Activation of protein kinase A (PKA) increases Na+ current derived from the human cardiac Na+ channel, hH1, in a slow, nonsaturable manner. This effect is prevented by compounds that disrupt plasma membrane recycling, implying enhanced trafficking of channels to the cell membrane as the mechanism responsible for Na+ current potentiation. To investigate the molecular basis of this effect, preferred consensus sites (serines 483, 571, and 593) and alternative sites phosphorylated by PKA in the rat heart isoform (serines 525 and 528) were removed in the I-II interdomain linker, a region in the channel previously implicated in the PKA response. Our results demonstrate that the presence of either serine 525 or 528 is required for Na+ current potentiation. The role of amino acid sequences that can mediate channel-protein interactions was also examined. Removal of a PDZ domain-binding motif at the carboxy terminus of hH1 did not alter the PKA response. The I-II interdomain linker of the channel contains 3 sites (479RKR481, 533RRR535, and 659RQR661) with the sequence RXR, a motif known to mediate retention of proteins in the endoplasmic reticulum (ER). The PKA-mediated increase in Na+ current was abolished when all 3 sites were eliminated, with RRR at position 533 to 535 primarily responsible for this effect. These results demonstrate that both alpha-subunit phosphorylation and the presence of putative ER retention signals are required for the PKA-mediated increase in cardiac Na+ current, an effect that likely involves interaction of the I-II interdomain linker with other proteins or regions of the channel.