Expression of Na(+)-Ca2+ exchanger with modified C-terminal hydrophobic domains and enhanced activity.
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.
Gabellini
N
N
Department of Biological Chemistry University of Padova, Italy.
Iwata
T
T
Carafoli
E
E
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D010446:Peptide Fragments; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium