Cardiomyocyte substructure reverts to an immature phenotype during heart failure.

KEY POINTS: As reactivation of the fetal gene program has been implicated in pathological remodelling during heart failure (HF), we examined whether cardiomyocyte subcellular structure and function revert to an immature phenotype during this disease. Surface and internal membrane structures appeared gradually during development, and returned to a juvenile state during HF. Similarly, dyadic junctions between the cell membrane and sarcoplasmic reticulum were progressively 'packed' with L-type Ca2+ channels and ryanodine receptors during development, and 'unpacked' during HF. Despite similarities in subcellular structure, dyads were observed to be functional from early developmental stages, but exhibited an impaired ability to release Ca2+ in failing cardiomyocytes. Thus, while immature and failing cardiomyocytes share similarities in subcellular structure, these do not fully account for the marked impairment of Ca2+ homeostasis observed in HF. ABSTRACT: Reactivation of the fetal gene programme has been implicated as a driver of pathological cardiac remodelling. Here we examined whether pathological remodelling of cardiomyocyte substructure and function during heart failure (HF) reflects a reversion to an immature phenotype. Using scanning electron microscopy, we observed that Z-grooves and t-tubule openings at the cell surface appeared gradually during cardiac development, and disappeared during HF. Confocal and super-resolution imaging within the cell interior revealed similar structural parallels; disorganization of t-tubules in failing cells was strikingly reminiscent of the late stages of postnatal development, with fewer transverse elements and a high proportion of longitudinal tubules. Ryanodine receptors (RyRs) were observed to be laid down in advance of developing t-tubules and similarly 'orphaned' in HF, although RyR distribution along Z-lines was relatively sparse. Indeed, nanoscale imaging revealed coordinated packing of L-type Ca2+ channels and RyRs into dyadic junctions during development, and orderly unpacking during HF. These findings support a 'last in, first out' paradigm, as the latest stages of dyadic structural development are reversed during disease. Paired imaging of t-tubules and Ca2+ showed that the disorganized arrangement of dyads in immature and failing cells promoted desynchronized and slowed Ca2+ release in these two states. However, while developing cells exhibited efficient triggering of Ca2+ release at newly formed dyads, dyadic function was impaired in failing cells despite similar organization of Ca2+ handling proteins. Thus, pathologically deficient Ca2+ homeostasis during HF is only partly linked to the re-emergence of immature subcellular structure, and additionally reflects lost dyadic functionality.

Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits.

To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D alpha 1 subunits were produced. Anti-CNC1 directed against class C immunoprecipitated 75% of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type alpha 1 subunit, LC1 and LC2, and two size forms of the class D L-type alpha 1 subunit, LD1 and LD2. The larger isoforms had apparent molecular masses of approximately 200-210 kD while the smaller isoforms were 180-190 kD, as estimated from electrophoresis in gels polymerized from 5% acrylamide. Immunocytochemical studies using CNC1 and CND1 antibodies revealed that the alpha 1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNC1, particularly in the dentate gyrus and the CA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calcium-dependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.

A beta2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2.

The existence of a large number of receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) raises the question of how a particular receptor selectively regulates specific targets. We provide insight into this question by identifying a prototypical macromolecular signaling complex. The beta(2) adrenergic receptor was found to be directly associated with one of its ultimate effectors, the class C L-type calcium channel Ca(v)1.2. This complex also contained a G protein, an adenylyl cyclase, cyclic adenosine monophosphate-dependent protein kinase, and the counterbalancing phosphatase PP2A. Our electrophysiological recordings from hippocampal neurons demonstrate highly localized signal transduction from the receptor to the channel. The assembly of this signaling complex provides a mechanism that ensures specific and rapid signaling by a G protein-coupled receptor.

Biochemical properties and subcellular distribution of an N-type calcium channel alpha 1 subunit.

