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.

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.

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.

Interaction with the NMDA receptor locks CaMKII in an active conformation.

Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-D-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.

SAP97 concentrates at the postsynaptic density in cerebral cortex.

SAP97, a PDZ-containing protein, is reported to concentrate in axon terminals, where its function remains unknown. Using highly specific new antibodies, we show that SAP97 in rat cerebral cortex is associated with heteromeric AMPA receptors via a selective biochemical interaction between SAP97 and the GluR1 subunit. Using light and electron microscopic immunocytochemistry, we demonstrate cellular and synaptic colocalization of SAP97 and GluR1, and show that SAP97 concentrates at synapses that contain GluR1 but not necessarily GluR2 or GluR3. Using quantitative postembedding immunogold electron microscopy, we find that SAP97 is at highest concentration within the postsynaptic density of asymmetric synapses. These data suggest that SAP97 may help to anchor GluR1-containing AMPA receptors at the synapse. As a multifunctional scaffolding protein, SAP97 may organize components of AMPA-related intracellular signalling pathways, including those associated with calcium-permeable homomeric GluR1 channels.

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.

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.

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.

Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-D-aspartate receptor.

The molecular basis of long-term potentiation (LTP), a long-lasting change in synaptic transmission, is of fundamental interest because of its implication in learning. Usually LTP depends on Ca2+ influx through postsynaptic N-methyl-D-aspartate (NMDA)-type glutamate receptors and subsequent activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). For a molecular understanding of LTP it is crucial to know how CaMKII is localized to its postsynaptic targets because protein kinases often are targeted to their substrates by adapter proteins. Here we show that CaMKII directly binds to the NMDA receptor subunits NR1 and NR2B. Moreover, activation of CaMKIIalpha by stimulation of NMDA receptors in forebrain slices increase this association. This interaction places CaMKII not only proximal to a major source of Ca2+ influx but also close to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, which become phosphorylated upon stimulation of NMDA receptors in these forebrain slices. Identification of the postsynaptic adapter for CaMKII fills a critical gap in the understanding of LTP because CaMKII-mediated phosphorylation of AMPA receptors is an important step during LTP.

SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit.

Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartate-type glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.

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.

Cyclin G2 is up-regulated during growth inhibition and B cell antigen receptor-mediated cell cycle arrest.

Human cyclin G2 together with its closest homolog cyclin G1 defines a novel family of cyclins (Horne, M. C., Goolsby, G. L., Donaldson, K. L., Tran, D., Neubauer, M., and Wahl, A. F. (1996) J. Biol. Chem. 271, 6050-6061). Cyclin G2 is highly expressed in the immune system where immunologic tolerance subjects self-reactive lymphocytes to negative selection and clonal deletion via apoptosis. Here we investigated the effect of growth inhibitory signals on cyclin G2 mRNA abundance in different maturation stage-specific murine B cell lines. Upon treatment of wild-type and p53 null B cell lines with the negative growth factor, transforming growth factor beta1, or the growth inhibitory corticosteroid dexamethasone, cyclin G2 mRNA levels were increased in a time-dependent manner 5-14-fold over control cell levels. Unstimulated immature B cell lines (WEHI-231 and CH31) and unstimulated or IgM B cell receptor (BCR) -stimulated mature B cell lines (BAL-17 and CH12) rapidly proliferate and express low levels of cyclin G2 mRNA. In contrast, BCR-stimulated immature B cell lines undergo growth arrest and coincidentally exhibit an approximately 10-fold increase in cyclin G2 transcripts and a decrease in cyclin D2 message. Costimulation of WEHI-231 and CH31 cells with calcium ionophores and protein kinase C agonists partially mimics anti-IgM stimulation and elicits a strong up-regulation of cyclin G2 mRNA and down-regulation of cyclin D2 mRNA. Signaling mutants of WEHI-231 that are deficient in the phosphoinositide signaling pathway and consequently resistant to the BCR stimulus-induced growth arrest did not display a significant increase in cyclin G2 or decrease in cyclin D2 mRNAs when challenged with anti-IgM antibodies. The two polyclonal activators lipopolysaccharide and soluble gp39, which inhibit the growth arrest response of immature B cells, suppressed cyclin G2 mRNA expression induced by BCR stimulation. These results suggest that in murine B cells responding to growth inhibitory stimuli cyclin G2 may be a key negative regulator of cell cycle progression.

Specific phosphorylation of a site in the full-length form of the alpha 1 subunit of the cardiac L-type calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase.

