Qu W, Zhu X O, Moorhouse A J, Bieri S, Cunningham A M, Barry P H
School of Physiology and Pharmacology, The University of New South Wales, Sydney 2052, Australia.
J Membr Biol. 2000 Nov 15;178(2):137-50. doi: 10.1007/s002320010021.
The permeation properties of adenosine 3', 5'-cyclic monophosphate (cAMP)-activated recombinant rat olfactory cyclic nucleotide-gated channels (rOCNC1) in human embryonic kidney (HEK 293) cells were investigated using inside-out excised membrane patches. The relative permeability of these rOCNC1 channels to monovalent alkali cations and organic cations was determined from measurements of the changes in reversal potential upon replacing sodium in the bathing solution with different test cations. The permeability ratio of Cl(-) relative to Na(+) (P(Cl)/P(Na)) was about 0.14, confirming that these channels are mainly permeable to cations. The sequence of relative permeabilities of monovalent alkali metal ions in these channels was P(Na) > or = P(K) > P(Li) > P(Cs) > or = P(Rb), which closely corresponds to a high-strength field sequence as previously determined for native rat olfactory receptor neurons (ORNs). The permeability sequence for organic cations relative to sodium was P(NH3OH) > P(NH4) > P(Na) > P(Tris) > P(Choline) > P(TEA), again in good agreement with previous permeability ratios obtained in native rat ORNs. Single-channel conductance sequences agreed surprisingly well with permeability sequences. These conductance measurements also indicated that, even in asymmetric bi-ionic cation solutions, the conductance was somewhat independent of current direction and dependent on the composition of both solutions. These results indicate that the permeability properties of rOCNC1 channels are similar to those of native rat CNG channels, and provide a suitable reference point for exploring the molecular basis of ion selectivity in recombinant rOCNC1 channels using site-directed mutagenesis.
利用内面向外式膜片，研究了3',5'-环磷酸腺苷（cAMP）激活的重组大鼠嗅觉环核苷酸门控通道（rOCNC1）在人胚肾（HEK 293）细胞中的渗透特性。通过测量用不同测试阳离子替代浴液中的钠时反转电位的变化，确定了这些rOCNC1通道对单价碱金属阳离子和有机阳离子的相对渗透率。Cl(-)相对于Na(+)的渗透率比值（P(Cl)/P(Na)）约为0.14，证实这些通道主要对阳离子通透。这些通道中单价碱金属离子的相对渗透率顺序为P(Na)≥P(K)>P(Li)>P(Cs)≥P(Rb)，这与先前在天然大鼠嗅觉受体神经元（ORN）中确定的高强度场顺序密切对应。有机阳离子相对于钠的渗透率顺序为P(NH3OH)>P(NH4)>P(Na)>P(Tris)>P(胆碱)>P(TEA)，同样与先前在天然大鼠ORN中获得的渗透率比值高度一致。单通道电导顺序与渗透率顺序惊人地吻合。这些电导测量还表明，即使在不对称双离子阳离子溶液中，电导也在一定程度上与电流方向无关，且取决于两种溶液的组成。这些结果表明，rOCNC1通道的渗透特性与天然大鼠CNG通道相似，并为利用定点诱变探索重组rOCNC1通道中离子选择性的分子基础提供了合适的参考点。
