CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions
CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions
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DOI：
10.1152/ajpcell.00037.2013
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发表时间：
2013-07-01
影响因子：
5.5
通讯作者：
Varnum, Michael D.
中科院分区：
文献类型：
--
作者：
Dai, Gucan;Varnum, Michael D.
环核苷酸门控（CNG）通道对于视网膜光感受器和嗅觉感受器细胞中的感觉转导至关重要;它们的活性由磷酸肌醇（PIPn）如磷脂酰肌醇4，5-二磷酸（PIP 2）和磷脂酰肌醇3，4，5-三磷酸（PIP 3）调节。视锥光感受器CNGA 3，L 633 P，无色相关突变位于羧基（COOH）末端亮氨酸拉链结构域之前显示是重要的通道组装和PIPn调节。我们使用电生理记录从表达野生型和突变型CNG通道亚基的细胞中切下的斑块来确定这种突变的功能后果。CNGA 3-L 633 P亚基在有或没有CNGB 3的情况下形成功能通道，产生表观cGMP亲和力的增加。令人惊讶的是，L 633 P显著增强PIPn对这些通道的表观cGMP亲和力的抑制。L 633 P对PIPn敏感性的影响依赖于完整的氨基（NH 2）末端PIPn调节模块。这些观察结果使我们假设L 633 P通过改变CNGA 3的NH 2-和COOH-末端区域之间的偶联来增强PIPn抑制。重组COOH-末端片段部分恢复正常PIPn的敏感性与COOH-末端截断通道，但L 633 P阻止了这种效果。此外，通道片段的共免疫沉淀和热力学连接分析也为NH 2-COOH相互作用提供了证据。最后，CNGA 3亚基的串联二聚体指定了含有L 633 P和其他突变的亚基的排列，表明推定的结构域间相互作用发生在通道亚基之间（亚基间），而不是仅发生在同一亚基内（亚基内）。总的来说，这些研究支持一个模型，其中亚基间的相互作用控制锥CNG通道的磷酸肌醇调节的敏感性。异常通道调节可能导致L 633 P突变患者的疾病进展。
Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the L633P mutation.