A site-directed anti-peptide antibody, CNB-1, that recognizes the alpha 1 subunit of rat brain class B calcium channels (rbB) immunoprecipitated 43% of the N-type calcium channels labeled by [125I]omega-conotoxin. CNB-1 recognized proteins of 240 and 210 kd, suggesting the presence of two size forms of this alpha 1 subunit. Calcium channels recognized by CNB-1 were localized predominantly in dendrites; both dendritic shafts and punctate synaptic structures upon the dendrites were labeled. The large terminals of the mossy fibers of the dentate gyrus granule neurons were heavily labeled, suggesting that the punctate labeling pattern represents calcium channels in nerve terminals. The pattern of immunostaining was cell specific. The cell bodies of some pyramidal cells in layers II, III, and V of the dorsal cortex, Purkinje cells, and scattered cell bodies elsewhere in the brain were also labeled at a low level. The results define complementary distributions of N- and L-type calcium channels in dendrites, nerve terminals, and cell bodies of most central neurons and support distinct functional roles in calcium-dependent electrical activity, intracellular calcium regulation, and neurotransmitter release for these two channel types.

Amino acid neurotransmission: spotlight on synaptic vesicles.

The vesicle hypothesis describing quantal release of neurotransmitter at the cholinergic neuromuscular junction was introduced in 1956. Since then, the concept of vesicular storage and release of acetylcholine has become firmly established and extended to include other synapses and neurotransmitters. However, for the amino acids, which are the major class of neurotransmitters in the mammalian CNS, there was no direct experimental evidence of the participation of synaptic vesicles in neurotransmission. This area of research has now moved out of the shadows and this article discusses recent findings which indicate that amino acid neurotransmitters are accumulated and stored by synaptic vesicles in presynaptic nerve endings.

Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C.

Voltage-dependent L-type Ca(2+) channels are multisubunit transmembrane proteins, which allow the influx of Ca(2+) (I:(Ca)) essential for normal excitability and excitation-contraction coupling in cardiac myocytes. A variety of different receptors and signaling pathways provide dynamic regulation of I:(Ca) in the intact heart. The present review focuses on recent evidence describing the molecular details of regulation of L-type Ca(2+) channels by protein kinase A (PKA) and protein kinase C (PKC) pathways. Multiple G protein-coupled receptors act through cAMP/PKA pathways to regulate L-type channels. ss-Adrenergic receptor stimulation results in a marked increase in I:(Ca), which is mediated by a cAMP/PKA pathway. Growing evidence points to an important role of localized signaling complexes involved in the PKA-mediated regulation of I:(Ca), including A-kinase anchor proteins and binding of phosphatase PP2a to the carboxyl terminus of the alpha(1C) (Ca(v)1.2) subunit. Both alpha(1C) and ss(2a) subunits of the channel are substrates for PKA in vivo. The regulation of L-type Ca(2+) channels by Gq-linked receptors and associated PKC activation is complex, with both stimulation and inhibition of I:(Ca) being observed. The amino terminus of the alpha(1C) subunit is critically involved in PKC regulation. Crosstalk between PKA and PKC pathways occurs in the modulation of I:(Ca). Ultimately, precise regulation of I:(Ca) is needed for normal cardiac function, and alterations in these regulatory pathways may prove important in heart disease.

A developmental change in NMDA receptor-associated proteins at hippocampal synapses.

