Crary J I, Dean D M, Nguitragool W, Kurshan P T, Zimmerman A L
Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island 02912, USA.
J Gen Physiol. 2000 Dec;116(6):755-68. doi: 10.1085/jgp.116.6.755.
Cyclic nucleotide-gated (CNG) channels are critical components in the visual and olfactory signal transduction pathways, and they primarily gate in response to changes in the cytoplasmic concentration of cyclic nucleotides. We previously found that the ability of the native rod CNG channel to be opened by cGMP was markedly inhibited by analogues of diacylglycerol (DAG) without a phosphorylation reaction (Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409-417). Here, we have studied cloned bovine rod and rat olfactory CNG channels expressed in Xenopus oocytes, and have determined that they are differentially inhibited by DAG. At saturating [cGMP], DAG inhibition of homomultimeric (alpha subunit only) rod channels was similar to that of the native rod CNG channel, but DAG was much less effective at inhibiting the homomultimeric olfactory channel, producing only partial inhibition even at high [DAG]. However, at low open probability (P(o)), both channels were more sensitive to DAG, suggesting that DAG is a closed state inhibitor. The Hill coefficients for DAG inhibition were often greater than one, suggesting that more than one DAG molecule is required for effective inhibition of a channel. In single-channel recordings, DAG decreased the P(o) but not the single-channel conductance. Results with chimeras of rod and olfactory channels suggest that the differences in DAG inhibition correlate more with differences in the transmembrane segments and their attached loops than with differences in the amino and carboxyl termini. Our results are consistent with a model in which multiple DAG molecules stabilize the closed state(s) of a CNG channel by binding directly to the channel and/or by altering bilayer-channel interactions. We speculate that if DAG interacts directly with the channel, it may insert into a putative hydrophobic crevice among the transmembrane domains of each subunit or at the hydrophobic interface between the channel and the bilayer.
环核苷酸门控（CNG）通道是视觉和嗅觉信号转导途径中的关键组成部分，它们主要根据细胞质中环核苷酸浓度的变化而开启或关闭。我们之前发现，在没有磷酸化反应的情况下，二酰基甘油（DAG）类似物能显著抑制天然视杆细胞CNG通道被cGMP开启的能力（Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409 - 417）。在此，我们研究了在非洲爪蟾卵母细胞中表达的克隆牛视杆细胞和大鼠嗅觉CNG通道，确定它们受到DAG的抑制作用存在差异。在饱和[cGMP]浓度下，DAG对同型多聚体（仅α亚基）视杆细胞通道的抑制作用与天然视杆细胞CNG通道相似，但DAG对同型多聚体嗅觉通道的抑制效果要差得多，即使在高[DAG]浓度下也只能产生部分抑制。然而，在低开放概率（P(o)）时，两种通道对DAG都更敏感，这表明DAG是一种关闭状态抑制剂。DAG抑制的希尔系数通常大于1，这表明有效抑制一个通道需要不止一个DAG分子。在单通道记录中，DAG降低了P(o)，但没有改变单通道电导。视杆细胞和嗅觉通道嵌合体的实验结果表明，DAG抑制的差异与跨膜片段及其连接环的差异相关性更大，而与氨基和羧基末端的差异相关性较小。我们的结果与一个模型一致，即多个DAG分子通过直接与通道结合和/或改变双层膜与通道的相互作用来稳定CNG通道处于关闭状态。我们推测，如果DAG直接与通道相互作用，它可能会插入每个亚基跨膜结构域之间的一个假定疏水裂缝中，或者插入通道与双层膜之间的疏水界面处。
