Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons are tetrameric membrane receptors that are composed of two A2 subunits, one A4 subunit, and one B1b subunit. Each subunit carries a cyclic nucleotide-binding domain in the carboxyl terminus, and the channels are activated by the binding of cyclic nucleotides. The mechanism of cooperative channel activation is still elusive. Using a complete set of engineered concatenated olfactory CNG channels, with all combinations of disabled binding sites and fit analyses with systems of allosteric models, the thermodynamics of microscopic cooperativity for ligand binding was subunit- and state-specifically quantified. We show, for the closed channel, that preoccupation of each of the single subunits increases the affinity of each other subunit with a Gibbs free energy (ΔΔG) of ∼−3.5 to ∼−5.5 kJ ⋅ mol⁻¹, depending on the subunit type, with the only exception that a preoccupied opposite A2 subunit has no effect on the other A2 subunit. Preoccupation of two neighbor subunits of a given subunit causes the maximum affinity increase with ΔΔG of ∼−9.6 to ∼−9.9 kJ ⋅ mol⁻¹. Surprisingly, triple preoccupation leads to fewer negative ΔΔG values for a given subunit as compared to double preoccupation. Channel opening increases the affinity of all subunits. The equilibrium constants of closed–open isomerizations systematically increase with progressive liganding. This work demonstrates, on the example of the heterotetrameric olfactory CNG channel, a strategy to derive detailed insights into the specific mutual control of the individual subunits in a multisubunit membrane receptor.

嗅觉神经元的环核苷酸门控 (CNG) 离子通道是四聚体膜受体，由两个 A2 亚基、一个 A4 亚基和一个 B1b 亚基组成。每个亚基在羧基末端携带一个环核苷酸结合域，通道通过环核苷酸的结合而被激活。协作通道激活的机制仍然难以捉摸。使用一套完整的工程串联嗅觉 CNG 通道，结合禁用结合位点的所有组合和变构模型系统的拟合分析，配体结合的微观协同热力学被亚基和状态特异性量化。我们表明，对于封闭通道，每个单个亚基的关注增加了具有吉布斯自由能 ( ΔΔG) 的其他亚基的亲和力) ∼−3.5 到∼−5.5 kJ ⋅ mol ^-1，取决于亚基类型，唯一的例外是一个被占据的相对 A2 亚基对另一个 A2 亚基没有影响。对给定亚基的两个相邻亚基的关注导致最大亲和力增加，ΔΔG从 ~ -9.6到 ~ -9.9 kJ ⋅ mol ^-1。令人惊讶的是，三重专注导致更少的负ΔΔG与双重关注相比，给定亚基的值。通道开放增加了所有亚基的亲和力。封闭-开放异构化的平衡常数随着渐进配体系统地增加。这项工作以异四聚体嗅觉 CNG 通道为例，展示了一种策略，可以详细了解多亚基膜受体中各个亚基的特定相互控制。