The membrane-associated guanylate kinases [Chapsyn-110/postsynaptic density-93 (PSD-93), synapse-associated protein-90 (SAP-90)/PSD-95, and SAP-102] are believed to cluster and anchor NMDA receptors at the synapse and to play a role in signal transduction. We have investigated the developmental changes in expression of these proteins in rat hippocampus using biochemical analyses and quantitative immunogold electron microscopy. At postnatal day 2 (P2), SAP-102 was highly expressed, whereas PSD-93 and PSD-95 were low. SAP-102 expression increased during the first week, stayed stable through P35, and showed a reduced expression at 6 months. From P2 through 6 months, PSD-93 and PSD-95 increased. For PSD-95, the percent of labeled synapses increased almost threefold with age, whereas the number of gold particles per labeled synapse did not change significantly, suggesting that the increase in PSD-95 is attributable primarily to an increase in the number of synapses containing PSD-95. In contrast, for SAP-102, both percent labeled synapses and the number of gold particles per labeled synapse decreased during this time. From Western blots of hippocampus and immunogold analysis of CA1 synapses, the high expression of NR2B at P2 coincides with the high level of SAP-102 at synapses, whereas the later expression of NR2A coincides with that of PSD-93 and PSD-95. To determine whether the changes in PSD-93/95 and SAP-102 reflect preferred associations with NR2A and NR2B, respectively, we measured co-immunoprecipitation in the adult hippocampus. These studies suggest that there is a preference for complexes of NR2A/PSD-93/95 and NR2B/SAP-102. These results indicate that individual receptor-associated proteins may have specific functions that are critical to synapse development.

Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability.

The Kv2.1 delayed rectifier potassium channel exhibits high-level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity-dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1(-/-) ) mice lacking this channel. Kv2.1(-/-) mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1(-/-) mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1(-/-) mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1(-/-) animals. Field recordings from hippocampal slices of Kv2.1(-/-) mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1(-/-) mice, long-term potentiation at the Schaffer collateral - CA1 synapse is decreased. Kv2.1(-/-) mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1(-/-) mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1(-/-) mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function.

Cyclic AMP-dependent protein kinase and protein kinase C phosphorylate N-methyl-D-aspartate receptors at different sites.

Ca2+ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors plays a pivotal role in synaptic plasticity during brain development as well as in mature brain. Cyclic AMP-dependent protein kinase (PKA) and members of the protein kinase C (PKC) family are also essential for various forms of synaptic plasticity and regulate the activity of different ion channels including NMDA and non-NMDA receptors. We now demonstrate that PKA and various PKC isoforms phosphorylate the NMDA receptor in vitro. The stoichiometry of [32P]phosphate incorporation per [3H]MK-801 binding site is greater than 1 for both PKA and PKC. Double immunoprecipitation experiments show that all three NMDA receptor subunits that are prevalent in the cortical structures, NR1, NR2A, and NR2B, are substrates for PKA as well as PKC. Two-dimensional phosphopeptide mapping reveals that the major phosphorylation sites for PKA and PKC differ for all three subunits. We provide evidence that some if not most of these sites are phosphorylated in the central nervous system of rats in vivo. The results presented in this article together with earlier electrophysiological experiments demonstrating that PKA and PKC activation increases the activity of NMDA receptors indicate that NMDA receptor potentiation can be mediated by direct phosphorylation by PKA and PKC. Collectively, these results strongly suggest that NMDA receptor functions such as control of neuronal development or expression of synaptic plasticity are modulated by PKA- and PKC-mediated phosphorylation of NMDA receptors.

Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase.

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.

Energy dependence and functional reconstitution of the gamma-aminobutyric acid carrier from synaptic vesicles.

The energy dependence of gamma-aminobutyric acid (GABA) uptake was characterized in rat brain synaptic vesicles and in proteoliposomes reconstituted with a new procedure from vesicular detergent extracts. The proteoliposomes displayed high ATP-dependent GABA uptake activity with properties virtually identical to those of intact vesicles. GABA uptake was similar at chloride concentrations of 0 and 150 mM, i.e. conditions under which either the membrane potential (delta psi) or the pH difference (delta pH) predominates. Delta psi was gradually dissipated by increasing the concentration of SCN-. GABA uptake was reduced by 10 mM SCN-, showing less sensitivity to delta psi reduction than glutamate uptake but more than dopamine uptake. Dissipation of delta pH with NH+4 abolished GABA uptake at pH 7.3, whereas no significant inhibition occurred at pH 6.5. In contrast, dopamine uptake was inhibited more strongly, even at pH 6.5, and glutamate uptake was not reduced in either condition. We conclude that GABA uptake is driven by both components of the proton electrochemical gradient, delta pH and delta psi, and that this is different from the uptake of both dopamine and glutamate, which is more strongly dependent on delta pH and delta psi, respectively. Thus, our data suggest that GABA uptake is electrogenic and occurs in exchange for protons.

