CACNA1S expression in mouse retina: Novel isoforms and antibody cross-reactivity with GPR179.
CACNA1S expression in mouse retina: Novel isoforms and antibody cross-reactivity with GPR179.
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DOI：
10.1017/s0952523816000055
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发表时间：
2016-01
影响因子：
1.9
通讯作者：
Gregg RG
中科院分区：
文献类型：
--
作者：
Hasan N;Ray TA;Gregg RG
Cacnals编码电压依赖性钙通道的α 1 S亚基（Cav1.1），是正常骨骼肌和心肌功能所必需的，在那里它与兰尼碱受体偶联以调节肌肉收缩。最近的研究发现，CACNA 1 S在视网膜去极化双极细胞（DBC）的顶端表达，并与代谢型谷氨酸受体6（mGluR 6）共定位，后者是DBC信号转导的关键。此外，在mGluR 6敲除小鼠中，该位置的表达下调。我们检查了来自小鼠视网膜的RNAseq数据，并发现了Cacnals的一种新亚型的表达。为了确定CACNA 1 S是否是DBC信号转导级联的功能组分，我们进行了免疫组织化学以观察其在缺乏DBC功能的几种小鼠系中的表达。CACNA 1 S抗体的免疫组织化学染色显示野生型（WT）视网膜中DBCs尖端的点状标记，这在Gpr 179 nob 5突变体视网膜中不存在，在Grm 6 −/−小鼠视网膜中减少。CACNA 1 S和瞬时受体电位阳离子通道，亚家族M，成员1（TRPM 1）染色也共定位于WT视网膜。用CACNA 1 S抗体免疫沉淀后，对视网膜裂解物或蛋白质的CACNA 1 S的Western印迹分析未能显示出预期的CACNA 1 S条带的存在。CACNA 1 S免疫沉淀蛋白质的质谱分析也未能检测到任何与CACNA 1 S匹配的肽。在HEK 293 T细胞中表达GPR 179后的免疫组织化学和蛋白质印迹表明，本文和迄今为止发表的视网膜研究中使用的CACNA 1 S抗体与GPR 179交叉反应。这些数据表明，应谨慎行使赋予的作用CACNA 1 S在DBC信号转导的基础上，仅免疫组化染色。
Cacnals encodes the α1S subunit (Cav1.1) of voltage-dependent calcium channels, and is required for normal skeletal and cardiac muscle function, where it couples with the ryanodine receptor to regulate muscle contraction. Recently CACNA1S was reported to be expressed on the tips of retinal depolarizing bipolar cells (DBCs) and colocalized with metabotropic glutamate receptor 6 (mGluR6), which is critical to DBC signal transduction. Further, in mGluR6 knockout mice, expression at this location is down regulated. We examined RNAseq data from mouse retina and found expression of a novel isoform of Cacnals. To determine if CACNA1S was a functional component of the DBC signal transduction cascade, we performed immunohistochemistry to visualize its expression in several mouse lines that lack DBC function. Immunohistochemical staining with antibodies to CACNA1S show punctate labeling at the tips of DBCs in wild type (WT) retinas that are absent in Gpr179nob5 mutant retinas and decreased in Grm6−/− mouse retinas. CACNA1S and transient receptor potential cation channel, subfamily M, member 1 (TRPM1) staining also colocalized in WT retinas. Western blot analyses for CACNA1S of either retinal lysates or proteins after immunoprecipitation with the CACNA1S antibody failed to show the presence of bands expected for CACNA1S. Mass spectrometric analysis of CACNA1S immunoprecipitated proteins also failed to detect any peptides matching CACNA1S. Immunohistochemistry and western blotting after expression of GPR179 in HEK293T cells indicate that the CACNA1S antibody used here and in the retinal studies published to date, cross-reacts with GPR179. These data suggest caution should be exercised in conferring a role for CACNA1S in DBC signal transduction based solely on immunohistochemical staining.