The absence of endogenous beta-endorphin selectively blocks phosphorylation and desensitization of mu opioid receptors following partial sciatic nerve ligation.

Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the beta-endorphin peptides derived from the proopiomelanocortin (Pomc) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid beta-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.

Decrease in density of INa is in the common final pathway to heart block in murine hearts overexpressing calcineurin.

Overexpression of calcineurin in transgenic mouse heart results in massive cardiac hypertrophy followed by sudden death. Sudden deaths are caused by abrupt transitions from sinus rhythm to heart block (asystole) in calcineurin-overexpressing (CN) mice. Preliminary studies showed decreased maximum change in potential over time (dV/dt(max)) of phase 0 of the action potential. Accordingly, the hypothesis was tested that decreased activity of the sodium channel contributes to heart block. Profound decreases in activity of sodium currents (I(Na)) paralleled the changes in action potential characteristics. Progressive age-dependent decreases were observed such that at 42-50 days of life little sodium channel function existed. However, this was not paralleled by decreased protein expression as assessed by immunocytochemistry or by Western blot. Since calcineurin can interact with the ryanodine receptor, we assessed whether chronic in vitro treatment with BAPTA-AM, thapsigargin, and ryanodine could rescue the decrease of I(Na). All of these treatments rescued I(Na) to levels indistinguishable from wild type. The nonspecific PKC inhibitor bisindolylmaleimide I also rescued the decrease of I(Na). To assess whether decreased sodium channel activity contributes to sudden death in vivo, the response to encainide (20 mg/kg) was assessed: 6 of 10 young CN mice died because of asystole, whereas 0 of 10 wild-type mice died (P < 0.01). Moreover, encainide produced exaggerated prolongation of the QRS width in sinus beats before the heart block. Catecholamine tone appears necessary to support life in older CN mice because propranolol (1 mg/kg) triggered asystolic death in five of six CN mice. We conclude that decrease in sodium channel activity is in the common final pathway to asystole in CN mice.

Altered localization of Cav1.2 (L-type) calcium channels in nerve fibers, Schwann cells, odontoblasts, and fibroblasts of tooth pulp after tooth injury.

We have determined the localization of Cav1.2 (L-Type) Ca2+ channels in the cells and nerve fibers in molars of normal or injured rats. We observed high levels of immunostaining of L-type Ca2+ channels in odontoblast cell bodies and their processes, in fibroblast cell bodies and in Schwann cells. Many Cav1.2-containing unmyelinated and myelinated axons were also present in root nerves and proximal branches in coronal pulp, but were usually missing from nerve fibers in dentin. Labeling in the larger fibers was present along the axonal membrane, localized in axonal vesicles, and in nodal regions. After focal tooth injury, there is a marked loss of Cav1.2 channels in injured teeth. Immunostaining of Cav1.2 channels was lost selectively in nerve fibers and local cells of the tooth pulp within 10 min of the lesion, without loss of other Cav channel or pulpal labels. By 60 min, Cav1.2 channels in odontoblasts were detected again but at levels below controls, whereas fibroblasts were labeled well above control levels, similar to upregulation of Cav1.2 channels in astrocytes after injury. By 3 days after the injury, Cav1.2 channels were again detected in nerve fibers and immunostaining of fibroblasts and odontoblasts had returned to control levels. These findings provide new insight into the localization of Cav1.2 channels in dental pulp and sensory fibers, and demonstrate unexpected plasticity of channel distribution in response to nerve injury.

Sodium channel beta1 and beta3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain.

Sequence homology predicts that the extracellular domain of the sodium channel beta1 subunit forms an immunoglobulin (Ig) fold and functions as a cell adhesion molecule. We show here that beta1 subunits associate with neurofascin, a neuronal cell adhesion molecule that plays a key role in the assembly of nodes of Ranvier. The first Ig-like domain and second fibronectin type III-like domain of neurofascin mediate the interaction with the extracellular Ig-like domain of beta1, confirming the proposed function of this domain as a cell adhesion molecule. beta1 subunits localize to nodes of Ranvier with neurofascin in sciatic nerve axons, and beta1 and neurofascin are associated as early as postnatal day 5, during the period that nodes of Ranvier are forming. This association of beta1 subunit extracellular domains with neurofascin in developing axons may facilitate recruitment and concentration of sodium channel complexes at nodes of Ranvier.