Glutamate uptake by brain synaptic vesicles. Energy dependence of transport and functional reconstitution in proteoliposomes.

The dependence of glutamate uptake on ATP-generated proton electrochemical potential was studied in a highly purified preparation of synaptic vesicles from rat brain. At low chloride concentration (4 mM), the proton pump present in synaptic vesicles generated a large membrane potential (inside-positive), associated with only minor acidification. Under these conditions, the rate of L-[3H]glutamate uptake was maximal. In addition, L-glutamate induced acidification of the vesicle interior. D-Glutamate produced only 40% of the effect, and L-aspartate or gamma-aminobutyric acid produced less than 5%. The initial rate of glutamate-induced acidification increased with increasing glutamate concentration. It was saturable and showed first-order kinetics (KM = 0.32 mM). Correspondingly, L-glutamate induced a small reduction in the membrane potential. The rate of ATP hydrolysis was unaffected. In comparison, glutamate had no effect on acidification or membrane potential in resealed membranes of chromaffin granules. At high chloride concentration (150 mM), the vesicular proton pump generated a large pH difference, associated with a small change in membrane potential. Under these conditions, uptake of L-[3H]glutamate by synaptic vesicles was low. For reconstitution, vesicle proteins were solubilized with the detergent sodium cholate, supplemented with brain phospholipids, and incorporated into liposomes. Proton pump and glutamate uptake activities of the proteoliposomes showed properties similar to those of intact vesicles indicating that the carrier was reconstituted in a functionally active form. It is concluded that glutamate uptake by synaptic vesicles is dependent on the membrane potential and that all components required for uptake are integral parts of the vesicle membrane.

Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure.

Uptake of GABA was demonstrated in rat brain synaptic vesicles which were prepared by a new and efficient procedure. The uptake activity co-purified with the synaptic vesicles during the isolation procedure. The purity of the vesicle fraction was rigorously examined by analysis of marker enzymes and marker proteins and also by immunogold electron microscopy using antibodies against p38 (synaptophysin). Contamination by other cellular components was negligible, indicating that GABA uptake by the synaptic vesicle fraction is specific for synaptic vesicles and not due to the presence of other structure possessing GABA uptake or binding activities. GABA uptake was ATP dependent and similar to the uptake of glutamate, which was assayed for a comparison. Both uptake activities were independent of sodium. They were inhibited by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, indicating that the energy for the uptake is provided by an electrochemical proton gradient. This gradient is generated by a proton ATPase of the vacuolar type as suggested by the effects of various ATPase inhibitors on neurotransmitter uptake and proton pumping. Competition experiments revealed that the transporters for GABA and glutamate are selective for the respective neurotransmitters.

Phosphorylation of presynaptic and postsynaptic calcium channels by cAMP-dependent protein kinase in hippocampal neurons.

Phosphorylation by cAMP-dependent protein kinase (PKA) and other second messenger-activated protein kinases modulates the activity of a variety of effector proteins including ion channels. Anti-peptide antibodies specific for the alpha 1 subunits of the class B, C or E calcium channels from rat brain specifically recognize a pair of polypeptides of 220 and 240 kDa, 200 and 220 kDa, and 240 and 250 kDa, respectively, in hippocampal slices in vitro. These calcium channels are localized predominantly on presynaptic and dendritic, somatic and dendritic, and somatic sites, respectively, in hippocampal neurons. Both size forms of alpha 1B and alpha 1E and the full-length form of alpha 1C are phosphorylated by PKA after solubilization and immunoprecipitation. Stimulation of PKA in intact hippocampal slices also induced phosphorylation of 25-50% of the PKA sites on class B N-type calcium channels, class C L-type calcium channels and class E calcium channels, as assessed by a back-phosphorylation method. Tetraethylammonium ion (TEA), which causes neuronal depolarization and promotes repetitive action potentials and neurotransmitter release by blocking potassium channels, also stimulated phosphorylation of class B, C and E alpha 1 subunits, suggesting that these three classes of channels are phosphorylated by PKA in response to endogenous electrical activity in the hippocampus. Regulation of calcium influx through these calcium channels by PKA may influence calcium-dependent processes within hippocampal neurons, including neurotransmitter release, calcium-activated enzymes and gene expression.

