Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
J Gen Physiol. 2011 Jun;137(6):591-603. doi: 10.1085/jgp.201010560.
Cyclic nucleotide-gated (CNG) channels bind cGMP or cAMP in a cytoplasmic ligand-binding domain (BD), and this binding typically increases channel open probability (P(o)) without inducing desensitization. However, the catfish CNGA2 (fCNGA2) subtype exhibits bimodal agonism, whereby steady-state P(o) increases with initial cGMP-binding events ("pro" action) up to a maximum of 0.4, but decreases with subsequent cGMP-binding events ("con" action) occurring at concentrations >3 mM. We sought to clarify if low pro-action efficacy was either necessary or sufficient for con action to operate. To find BD residues responsible for con action or low pro-action efficacy or both, we constructed chimeric CNG channels: subregions of the fCNGA2 BD were substituted with corresponding sequence from the rat CNGA4 BD, which does not support con action. Constructs were expressed in frog oocytes and tested by patch clamp of cell-free membranes. For nearly all BD elements, we found at least one construct where replacing that element preserved robust con action, with a ratio of steady-state conductances, g((10 mM cGMP))/g((3 mM cGMP)) < 0.75. When all of the BD sequence C terminal of strand β6 was replaced, g((10 mM cGMP))/g((3 mM cGMP)) was increased to 0.95 ± 0.05 (n = 7). However, this apparent attenuation of con action could be explained by an increase in the efficacy of pro action for all agonists, controlled by a conserved "phosphate-binding cassette" motif that contacts ligand; this produces high P(o) values that are less sensitive to shifts in gating equilibrium. In contrast, substituting a single valine in the N-terminal helix αA abolished con action (g((30 mM cGMP))/g((3 mM cGMP)) increased to 1.26 ± 0.24; n = 7) without large increases in pro-action efficacy. Our work dissociates the two functional features of low pro-action efficacy and con action, and moreover identifies a separate structural determinant for each.
环核苷酸门控 (CNG) 通道在细胞质配体结合域 (BD) 结合 cGMP 或 cAMP，这种结合通常会增加通道的开放概率 (P(o))，而不会引起脱敏。然而，鲶鱼 CNGA2（fCNGA2）亚基表现出双模态激动作用，其中稳态 P(o)随着初始 cGMP 结合事件（“前”作用）增加到 0.4，但随着随后发生在 >3 mM 浓度的 cGMP 结合事件（“后”作用）而降低。我们试图澄清低前作用效力是否是后作用发挥作用的必要条件或充分条件。为了确定负责后作用或低前作用效力或两者的 BD 残基，我们构建了嵌合 CNG 通道：fCNGA2 BD 的亚区被大鼠 CNGA4 BD 的相应序列取代，该序列不支持后作用。构建体在蛙卵母细胞中表达，并通过细胞游离膜的膜片钳进行测试。对于几乎所有的 BD 元素，我们发现至少有一种构建体，其中取代该元素保留了强大的后作用，稳态电导比，g((10 mM cGMP))/g((3 mM cGMP)) < 0.75。当β6 链 C 末端的所有 BD 序列都被替换时，g((10 mM cGMP))/g((3 mM cGMP))增加到 0.95 ± 0.05（n = 7）。然而，这种后作用的明显衰减可以通过所有激动剂的前作用效力的增加来解释，这种增加由一个保守的“磷酸结合盒”基序控制，该基序与配体接触；这产生了高 P(o)值，对门控平衡的变化不那么敏感。相比之下，在αA 螺旋的 N 端替换单个缬氨酸会消除后作用（g((30 mM cGMP))/g((3 mM cGMP))增加到 1.26 ± 0.24；n = 7），而前作用效力没有大幅增加。我们的工作将低前作用效力和后作用的两个功能特征分开，并且还确定了每个特征的单独结构决定因素。