Axonal L-type Ca2+ channels and anoxic injury in rat CNS white matter.

We studied the magnitude and route(s) of Ca2+ flux from extra- to intracellular compartments during anoxia in adult rat optic nerve (RON), a central white matter tract, using Ca2+ sensitive microelectrodes to monitor extracellular [Ca2+] ([Ca2+]o). One hour of anoxia caused a rapid loss of the stimulus-evoked compound action potential (CAP), which partially recovered following re-oxygenation, indicating that irreversible injury had occurred. After an initial increase caused by extracellular space shrinkage, anoxia produced a sustained decrease of 0.42 mM (29%) in [Ca2+]o. We quantified the [Ca2+]o decrease as the area below baseline [Ca2+]o during anoxia and used this as a qualitative index of suspected Ca2+ influx. The degree of RON injury was predicted by the amount of Ca2+ leaving the extracellular space. Bepridil, 0 Na+ artificial cerebrospinal fluid or tetrodotoxin reduced suspected Ca2+ influx during anoxia implicating reversal of the Na+/Ca2+ exchanger as a route of Ca2+ influx. Diltiazem reduced suspected Ca2+ influx during anoxia, suggesting that Ca2+ influx via L-type Ca2+ channels is a route of toxic Ca2+ influx into axons during anoxia. Immunocytochemical staining was used to demonstrate and localize high-threshold Ca2+ channels. Only alpha1(C) and alpha1(D) subunits were detected, indicating that only L-type Ca2+ channels were present. Double labeling with anti-neurofilament antibodies or anti-glial fibrillary acidic protein antibodies localized L-type Ca2+ channels to axons and astrocytes.

CaV2.2 and CaV2.3 (N- and R-type) Ca2+ channels in depolarization-evoked entry of Ca2+ into mouse sperm.

As sperm prepare for fertilization, surface Ca(2+) channels must open to initiate required, Ca(2+)-mediated events. However, the molecular identity and functional properties of sperm Ca(2+) channels remain uncertain. Here, we use rapid local perfusion and single-cell photometry to examine the kinetics of calcium responses of mouse sperm to depolarizing stimuli. The linear rise of intracellular [Ca(2+)] evoked by approximately 10-s applications of an alkaline high [K(+)] medium directly reports activity of voltage-gated Ca(2+) channels. Little response occurs if external Ca(2+) is removed or if external or internal pH is elevated without depolarization. Responses are inhibited 30-40% by 30-100 micrometer Ni(2+) and more completely by 100-300 micrometer Cd(2+). They resist the dihydropyridines nitrendipine and PN200-110, but 1-10 micrometer mibefradil inhibits reversibly. They also resist the venom toxins calciseptine, omega-conotoxin MVIIC, and kurtoxin, but omega-conotoxin GVIA (5 micrometer) inhibits approximately 50%. GVIA also partially blocks transient, low voltage activated Ca(2+) currents of patch-clamped spermatids. Differential sensitivity of sperm responses to Ni(2+) and Cd(2+) and partial blockade by GVIA indicate that depolarization opens at least two types of voltage-gated Ca(2+) channels in epididymal sperm examined prior to capacitation. Involvement of a previously undetected Ca(V)2.2 (N-type) channel, suggested by the action of GVIA, is substantiated by immunodetection of Ca(2+) channel alpha(1B) subunits in sperm and sperm extracts. Resistance to dihydropyridines, calciseptine, MVIIC, and kurtoxin indicates that Ca(V)1, Ca(V)2.1, and Ca(V)3 (L-, P/Q-, and T-type) channels contribute little to this evoked response. Partial sensitivity to 1 micrometer mibefradil and an enhanced sensitivity of the GVIA-resistant component of response to Ni(2+) suggest participation of a Ca(V)2.3 (R-type) channel specified by previously found alpha(1E) subunits. Our examination of depolarization-evoked Ca(2+) entry indicates that mature sperm possess a larger palette of voltage-gated Ca(2+) channels than previously thought. Such diversity may permit specific responses to multiple cues encountered on the path to fertilization.

Dopaminergic modulation of voltage-gated Na+ current in rat hippocampal neurons requires anchoring of cAMP-dependent protein kinase.

