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Received November 26, 2002
Accepted after revision February 7, 2003
1 Georgetown University, 4000 Reservoir Road NW, Washington, DC 20007, USA
2 National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA
* To whom correspondence should be addressed. E-mail: cleemanl{at}georgetown.edu.
The cardiac Na+-Ca2+ exchanger (NCX1) is one of the major sarcolemmal Ca2+ transporters of cardiomyocytes. Structure-function studies suggest that 
-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 established four stably transfected human cell lines (human embryonic kidney HEK293 and BHK 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 (INa-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 µM 8-bromoadenosine 3â,5â cyclic monophosphate (8-Br-cAMP) suppressed INa-Ca of frog and frog/dog NCX1 by 60-80 %. The suppression of INa-Ca was smaller and transient in cells expressing S374G frog NCX1, and absent in cells expressing dog NCX1. Intracellular Ca2+ (Ca2+i)-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 -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 be critical in the cAMP-mediated regulation of the exchanger.
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