Ramakrishnan Neeliyath A, Drescher Marian J, Barretto Roberto L, Beisel Kirk W, Hatfield James S, Drescher Dennis G
Department of Otolaryngology, Wayne State University School of Medicine, Detroit, Michigan 48201.
Department of Otolaryngology, Wayne State University School of Medicine, Detroit, Michigan 48201.
J Biol Chem. 2009 Jan 30;284(5):3227-3238. doi: 10.1074/jbc.M806177200. Epub 2008 Nov 13.
The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform expressed in trout saccular hair cells, was found by yeast two-hybrid protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin 15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair cell stereocilia, consistent with gradated distribution expected for tip link sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries of rat cochlear inner and outer hair cells, and HCN1 protein was immunolocalized to cochlear hair cell stereocilia. As predicted by the trout hair cell model, the amino terminus of rat organ of Corti HCN1 was found by yeast two-hybrid analysis to bind the carboxyl terminus of protocadherin 15 CD3, a tip link protein implicated in mechanosensory transduction. Specific binding between HCN1 and protocadherin 15 CD3 was confirmed with pull-down assays and surface plasmon resonance analysis, both predicting dependence on Ca(2+). In the presence of calcium chelators, binding between HCN1 and protocadherin 15 CD3 was characterized by a K(D) = 2.39 x 10(-7) m. Ca(2+) at 26.5-68.0 microm promoted binding, with K(D) = 5.26 x 10(-8) m (at 61 microm Ca(2+)). Binding by deletion mutants of protocadherin 15 CD3 pointed to amino acids 158-179 (GenBank accession number XP_238200), with homology to the comparable region in trout hair cell protocadherin 15a-like protein, as necessary for binding to HCN1. Amino terminus binding of HCN1 to HCN1, hypothesized to underlie HCN1 channel formation, was also found to be Ca(2+)-dependent, although the binding was skewed toward a lower effective maximum [Ca(2+)] than for the HCN1 interaction with protocadherin 15 CD3. Competition may therefore exist in vivo between the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3 favored between 26.5 and 68 microm Ca(2+). Taken together, the evidence supports a role for HCN1 in mechanosensory transduction of inner ear hair cells.
通过酵母双杂交实验发现，在鳟鱼球囊毛细胞中表达的主要全长HCN亚型HCN1的胞质氨基末端，可与原钙黏蛋白15a样蛋白的胞质羧基末端结构域结合。HCN1免疫定位在球囊毛细胞静纤毛上的离散位点，这与原钙黏蛋白15a的顶连接位点预期的分级分布一致。在大鼠耳蜗内、外毛细胞的cDNA文库中也检测到了HCN1信息，并且HCN1蛋白免疫定位在耳蜗毛细胞静纤毛上。正如鳟鱼毛细胞模型所预测的，通过酵母双杂交分析发现大鼠柯蒂氏器HCN1的氨基末端可与原钙黏蛋白15 CD3的羧基末端结合，原钙黏蛋白15 CD3是一种与机械感觉转导有关的顶连接蛋白。通过下拉实验和表面等离子体共振分析证实了HCN1与原钙黏蛋白15 CD3之间的特异性结合，两者均预测其依赖于Ca(2+)。在存在钙螯合剂的情况下，HCN1与原钙黏蛋白15 CD3之间的结合特征为解离常数K(D)=2.39×10(-7) m。26.5 - 68.0 μmol的Ca(2+)促进结合，K(D)=5.26×10(-8) m（在61 μmol Ca(2+)时）。原钙黏蛋白15 CD3缺失突变体的结合指向氨基酸158 - 179（GenBank登录号XP_238200），其与鳟鱼毛细胞原钙黏蛋白15a样蛋白中的可比区域具有同源性，是与HCN1结合所必需的。HCN1的氨基末端与HCN1的结合，被认为是HCN1通道形成的基础，也被发现是依赖于Ca(2+)的，尽管这种结合倾向于比HCN1与原钙黏蛋白15 CD3相互作用更低的有效最大[Ca(2+)]。因此，体内可能存在HCN1两个结合位点之间的竞争，在26.5至68 μmol Ca(2+)之间，HCN1与原钙黏蛋白15 CD3的结合更受青睐。综上所述，这些证据支持HCN1在内耳毛细胞机械感觉转导中的作用。