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.

Up-regulation of class A Ca2+ channels in trigeminal ganglion after pulp exposure.

We have studied changes in the level of calcium channel expression in the cell bodies of neurons located in the maxillary division of the trigeminal ganglion following induction of persistent pulpitis by pulp exposure in the right maxillary molars. Using anti-peptide antibodies to the alpha1 subunit of class A (P-/Q-type) voltage-gated calcium channels, we observed slight increases in the expression level three days following surgery and approximately 4 fold increase by eight days following the lesion. These changes in the expression of the alpha1 subunit of class A calcium channels may have functional implications in the responses of nociceptive neurons to chronic inflammation.

Discrete regional distributions suggest diverse functional roles of calcium channel alpha1 subunits in sperm.

The Ca channels of male germ-line cells are partially characterized, but the molecular properties and subcellular localization of the Ca channels of mature sperm are unknown. Here, we probe rodent sperm with anti-peptide antibodies directed to cytosolic domains of cloned rat brain alpha1A, alpha1C, and alpha1E Ca channel subunits. Each recognizes a 200- to 245-kDa band on immunoblots of whole rat sperm extracts. A smaller ( approximately 110-kDa) alpha1C band also is detected. Confocal fluorescence images of mouse sperm show characteristic patterns of punctate alpha1A-, alpha1C-, and alpha1E-immunoreactivity. For alpha1A, the puncta are larger, less numerous, and more variable in distribution than for alpha1C and alpha1E. They are absent from the acrosomal crescent, but are present elsewhere over the sperm head, often at the apical tip and equatorial segment. They also are found at irregular intervals along both the midpiece and the principal piece of the flagellum. For alpha1C and alpha1E, puncta are dense along dorsal and ventral aspects of the acrosomal cap. For alpha1E but not alpha1C, the remainder of the acrosomal region also is labeled. Neither is found in the postacrosomal region or on the midpiece. Puncta of alpha1C and alpha1E occur at regular intervals each in two parallel rows, at the dorsal and ventral aspects of the proximal segment of the flagellar principal piece. The puncta in these arrays become less abundant and intense in the distal flagellum. These results demonstrate that multiple Ca channel proteins are present in mature sperm and are regionally localized in ways that may give them different regulatory roles.

Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses.

We studied how Ca2+ influx through different subtypes of Ca2+ channels couples to release at a calyx-type terminal in the rat medial nucleus of the trapezoid body by simultaneously measuring the presynaptic Ca2+ influx evoked by a single action potential and the EPSC. Application of subtype-specific toxins showed that Ca2+ channels of the P/Q-, N-, and R-type controlled glutamate release at a single terminal. The Ca2+ influx through the P/Q-type channels triggered release more effectively than Ca2+ influx through N- or R-type channels. We investigated mechanisms that contributed to these differences in effectiveness. Electrophysiological experiments suggested that individual release sites were controlled by all three subtypes of Ca2+ channels. Immunocytochemical staining indicated, however, that a substantial fraction of N- and R-type channels was located distant from release sites. Although these distant channels contributed to the Ca2+ influx into the terminal, they may not contribute to release. Taken together, the results suggest that the Ca2+ influx into the calyx via N- and R-type channels triggers release less effectively than that via P/Q-type because a substantial fraction of the N- and R-type channels in the calyx is localized distant from release sites.

Physical link and functional coupling of presynaptic calcium channels and the synaptic vesicle docking/fusion machinery.

N- and P/Q-type calcium channels are localized in high density in presynaptic nerve terminals and are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. As outlined in the preceding article, these calcium channels can be purified from brain as a complex with SNARE proteins which are involved in exocytosis. In addition, N-type and P/Q-type calcium channels are co-localized with syntaxin in high-density clusters in nerve terminals. Here we review the role of the synaptic protein interaction (synprint) sites in the intracellular loop II-III (L(II-III)) of both alpha1B and alpha1A subunits of N-type and P/Q-type calcium channels, which bind to syntaxin, SNAP-25, and synaptotagmin. Calcium has a biphasic effect on the interactions of N-type calcium channels with SNARE complexes, stimulating optimal binding in the range of 10-20 microM. PKC or CaM KII phosphorylation of the N-type synprint peptide inhibits interactions with native brain SNARE complexes containing syntaxin and SNAP-25. Introduction of the synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibits EPSPs from synchronous transmitter release by 42%. At physiological Ca2+ concentrations, synprint peptides cause an approximate 25% reduction in transmitter release of injected frog neuromuscular junction in cultures, consistent with detachment of 70% of the docked vesicles from calcium channels based on a theoretical model. Together, these studies suggest that presynaptic calcium channels not only provide the calcium signal required by the exocytotic machinery, but also contain structural elements that are integral to vesicle docking, priming, and fusion processes.

