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Expression of Na + ‐Ca 2+ Exchanger with Modified C‐Terminal Hydrophobic Domains and Enhanced Activity
Author(s) -
GABELLINI N.,
IWATA T.,
CARAFOLI E.
Publication year - 1996
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.1996.tb44775.x
Subject(s) - library science , chemistry , computer science
A 6-Kb canine cDNA fragment complementary to the 5' region of the 7-Kb mRNA encoding the cardiac Na+-Ca2+ exchanger was expressed in human kidney 293 cells. The mRNA products were reverse transcribed and amplified by PCR. The determined DNA sequence of the amplified DNA fragments revealed the presence of an intron that was alternatively spliced. The partial exon sequence, located at the 3' end of the 6-Kb cDNA, was alternatively connected to bases 3198, 2821, 2620 and 1844 in four types of splicing products identified. In the largest product the adjoining exon was located after the putative stop codon of the regular sequence. In a second and third type of shortened transcripts, a hydrophobic sequence encoded by the spliced-in exon was linked with the 4th or the 5th extracellular loops, and could possibly replace transmembrane segments 9 or 11. In the fourth type of spliced transcript the in-frame exon sequence introduced one Leu followed by a stop codon in the large hydrophilic loop. Measurements of Ca2+ uptake in 293 cells expressing the modified exchanger indicated a higher activity in comparison with 293 cells expressing the 3.7-Kb cDNA, in which this alternative splicing does not occur. Deletion mutagenesis of the C-terminal region encoded by the spliced-in exon was performed to investigate its role in the enhancement of the transport activity.