Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA 99164, USA.
J Gen Physiol. 2013 Apr;141(4):413-30. doi: 10.1085/jgp.201210944.
Cyclic nucleotide-gated (CNG) channels in retinal photoreceptors play a crucial role in vertebrate phototransduction. The ligand sensitivity of photoreceptor CNG channels is adjusted during adaptation and in response to paracrine signals, but the mechanisms involved in channel regulation are only partly understood. Heteromeric cone CNGA3 (A3) + CNGB3 (B3) channels are inhibited by membrane phosphoinositides (PIP(n)), including phosphatidylinositol 3,4,5-triphosphate (PIP(3)) and phosphatidylinositol 4,5-bisphosphate (PIP(2)), demonstrating a decrease in apparent affinity for cyclic guanosine monophosphate (cGMP). Unlike homomeric A1 or A2 channels, A3-only channels paradoxically did not show a decrease in apparent affinity for cGMP after PIP(n) application. However, PIP(n) induced an ∼2.5-fold increase in cAMP efficacy for A3 channels. The PIP(n)-dependent change in cAMP efficacy was abolished by mutations in the C-terminal region (R643Q/R646Q) or by truncation distal to the cyclic nucleotide-binding domain (613X). In addition, A3-613X unmasked a threefold decrease in apparent cGMP affinity with PIP(n) application to homomeric channels, and this effect was dependent on conserved arginines within the N-terminal region of A3. Together, these results indicate that regulation of A3 subunits by phosphoinositides exhibits two separable components, which depend on structural elements within the N- and C-terminal regions, respectively. Furthermore, both N and C regulatory modules in A3 supported PIP(n) regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIP(n) sensitivity to heteromeric channels formed with PIP(n)-insensitive A subunits. Finally, channels formed by mixtures of PIP(n)-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIP(n) regulation, implying that intersubunit N-C interactions help control the phosphoinositide sensitivity of cone CNG channels.
视网膜光感受器中的环核苷酸门控 (CNG) 通道在脊椎动物光转导中发挥着关键作用。光感受器 CNG 通道的配体敏感性在适应过程中以及对外源信号的反应中进行调整，但涉及通道调节的机制仅部分被理解。异源三聚体视锥 CNGA3（A3）+CNGB3（B3）通道被膜磷肌醇（PIP(n)）抑制，包括磷脂酰肌醇 3,4,5-三磷酸（PIP(3)）和磷脂酰肌醇 4,5-二磷酸（PIP(2)），表现出对环鸟苷酸单磷酸（cGMP）的表观亲和力降低。与同型 A1 或 A2 通道不同，A3 仅通道在用 PIP(n) 处理后，cGMP 的表观亲和力没有降低。然而，PIP(n) 使 A3 通道的 cAMP 效力增加了约 2.5 倍。在 C 末端区域（R643Q/R646Q）突变或在环核苷酸结合域下游截断时，PIP(n) 依赖性 cAMP 效力变化被消除。此外，A3-613X 揭示了 PIP(n) 应用于同型通道时 cGMP 亲和力的三倍降低，并且这种效应依赖于 A3 的 N 末端区域内的保守精氨酸。总之，这些结果表明，磷肌醇对 A3 亚基的调节表现出两个可分离的成分，它们分别依赖于 N 和 C 末端区域内的结构元件。此外，A3 中的 N 和 C 调节模块都支持 PIP(n) 对异源 A3+B3 通道的调节。B3 亚基不足以使异源通道形成 PIP(n) 敏感性，这些异源通道形成于 PIP(n) 不敏感的 A 亚基。最后，由具有互补 N 和/或 C 末端区域突变的 PIP(n) 不敏感 A3 亚基混合物形成的通道恢复了 PIP(n) 调节，这意味着亚基间的 N-C 相互作用有助于控制视锥 CNG 通道的磷肌醇敏感性。
