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Molecular determinants of cAMP‐mediated regulation of the Na + –Ca 2+ exchanger expressed in human cell lines
Author(s) -
He LiPing,
Cleemann L.,
Soldatov N. M.,
Morad M.
Publication year - 2003
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2003.00677.x
Subject(s) - microbiology and biotechnology , transfection , baby hamster kidney cell , biology , hek 293 cells , cell culture , chemistry , genetics
The cardiac Na + –Ca 2+ exchanger (NCX1) is one of the major sarcolemmal Ca 2+ transporters of cardiomyocytes. Structure–function studies suggest that β‐adrenergic inhibition of NCX1, as reported for frog, but not mammalian hearts, may be associated with a unique splice variant of frog cardiac NCX1 where insertion of an extra exon completes the coding of a nucleotide binding P‐loop. To test the involvement of the P‐loop in cAMP‐mediated regulation of NCX1 we used four stably transfected human cell lines (a previously established line of baby hamster kidney (BHK) cells and three new lines of human embryonic kidney (HEK) cells) expressing: (1) wild‐type dog NCX1 (dog NCX1); (2) wild‐type frog NCX1 (frog NCX1); (3) chimeric frog‐dog NCX1 incorporating the completed P‐loop from the frog NCX1 into the dog NCX1 sequence (frog/dog NCX1); and (4) a mutated frog NCX1 where a putative protein kinase A (PKA) site was disrupted by substitution of a single serine residue with glycine (S374G frog NCX1). Structural expression of these NCX1 constructs was confirmed using Western blot analysis of extracted proteins and immunofluorescence imaging. The NCX1‐generated current ( I Na–Ca ) was reliably measured in cells expressing dog (2.0 ± 0.15 pA pF −1 ), frog (0.6 ± 0.1 pA pF −1 ) and frog/dog (0.6 ± 0.1 pA pF −1 ) NCX1, but less so in those expressing S374G frog NCX1 (0.3 ± 0.1 pA pF −1 ). Addition of 100 μ m 8‐bromoadenosine 3′,5′ cyclic monophosphate (8‐Br‐cAMP) suppressed I Na–Ca of frog and frog/dog NCX1 by 60–80 %. The suppression of I Na–Ca was smaller and transient in cells expressing S374G frog NCX1, and absent in cells expressing dog NCX1. Intracellular Ca 2+ (Ca 2+ 1 )‐transients, activated by rapid withdrawal of Na + , were also downregulated in the frog and frog/dog NCX1 and to a smaller and transient extent in S374G frog NCX1. Our findings suggest that the suppressive effect of β‐adrenergic agonists requires the presence of the P‐loop domain of the frog NCX1, and provide evidence that the putative PKA site, present in both dog and frog NCX1, might also be critical in the cAMP‐mediated regulation of the exchanger.