Expression of the alpha1F calcium channel subunit by photoreceptors in the rat retina.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Morgans
C W
CW
John Curtin School of Medical Research, Australian National University, Canberra, Australia. catherine.morgans@anu.edu.au
Gaughwin
P
P
Maleszka
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D020746:Calcium Channels, L-Type; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis; D005136:Eye Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009755:Night Blindness / Q000151:congenital; D010446:Peptide Fragments / Q000276:immunology; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017729:Presynaptic Terminals / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011921:Rats, Inbred WKY; D017386:Sequence Homology, Amino Acid; D012756:Sheep
D000906:Antibodies; C113330:CACNA1F protein, human; C527361:Cacna1f protein, rat; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D017931:DNA Primers; D018076:DNA, Complementary; D005136:Eye Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D002118:Calcium