Department of Otolaryngology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Biochem J. 2012 Apr 15;443(2):463-76. doi: 10.1042/BJ20111255.
The molecular characteristics of CNG (cyclic nucleotide-gated) channels in auditory/vestibular hair cells are largely unknown, unlike those of CNG mediating sensory transduction in vision and olfaction. In the present study we report the full-length sequence for three CNGA3 variants in a hair cell preparation from the trout saccule with high identity to CNGA3 in olfactory receptor neurons/cone photoreceptors. A custom antibody targeting the N-terminal sequence immunolocalized CNGA3 to the stereocilia and subcuticular plate region of saccular hair cells. The cytoplasmic C-terminus of CNGA3 was found by yeast two-hybrid analysis to bind the C-terminus of EMILIN1 (elastin microfibril interface-located protein 1) in both the vestibular hair cell model and rat organ of Corti. Specific binding between CNGA3 and EMILIN1 was confirmed with surface plasmon resonance analysis, predicting dependence on Ca2+ with Kd=1.6×10-6 M for trout hair cell proteins and Kd=2.7×10-7 M for organ of Corti proteins at 68 μM Ca2+. Pull-down assays indicated that the binding to organ of Corti CNGA3 was attributable to the EMILIN1 intracellular sequence that follows a predicted transmembrane domain in the C-terminus. Saccular hair cells also express the transcript for PDE6C (phosphodiesterase 6C), which in cone photoreceptors regulates the degradation of cGMP used to gate CNGA3 in phototransduction. Taken together, the evidence supports the existence in saccular hair cells of a molecular pathway linking CNGA3, its binding partner EMILIN1 (and β1 integrin) and cGMP-specific PDE6C, which is potentially replicated in cochlear outer hair cells, given stereociliary immunolocalizations of CNGA3, EMILIN1 and PDE6C.
与介导视觉和嗅觉感觉转导的 CNG 不同，听觉/前庭毛细胞中的 CNG（环核苷酸门控）通道的分子特征在很大程度上是未知的。在本研究中，我们报道了从鳟鱼囊斑的毛细胞制备物中三种 CNGA3 变体的全长序列，其与嗅觉受体神经元/视锥光感受器中的 CNGA3 具有高度同一性。针对 N 端序列的定制抗体将 CNGA3 免疫定位到囊斑毛细胞的静纤毛和基板区域。通过酵母双杂交分析发现，CNGA3 的细胞质 C 端与前庭毛细胞模型和大鼠柯蒂氏器中的 EMILIN1（弹性微纤维界面定位蛋白 1）的 C 端结合。通过表面等离子体共振分析证实了 CNGA3 和 EMILIN1 之间的特异性结合，预测其依赖于 Ca2+，鳟鱼毛细胞蛋白的 Kd 值为 1.6×10-6 M，柯蒂氏器蛋白的 Kd 值为 2.7×10-7 M，Ca2+浓度为 68 μM。下拉测定表明，与柯蒂氏器 CNGA3 的结合归因于 EMILIN1 胞内序列，该序列紧随 C 端预测的跨膜结构域。囊斑毛细胞还表达 PDE6C（磷酸二酯酶 6C）的转录本，该蛋白在视锥光感受器中调节用于门控光转导中 CNGA3 的 cGMP 的降解。综上所述，证据支持 CNGA3、其结合伴侣 EMILIN1（和β1 整合素）和 cGMP 特异性 PDE6C 的分子途径存在于囊斑毛细胞中，鉴于 CNGA3、EMILIN1 和 PDE6C 的静纤毛免疫定位，该途径可能在耳蜗外毛细胞中复制。
