and the K+ channel protein must be in a phosphorylated state to respond to intracellular concentrations of Ca2+. As a step towards purification of the K+ channel proteins, procedures for solubilization and reconstitution of the K+ channels have been developed. The observation that the epithelial Ca(2+)-activated K+ channels bind calmodulin in the presence of Ca2+ have allowed for partial purification of the K+ channel proteins by calmodulin affinity chromatography. In the sequences for the two cloned Ca(2+)-activated K+ channels, the mSlo channel and the slowpoke channel, putative calmodulin binding regions can be identified.
肾脏中亨利氏袢厚升支以及远端结肠表面细胞对氯化钠的重吸收涉及多种膜转运系统的整合功能，包括离子通道、钠钾氯共转运系统和钠钾泵。为了确定它们的特性是否与跨上皮转运调节作用相符，通过质膜囊泡制剂中的通量研究以及脂质双层中的单通道测量，对来自亨利氏袢升支粗段细胞腔膜和远端结肠细胞基底外侧膜的钙激活钾通道进行了表征。发现这些通道在生理浓度范围内被钙离子激活，且强烈依赖细胞内pH值和膜电位。钾通道的钙敏感性受磷酸化和去磷酸化调节，钾通道蛋白必须处于磷酸化状态才能对细胞内钙离子浓度作出反应。作为纯化钾通道蛋白的一个步骤，已经开发了钾通道的溶解和重组程序。上皮钙激活钾通道在钙离子存在下与钙调蛋白结合这一观察结果，使得通过钙调蛋白亲和层析对钾通道蛋白进行部分纯化成为可能。在两个克隆的钙激活钾通道（mSlo通道和慢poke通道）的序列中，可以识别出假定的钙调蛋白结合区域。