Voltage-gated L-type Ca2+ channels mediate Ca2+ entry into cells in response to membrane depolarization. Ca2+ entry through the cardiac Ca2+ channel determines the rate and force of contraction, and modulation of Ca2+ channel activity by beta-adrenergic agents acting through adenosine 3',5'-cyclic monophosphate-(cAMP)-dependent protein phosphorylation contributes to physiological regulation of cardiac function by the sympathetic nervous system. Immunoblotting experiments using site-directed anti-peptide antibodies against different peptide segments indicate that the alpha 1 subunit of the cardiac L-type Ca2+ channel exists in two size forms with apparent molecular masses of 240 and 210 kDa, which we call alpha 1(242) and alpha 1(210), Alpha 1(242) corresponds to the full-length cardiac alpha 1 subunit predicted from its cDNA sequence, while alpha 1(210) is truncated at its COOH terminus. Only alpha 1(242) is phosphorylated in vitro by cAMP-dependent protein kinase. Protein microsequencing and peptide mapping of wild-type and mutant fusion proteins show that this phosphorylation occurs at serine 1928 near the COOH terminus. Phosphorylation of this residue can be detected by phosphospecific antibodies raised against the corresponding phosphopeptide. Experiments with these antibodies show that alpha 1(242) is phosphorylated in intact cells expressing the cardiac alpha 1 subunit in response to increased intracellular levels of cAMP. These results identify serine 1928 on the alpha 1 subunit as a possible site of regulation by cAMP-dependent phosphorylation.

N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.

Immunochemical identification and subcellular distribution of the alpha 1A subunits of brain calcium channels.

A site-directed anti-peptide antibody (anti-CNA1) directed against the alpha 1 subunit of class A calcium channels (alpha 1A) recognized a protein of approximately 190-200 kDa in immunoblot and immunoprecipitation analyses of rat brain glycoproteins. Calcium channels recognized by anti-CNA1 were distributed throughout the brain with a high concentration in the cerebellum. Calcium channels having alpha 1A subunits were concentrated in presynaptic terminals making synapses on cell bodies and on dendritic shafts and spines of many classes of neurons and were especially prominent in the synapses of the parallel fibers of cerebellar granule cells on Purkinje neurons where their localization in presynaptic terminals was confirmed by double labeling with the synaptic membrane protein syntaxin or the microinjected postsynaptic marker Neurobiotin. They were present in lower density in the surface membrane of dendrites of most major classes of neurons. There was substantial labeling of Purkinje cell bodies, but less intense staining of the cell bodies of hippocampal pyramidal neurons, layer V pyramidal neurons in the dorsal cortex, and most other classes of neurons in the forebrain and cerebellum. Scattered cell bodies elsewhere in the brain were labeled at low levels. These results define a unique pattern of localization of class A calcium channels in the cell bodies, dendrites, and presynaptic terminals of most central neurons. Compared to class B N-type calcium channels, class A calcium channels are concentrated in a larger number of presynaptic nerve terminals implying a more prominent role in neurotransmitter release at many central synapses.

Biochemical properties and subcellular distribution of the neuronal class E calcium channel alpha 1 subunit.

Anti-peptide antibodies specific for the neuronal calcium channel alpha 1E subunit (anti-CNE1 and anti-CNE2) were produced to study the biochemical properties and subcellular distribution of the alpha 1E polypeptide from rat brain. Immunoblotting identified a single size form of 245-255 kDa which was a substrate for phosphorylation by cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II. Ligand-binding studies of alpha 1E indicate that it is not a high affinity receptor for the dihydropyridine isradipine or the peptide toxins omega-conotoxin GVIA or omega-conotoxin MVIIC at concentrations which elicit high affinity binding to other channel types in the same membrane preparation. The alpha 1E subunit is widely distributed in the brain with the most prominent immunocytochemical staining in deep midline structures such as caudate-putamen, thalamus, hypothalamus, amygdala, cerebellum, and a variety of nuclei in the ventral midbrain and brainstem. Staining is primarily in the cell soma but is also prominent in the dendritic field of a discrete subset of neurons including the mitral cells of the olfactory bulb and the distal dendritic branches of the cerebellar Purkinje cells. Our observations indicate that the 245-255 kDa alpha 1E subunit is localized in cell bodies, and in some cases in dendrites, of a broad range of central neurons and is potentially modulated by multiple second messenger-activated protein kinase.

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.