Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.
Channels (Austin). 2009 Nov;3(6):427-36. doi: 10.4161/chan.3.6.10053. Epub 2009 Nov 11.
Direct binding of cGMP or cAMP to tetrameric cyclic nucleotide-gated (CNG) channels will normally promote the open (conductive) conformation. However, the catfish CNGA2 subtype exhibits bimodal agonism, whereby open probability (P(o)) increases with initial cGMP binding events ("pro" action) but decreases with subsequent cGMP binding events ("con" action) that occur at concentrations above 3 mM. We constructed, and heterologously expressed, chimeric CNG channel subunits with sequence substitutions in the binding domain (BD), and tested their activation using patch-clamp of cell-free membranes. A normal subunit with the rat CNGA4 BD (with only pro action) could be converted into a bimodal subunit (both pro and con action) by replacing the N-terminal portion of the BD with catfish CNGA2 sequence. We then fused two bimodal and two normal subunits in tandem tetramers, to form heteromeric CNG channels with bimodal pseudosubunits either adjacent (cis) or diagonally opposite (trans). The cis tetramer showed con action, with a mean ratio of steady-state conductances g((30 mM cGMP))/g((3 mM cGMP)) = 0.87, demonstrating bimodal agonism in a heteromeric CNG channel for the first time. In contrast, trans tetramers showed normal cGMP agonism up to 30 mM cGMP with mean g((30 mM cGMP))/g((3mM cGMP))= 1.02, although a minority of oocytes (4 of 15) expressed anomalous channel populations with con action. Rearranging subunits in a heteromer thus influences a channel's P(o) at high cGMP concentration. The sensitivity of con action to neighbouring subunits implies a cooperative mechanism.
直接将 cGMP 或 cAMP 与四聚体环核苷酸门控 (CNG) 通道结合通常会促进开放（传导）构象。然而，鲶鱼 CNGA2 亚型表现出双模态激动作用，即开放概率 (P(o)) 随着初始 cGMP 结合事件（“前”作用）增加，但随着随后发生在高于 3mM 的浓度的 cGMP 结合事件（“后”作用）而降低。我们构建了并异源表达了具有结合域（BD）序列取代的嵌合 CNG 通道亚基，并使用无细胞膜片钳技术测试了它们的激活情况。具有正常大鼠 CNGA4 BD（仅具有前作用）的亚基可以通过用鲶鱼 CNGA2 序列取代 BD 的 N 端部分而被转化为双模态亚基（前作用和后作用）。然后，我们将两个双模态和两个正常亚基串联融合形成四聚体，形成具有双模态拟亚基的异源二聚体 CNG 通道，这些拟亚基要么相邻（顺式）要么对角相对（反式）。顺式四聚体显示出后作用，稳态电导 g((30mM cGMP))/g((3mM cGMP)) 的平均值为 0.87，首次在异源 CNG 通道中证明了双模态激动作用。相比之下，反式四聚体在高达 30mM cGMP 的浓度下表现出正常的 cGMP 激动作用，平均 g((30mM cGMP))/g((3mM cGMP))=1.02，尽管少数卵母细胞（15 个中有 4 个）表达了具有后作用的异常通道群体。在异源体中重新排列亚基会影响通道在高 cGMP 浓度下的 P(o)。后作用对相邻亚基的敏感性表明存在协同机制。