Immunochemical identification and differential phosphorylation of alternatively spliced forms of the alpha 1A subunit of brain calcium channels.

Biochemical properties of the alpha 1 subunits of class A brain calcium channels (alpha 1A) were examined in adult rat brain membrane fractions using a site-directed anti-peptide antibody (anti-CNA3) specific for alpha 1A. Anti-CNA3 specifically immunoprecipitated high affinity receptor sites for omega-conotoxin MVIIC (Kd approximately 100 pM), but not receptor sites for the dihydropyridine isradipine or for omega-conotoxin GVIA. In immunoblotting and immunoprecipitation experiments, anti-CNA3 recognized at least two distinct immunoreactive alpha 1A polypeptides, a major form with an apparent molecular mass of 190 kDa and a minor, full-length form with an apparent molecular mass of 220 kDa. The 220- and 190-kDa alpha 1A polypeptides were also specifically recognized by both anti-BI-Nt and anti-BI-1-Ct antibodies, which are directed against the NH2- and COOH-terminal ends of alpha 1A predicted from cDNA sequence, respectively. These data indicate that the predicted NH2 and COOH termini are present in both size forms and therefore that these isoforms of alpha 1A are created by alternative RNA splicing rather than post-translational proteolytic processing of the NH2 or COOH termini. The 220-kDa form was phosphorylated preferentially by cAMP-dependent protein kinase, whereas protein kinase C and cGMP-dependent protein kinase preferentially phosphorylated the 190-kDa form. Our results identify at least two distinct alpha 1A subunits with different molecular mass, demonstrate that they may result from alternative mRNA splicing, and suggest that they may be differentially regulated by protein phosphorylation.

The A-kinase anchor protein MAP2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons.

Phosphorylation by cAMP-dependent protein kinase (PKA) increases the activity of class C L-type Ca(2+) channels which are clustered at postsynaptic sites and are important regulators of neuronal functions. We investigated a possible mechanism that could ensure rapid and efficient phosphorylation of these channels by PKA upon stimulation of cAMP-mediated signaling pathways. A kinase anchor proteins (AKAPs) bind to the regulatory R subunits of PKA and target the holoenzyme to defined subcellular compartments and substrates. Class C channels isolated from rat brain extracts by immunoprecipitation contain an endogenous kinase that phosphorylates kemptide, a classic PKA substrate peptide, and also the main phosphorylation site for PKA in the pore-forming alpha(1) subunit of the class C channel complex, serine 1928. The kinase activity is inhibited by the PKA inhibitory peptide PKI(5-24) and stimulated by cAMP. Physical association of the catalytic C subunit of PKA with the immunoisolated class C channel complex was confirmed by immunoblotting. A direct protein overlay binding assay performed with (32)P-labeled RIIbeta revealed a prominent AKAP with an M(r) of 280,000 in class C channel complexes. The protein was identified by immunoblotting as the microtubule-associated protein MAP2B, a well established AKAP. Class C channels did not contain tubulin and MAP2B association was not disrupted by dilution or addition of nocodazole, two treatments that cause dissociation of microtubules. In vitro experiments show that MAP2B can directly bind to the alpha(1) subunit of the class C channel. Our findings indicate that PKA is an integral part of neuronal class C L-type Ca(2+) channels and suggest that the AKAP MAP2B may mediate this interaction. Neither PKA nor MAP2B were detected in immunoprecipitates of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-type glutamate receptors or class B N-type Ca(2+) channels. Accordingly, MAP2B docked at class C Ca(2+) channels may be important for recruiting PKA to postsynaptic sites.