Voltage-gated sodium channels in brain neurons were found to associate with receptor protein tyrosine phosphatase beta (RPTPbeta) and its catalytically inactive, secreted isoform phosphacan, and this interaction was regulated during development. Both the extracellular domain and the intracellular catalytic domain of RPTPbeta interacted with sodium channels. Sodium channels were tyrosine phosphorylated and were modulated by the associated catalytic domains of RPTPbeta. Dephosphorylation slowed sodium channel inactivation, positively shifted its voltage dependence, and increased whole-cell sodium current. Our results define a sodium channel signaling complex containing RPTPbeta, which acts to regulate sodium channel modulation by tyrosine phosphorylation.

Voltage-dependent potentiation of skeletal muscle Ltype calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) that is localized by binding to a cAMP-dependent protein kinase-anchoring protein (AKAP). L-type calcium channels purified from rabbit skeletal muscle contain an endogenous copurifying protein kinase activity that phosphorylates the ␣1 and ␤ subunits of the channel. The co-purifying kinase also phosphorylates a known PKA peptide substrate, is stimulated by cAMP, and is inhibited by PKA inhibitor peptide-(5-24), indicating that it is PKA. PKA activity co-immunoprecipitates with the calcium channel, suggesting that the channel and the kinase are physically associated. Using biotinylated type II regulatory subunit of PKA (RII) as a probe, we have identified a 15-kDa RII-binding protein in purified calcium channel preparations, which we have designated AKAP-15. Anti-peptide antibodies directed against the ␣1 subunit of the calcium channel co-immunoprecipitate AKAP-15. Together, these findings demonstrate a physical link between PKA and the calcium channel and suggest that AKAP-15 may mediate their interaction.The activation of PKA 1 following transient increases in intracellular levels of cAMP represents a fundamental mechanism for regulating protein function via phosphorylation (1, 2). A wide variety of proteins including enzymes, membrane receptors, ion channels, and transcription factors have been shown to be PKA substrates with activities reversibly modulated by phosphorylation and dephosphorylation. It is clear that despite its broad substrate specificity, PKA activity is highly selective in a physiological setting and that specific hormones, each capable of raising intracellular cAMP, can result in the preferential phosphorylation of different target substrates (3). Understanding how the activation of a single signaling pathway can lead to multiple but varying cellular effects has become an important goal in cAMP signaling research. Recent work has demonstrated that both cAMP and its target kinase are specifically localized within the cell (4, 5). Together, these findings emphasize that PKA phosphorylation of various target substrates depends not only on whether cAMP levels are increased, but also on where within the cell this increase occurs and whether PKA and its substrates are localized at the site.The PKA holoenzyme consists of two regulatory subunits and two catalytic subunits forming an inactive heterotetramer (6). Each regulatory subunit binds two cAMP molecules, causing the release of active catalytic subunits. Two classes of regulatory subunits exist, giving rise to type I and II holoenzymes. While type I PKA is generally soluble and cytoplasmic, the type II isoform is predominantly associated with the cell particulate fraction (7). Type II PKA activity has been shown to be associated with a variety of subcellular structures including the plasma membrane, cystoskeleton, Golgi apparatus, and nucleus (8). The type II regulatory subunit (RII) mediates kinase localizati...

No abstract

The voltage-sensitive sodium channel is regulated by cAMP-dependent protein kinase (PKA) phosphorylation. Using purified preparations of rat brain sodium channels, we have shown that the ␣ subunit was phosphorylated by a co-purifying protein kinase. The copurifying kinase was stimulated by cAMP and phosphorylated PKA substrate peptides. Both the regulatory and catalytic subunits of PKA were detected by immunoblotting in purified sodium channel preparations. Bound PKA was immunoprecipitated with anti-SP19 antibodies directed against the sodium channel ␣ subunit. PKA bound to sodium channels phosphorylated the sodium channel ␣ subunit on the same four serine residues as observed with exogenously added PKA, indicating that association with the sodium channel does not restrict the sites of phosphorylation. Analysis of proteins with high affinity for the type II ␣ regulatory subunit of PKA in a gel overlay assay identified a 15-kDa cAMP-dependent protein kinase-anchoring protein (AKAP) in these preparations. Determination of its amino acid sequence by mass spectrometry revealed two peptides identical to AKAP15, a recently described AKAP that targets PKA to skeletal muscle calcium channels. The co-purifying AKAP was also immunoreactive with antibodies generated against AKAP15, and antibodies directed against AKAP15 co-immunoprecipitated the sodium channel. Our results indicate that PKA is bound to brain sodium channels through interaction with AKAP15. Association of AKAP15 with both skeletal muscle calcium channels and brain sodium channels suggests that it may have broad specificity in targeting PKA to ion channels for regulation.Voltage-sensitive sodium channels are responsible for the generation of action potentials in nerve cells. The sodium channel purified from adult rat brain is composed of three glycoprotein subunits: ␣ (260 kDa), ␤1 (36 kDa), and ␤2 (33 kDa) (1). The ␣ subunit is sufficient to form voltage-sensitive sodium channels when expressed in mammalian cells or Xenopus oocytes (2-5). The brain sodium channel is rapidly phosphorylated by PKA 1 in purified preparations (6 -8), in synaptosomes (9), and in intact neurons or transfected mammalian cells expressing the type IIA sodium channel (8, 10) on four serine residues in the intracellular loop connecting homologous domains I and II of the ␣ subunit (8). In neurons and transfected cells expressing type IIA brain sodium channels, phosphorylation of the ␣ subunit by PKA reduces peak sodium current by 20 -50%, with little change in the voltage dependence of activation or inactivation (11). This effect is blocked by a specific peptide inhibitor of PKA (PKI) and reversed by a mixture of catalytic subunits of phosphatases 1 and 2A (11). Similarly, activation of D1-like dopamine receptors in acutely isolated hippocampal neurons reversibly reduces peak sodium current by activation of the cAMP pathway (12). The intracellular loop between domains I and II is necessary for regulation of sodium channels expressed in Xenopus oocytes (13), and phosphorylation of serin...