Activation of D1-like dopamine (DA) receptors reduces peak Na(+) current in acutely isolated hippocampal neurons via a modulatory mechanism involving phosphorylation of the Na(+) channel alpha subunit by cAMP-dependent protein kinase (PKA). Peak Na(+) current is reduced 20-50% in the presence of the D1 agonist SKF 81297 or the PKA activator Sp-5,6-dichloro-l-beta-d-ribofuranosyl benzimidazole-3',5'-cyclic monophosphorothionate (cBIMPS). Co-immunoprecipitation experiments show that Na(+) channels are associated with PKA and A-kinase-anchoring protein 15 (AKAP-15), and immunocytochemical labeling reveals their co-localization in the cell bodies and proximal dendrites of hippocampal pyramidal neurons. Anchoring of PKA near the channel by an AKAP, which binds the RII alpha regulatory subunit, is necessary for Na(+) channel modulation in acutely dissociated hippocampal pyramidal neurons. Intracellular dialysis with the anchoring inhibitor peptides Ht31 from a human thyroid AKAP and AP2 from AKAP-15 eliminated the modulation of the Na(+) channel by the D1-agonist SKF 81297 and the PKA activator cBIMPS. In contrast, dialysis with the inactive proline-substituted control peptides Ht31-P and AP2-P had little effect on the D1 and PKA modulation. Therefore, we conclude that modulation of the Na(+) channel by activation of D1-like DA receptors requires targeted localization of PKA near the channel to achieve phosphorylation of the alpha subunit and to modify the functional properties of the channel.