Morgans C W, Gaughwin P, Maleszka R
John Curtin School of Medical Research, Australian National University, Canberra, Australia.
Mol Vis. 2001 Aug 22;7:202-9.
The CACNA1F gene encodes a voltage-gated calcium channel alpha1 subunit, alpha1F, which is expressed in the human retina. Mutations in this gene cause incomplete X-linked congenital stationary night blindness (CSNB2). The aim of this study was to obtain the sequence of the rat alpha1F cDNA and localize the encoded polypeptide in the rat retina.
The full-length rat alpha1F sequence was compiled from sequencing of overlapping alpha1F PCR fragments amplified from rat retinal cDNA. Antiserum was raised against a human alpha1F peptide. It was found that the human alpha1F peptide used to generate the antiserum was conserved at only 11 out of 19 residues in the cloned rat sequence. Therefore, antibodies were affinity purified against either the human alpha1F peptide or the equivalent rat peptide and used for immunofluorescent staining of rat retina sections.
The rat alpha1F amino acid sequence was found to be 91% and 95% identical to the human and mouse alpha1F sequences, respectively. Antibodies affinity purified against the human alpha1F peptide stained both the outer nuclear layer (ONL) and outer plexiform layer of rat retina sections. In contrast, staining with antibodies affinity purified against the corresponding rat alpha1F peptide was restricted to the ONL.
The rat alpha1F amino acid sequence is highly homologous to the human and mouse sequences. The immunohistochemical results indicate the existence of distinct alpha1F isoforms or alpha1F-like channels, which are differentially distributed in the cell bodies and synaptic terminals of photoreceptors in the rat retina.
CACNA1F基因编码一种电压门控钙通道α1亚基，即α1F，其在人类视网膜中表达。该基因的突变会导致不完全X连锁先天性静止性夜盲（CSNB2）。本研究的目的是获取大鼠α1F cDNA的序列，并将编码的多肽定位在大鼠视网膜中。
通过对从大鼠视网膜cDNA扩增的重叠α1F PCR片段进行测序，编译出大鼠α1F的全长序列。制备针对人α1F肽的抗血清。结果发现，用于产生抗血清的人α1F肽在克隆的大鼠序列的19个残基中仅11个保守。因此，针对人α1F肽或等效大鼠肽对抗体进行亲和纯化，并用于大鼠视网膜切片的免疫荧光染色。
发现大鼠α1F氨基酸序列与人及小鼠α1F序列分别具有91%和95%的同一性。针对人α1F肽亲和纯化的抗体对大鼠视网膜切片的外核层（ONL）和外网状层均有染色。相比之下，用针对相应大鼠α1F肽亲和纯化的抗体染色仅限于ONL。
大鼠α1F氨基酸序列与人及小鼠序列高度同源。免疫组化结果表明存在不同的α1F同工型或α1F样通道，它们在大鼠视网膜光感受器的细胞体和突触终末中分布不同。
