Dai Xiao-Qing, Karpinski Edward, Chen Xing-Zhen
Membrane Protein Research Group, Department of Physiology, University of Alberta, 7-29 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7.
Biochim Biophys Acta. 2006 Feb;1758(2):197-205. doi: 10.1016/j.bbamem.2006.01.020. Epub 2006 Feb 24.
Polycystin-L (PCL), homologous to polycystin-2 (71% similarity in protein sequence), is the third member of the polycystin family of proteins. Polycystin-1 and -2 are mutated in autosomal dominant polycystic kidney disease, but the physiological role of PCL has not been determined. PCL acts as a Ca-regulated non-selective cation channel permeable to mono- and divalent cations. To further understand the biophysical and pharmacological properties of PCL, we examined a series of organic cations for permeation and inhibition, using single-channel patch clamp and whole-cell two-microelectrode voltage clamp techniques in conjunction with Xenopus oocyte expression. We found that PCL is permeable to organic amines, methlyamine (MA, 3.8 A), dimethylamine (DMA, 4.6 A) and triethylamine (TriEA, 6 A), and to tetra-alkylammonium cation (TAA) tetra-methylammonium (TMA, 5.5-6.4 A). TAA compounds tetra-ethylammonium (TEA, 6.1-8.2 A) and tetra-propylammonium (TPA, 9.8 A) were impermeable through PCL and exhibited weak inhibition on PCL (IC50 values>13 mM). Larger TAA cations tetra-butylammonium (TBA, 11.6 A) and tetra-pentylammonium (TPeA, 13.2 A) were impermeable through PCL as well and showed strong inhibition (IC50 values of 2.7 mM and 1.3 microM, respectively). Inhibition by TBA was on decreasing the single-channel current amplitude and exhibited no effect on open probability (NPo) or mean open time (MOT), suggesting that it blocks the PCL permeation pathway. In contract, TEA, TPA and TPeA reduced NPo and MOT values but had no effect on the amplitude, suggesting their binding to a different site in PCL, which affects the channel gating. Taken together, our studies revealed that PCL is permeable to organic amines and TAA cation TMA, and that inhibition of PCL by large TAA cations exhibits two different mechanisms, presumably through binding either to the pore pathway to reduce permeant flux or to another site to regulate the channel gating. These data allow to estimate a channel pore size of approximately 7 A for PCL.
多囊蛋白-L（PCL）与多囊蛋白-2同源（蛋白质序列相似度为71%），是多囊蛋白家族的第三个成员。多囊蛋白-1和-2在常染色体显性多囊肾病中发生突变，但PCL的生理作用尚未确定。PCL作为一种钙调节的非选择性阳离子通道，对单价和二价阳离子具有通透性。为了进一步了解PCL的生物物理和药理特性，我们使用单通道膜片钳和全细胞双微电极电压钳技术结合非洲爪蟾卵母细胞表达，检测了一系列有机阳离子的通透性和抑制作用。我们发现PCL对有机胺、甲胺（MA，3.8 Å）、二甲胺（DMA，4.6 Å）和三乙胺（TriEA，6 Å）以及四烷基铵阳离子（TAA）四甲基铵（TMA，5.5 - 6.4 Å）具有通透性。TAA化合物四乙铵（TEA，6.1 - 8.2 Å）和四丙铵（TPA，9.8 Å）不能透过PCL，且对PCL表现出较弱的抑制作用（IC50值>13 mM）。更大的TAA阳离子四丁铵（TBA，11.6 Å）和四戊铵（TPeA，13.2 Å）也不能透过PCL，并表现出较强的抑制作用（IC50值分别为2.7 mM和1.3 μM）。TBA的抑制作用是降低单通道电流幅度，对开放概率（NPo）或平均开放时间（MOT）没有影响，这表明它阻断了PCL的通透途径。相反，TEA、TPA和TPeA降低了NPo和MOT值，但对电流幅度没有影响，这表明它们与PCL中的不同位点结合，从而影响通道门控。综上所述，我们的研究表明PCL对有机胺和TAA阳离子TMA具有通透性，大TAA阳离子对PCL的抑制作用表现出两种不同的机制，可能是通过与孔道途径结合以减少通透通量，或者与另一个位点结合以调节通道门控。这些数据使得我们能够估计PCL的通道孔径约为7 Å。
