Functionally Important Calmodulin-binding Sites in Both NH2- and COOH-terminal Regions of the Cone Photoreceptor Cyclic Nucleotide-gated Channel CNGB3 Subunit*
Functionally Important Calmodulin-binding Sites in Both NH2- and COOH-terminal Regions of the Cone Photoreceptor Cyclic Nucleotide-gated Channel CNGB3 Subunit*
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DOI：
10.1074/jbc.m301699200
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发表时间：
2003-07
影响因子：
4.8
通讯作者：
Changhong Peng;E. D. Rich;C. Thor;M. Varnum
中科院分区：
文献类型：
--
作者：
Changhong Peng;E. D. Rich;C. Thor;M. Varnum
尽管视杆光感受器和嗅觉感受器神经元中感觉适应的一个重要方面被认为是钙-钙调蛋白（Ca 2 +-CaM）对环核苷酸门控（CNG）通道活性的调节，但尚不清楚视锥光感受器CNG通道是否受到类似的调节。锥体CNG通道由至少两种不同的亚基类型CNGA 3和CNGB 3组成。我们已经研究了钙调蛋白是否通过直接结合CNGB 3亚基来调节这些通道的活性。异聚体通道形成的共同表达的人CNGB 3与人CNGA 3亚基在爪蟾卵母细胞; CNGB 3亚基赋予调节的敏感性Ca 2 +-CaM，而CaM调节同源CNGA 3通道未检测到。为了探索这种调节的机制，我们使用凝胶覆盖和谷胱甘肽S-转移酶下拉分析在CNGB 3的NH 2-和COOH-末端胞质结构域中定位潜在的CaM结合位点。对于这两个网站，钙调素的结合依赖于Ca 2+的存在。CNGB 3中任一CaM结合位点的单独缺失产生对Ca 2 +-CaM调节保持敏感的通道，但两者一起缺失导致不受调节的异聚体通道。因此，CNGB 3中的NH 2-和COOH-末端CaM结合位点对于重组锥CNG通道的调节在功能上是重要的。这些研究表明，在视锥细胞的适应和恢复过程中的直接结合和脱结合的Ca 2 +-CaM的人CNGB 3的潜在作用。
Whereas an important aspect of sensory adaptation in rod photoreceptors and olfactory receptor neurons is thought to be the regulation of cyclic nucleotide-gated (CNG) channel activity by calcium-calmodulin (Ca2+-CaM), it is not clear that cone photoreceptor CNG channels are similarly modulated. Cone CNG channels are composed of at least two different subunit types, CNGA3 and CNGB3. We have investigated whether calmodulin modulates the activity of these channels by direct binding to the CNGB3 subunit. Heteromeric channels were formed by co-expression of human CNGB3 with human CNGA3 subunits in Xenopus oocytes; CNGB3 subunits conferred sensitivity to regulation by Ca2+-CaM, whereas CaM regulation of homomeric CNGA3 channels was not detected. To explore the mechanism underlying this regulation, we localized potential CaM-binding sites in both NH2- and COOH-terminal cytoplasmic domains of CNGB3 using gel-overlay and glutathione S-transferase pull-down assays. For both sites, binding of CaM depended on the presence of Ca2+. Individual deletions of either CaM-binding site in CNGB3 generated channels that remained sensitive to regulation by Ca2+-CaM, but deletion of both together resulted in heteromeric channels that were not modulated. Thus, both NH2- and COOH-terminal CaM-binding sites in CNGB3 are functionally important for regulation of recombinant cone CNG channels. These studies suggest a potential role for direct binding and unbinding of Ca2+-CaM to human CNGB3 during cone photoreceptor adaptation and recovery.