Alternative splicing of exons in the alpha1 subunit of the rat testis L-type voltage-dependent calcium channel generates germ line-specific dihydropyridine binding sites.
Cell-specific isoforms of the alpha1 subunit of the L-type voltage-dependent calcium channel (VDCC) have unique pharmacological reactivities. Prior sequence analysis of nucleotide bases 3908-6077 of the VDCC alpha1 subunit expressed in rat testis differed from cardiac sequences only in a 84 base pair region corresponding to exons 31/32 encoding a putative dihydropyridine binding region. We now report that sequence analysis of bases 3048-3936 identifies a second difference between the rat testis and rat cardiac alpha1 sequence in a 60 base pair region corresponding to exons 21/22 and encoding another putative dihydropyridine binding site. Variable VDCC exons 21/22 and 31/32 and their linking introns were sequenced using genomic DNA from rat lung as template, providing evidence that the rat testis and cardiac alpha1 isoforms are products of the same gene. Reverse transcription in-situ polymerase chain reaction (PCR) with frozen sections of rat testis was carried out with primers identifying the testis-specific exon 32 of the VDCC alpha1 subunit. PCR products were confined to seminiferous tubules and were associated with the germ cell lineage from Type A spermatogonia to mature spermatozoa. Close coupling of testis alpha1 VDCC gene transcription and translation was established by in-situ immunolabelling of serial frozen sections with a monoclonal antibody (IIF7) directed against epitopes on rabbit skeletal muscle L-type VDCC alpha1. Western blot analysis of rat proteins extracted from heart, skeletal muscle, testis and spermatozoa which were reactive with the IIF7 antibody detected primarily 175-220 kDa proteins in the size range of VDCC. These data unequivocally demonstrate that an L-type VDCC is expressed in rat testis and that VDCC isoforms from rat testis and heart differ in deduced amino acid composition in and around potential binding sites for calcium channel blocking drugs such as the dihydropyridines.
Goodwin
L O
LO
Department of Research, North Shore University Hospital-New York University School of Medicine, Manhasset, USA.
Leeds
N B
NB
Hurley
I
I
Cooper
G W
GW
Pergolizzi
R G
RG
Benoff
S
S
D017398:Alternative Splicing / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels / Q000032:analysis / Q000235:genetics*; D004095:Dihydropyridines / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal; D009206:Myocardium / Q000378:metabolism; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D017422:Sequence Analysis, DNA; D013094:Spermatozoa / Q000737:chemistry; D013737:Testis / Q000737:chemistry* / Q000502:physiology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D012333:RNA, Messenger; C038806:1,4-dihydropyridine