Effects of hydrophobic compounds on anion channels expressed in pancreatic duct cells

胰管细胞的负离子电导主要受囊性纤维化跨膜电导调节(CFTR)阴离子通道的影响。在本研究中，我们利用膜片钳技术研究了疏水化合物对CFTR通道的影响。芳香族羧酸等带负电荷的小分子疏水芳香族化合物同时引起单通道电导的降低和开放概率(Po)的增加。另一方面，小分子电中性疏水芳香族化合物在不降低单通道电导的情况下增加了Po。这些结果表明，小分子芳香族化合物以较快的动力学速度结合到通道孔内的疏水结合部位，导致单通道电导降低，同时通过与位于CFTR通道胞内侧的另一个部位结合，增加了Po。我们还研究了cftr通道依赖于ATP的门控机制以及胆固醇对CFtr门控的影响。我们的…更多关于ATP依赖的门控的结果有力地支持了“核苷酸结合结构域(NBD)二聚化假说”，即两个NbD与其界面上的两个ATP分子形成二聚体，导致通道孔打开，而与Nbd2结合的ATP的顺序水解破坏了二聚体，导致通道关闭。我们还发现质膜中的胆固醇含量显著改变了CFTR通道的门控动力学。总之，我们的结果表明，胆固醇通过改变跨膜结构域在通道孔道形成过程中的构象变化的机械阻力来影响核苷酸结合域的ATP水解率。综上所述，本研究表明，疏水化合物对cftr的影响取决于每种化合物的特殊特性，而不是它们的共同特征，这表明我们存在一个复杂的脂代谢反馈系统。我们在ATP依赖门控方面的新发现有助于理解cftr本身和其他ABC转运蛋白的门控机制。我们还对外周组织中表达的上皮性BK_lt；Ca&gt；通道和GABA_A通道进行了一些基础研究，这对下一步的研究领域具有重要意义。较少

Anion conductance in pancreatic duct cells is mainly underlied by Cystic Fibrosis Transmembrane-conductance Regulator (CFTR) anion channels. In this study, we investigated the effects of hydrophobic compounds on CFTR channels using patch-clamp technique.Small negatively-charged hydrophobic aromatic compounds like aromatic carboxylic acids induced both a decrease in single channel conductance and an increase in open probability (Po) simultaneously. On the other hand, small electro-neutral hydrophobic aromatic compounds increased Po without decreasing single channel conductance. These results suggested that the small aromatic compounds bind to a hydrophobic binding site inside the channel pore with a fast kinetics leading to reduce the single channel conductance, and also increase Po by binding to another site located in the intracellular side of CFTR channel.We also investigated mechanisms of ATP-dependent gating of CFTR channel as well as effects of cholesterol on the CFTR gating. Our … More results for the ATP-dependent gating strongly supported the "nucleotide binding domain (NBD) dimmerization hypothesis" that two NBDs make a dimmer with two ATP molecules in their interface, leading to open the channel pore, and the sequential hydrolysis of ATP binding to NBD2 breaks the dimmer, leading to channel closing. We also found that cholesterol content in plasma membrane changed the gating kinetics of CFTR channel significantly. Totally our results suggested that cholesterol affects rate of the ATP hydrolysis at the nucleotide binding domain via changing the mechanical resistance to the conformational change of the membrane spanning domains during channel pore gating.Taken together, the present study showed that effects of hydrophobic compounds on CFTR widely varied dependent on its peculiar characteristics of each compound, not their common feature of ‘hydrophobicity', which suggested us the existence of a complicated feed back system via lipid metabolism. Our novel findings for ATP-dependent gating significantly contribute to understanding of gating mechanisms of CFTR itself and other ABC transporters as well.We also performed some basic studies for epithelial BK_<Ca> channels and GABA_A channels expressed in peripheral tissue, which are important for the next step in this research field. Less