Localization of Ca2+ channel subtypes on rat spinal motor neurons, interneurons, and nerve terminals.

Ca2+ channels in distinct subcellular compartments of neurons mediate voltage-dependent Ca2+ influx, which integrates synaptic responses, regulates gene expression, and initiates synaptic transmission. Antibodies that specifically recognize the alpha1 subunits of class A, B, C, D, and E Ca2+ channels have been used to investigate the localization of these voltage-gated ion channels on spinal motor neurons, interneurons, and nerve terminals of the adult rat. Class A P/Q-type Ca2+ channels were present mainly in a punctate pattern in nerve terminals located along the cell bodies and dendrites of motor neurons. Both smooth and punctate staining patterns were observed over the surface of the cell bodies and dendrites with antibodies to class B N-type Ca2+ channels, indicating the presence of these channels in the cell surface membrane and in nerve terminals. Class C and D L-type and class E R-type Ca2+ channels were distributed mainly over the cell soma and proximal dendrites. Class A P/Q-type Ca2+ channels were present predominantly in the presynaptic terminals of motor neurons at the neuromuscular junction. Occasional nerve terminals innervating skeletal muscles from the hindlimb were labeled with antibodies against class B N-type Ca2+ channels. Staining of the dorsal laminae of the rat spinal cord revealed a complementary distribution of class A and class B Ca2+ channels in nerve terminals in the deeper versus the superficial laminae. Many of the nerve terminals immunoreactive for class B N-type Ca2+ channels also contained substance P, an important neuropeptide in pain pathways, suggesting that N-type Ca2+ channels are predominant at synapses that carry nociceptive information into the spinal cord.

Primary structure and function of an A kinase anchoring protein associated with calcium channels.

Rapid, voltage-dependent potentiation of skeletal muscle L-type calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A kinase anchoring protein (AKAP). Here we report the isolation, primary sequence determination, and functional characterization of AKAP15, a lipid-anchored protein of 81 amino acid residues with a single amphipathic helix that binds PKA. AKAP15 colocalizes with L-type calcium channels in transverse tubules and is associated with L-type calcium channels in transfected cells. A peptide fragment of AKAP15 encompassing the RII-binding domain blocks voltage-dependent potentiation. These results indicate that AKAP15 targets PKA to the calcium channel and plays a critical role in voltage-dependent potentiation and regulation of skeletal muscle contraction. The expression of AKAP15 in the brain and heart suggests that it may mediate rapid PKA regulation of L-type calcium channels in neurons and cardiac myocytes.

Upregulation of L-type Ca2+ channels in reactive astrocytes after brain injury, hypomyelination, and ischemia.

Anti-peptide antibodies that specifically recognize the alpha1 subunit of class A-D voltage-gated Ca2+ channels and a monoclonal antibody (MANC-1) to the alpha2 subunit of L-type Ca2+ channels were used to investigate the distribution of these Ca2+ channel subtypes in neurons and glia in models of brain injury, including kainic acid-induced epilepsy in the hippocampus, mechanical and thermal lesions in the forebrain, hypomyelination in white matter, and ischemia. Immunostaining of the alpha2 subunit of L-type Ca2+ channels by the MANC-1 antibody was increased in reactive astrocytes in each of these forms of brain injury. The alpha1C subunits of class C L-type Ca2+ channels were upregulated in reactive astrocytes located in the affected regions in each of these models of brain injury, although staining for the alpha1 subunits of class D L-type, class A P/Q-type, and class B N-type Ca2+ channels did not change from patterns normally observed in control animals. In all of these models of brain injury, there was no apparent redistribution or upregulation of the voltage-gated Ca2+ channels in neurons. The upregulation of L-type Ca2+ channels in reactive astrocytes may contribute to the maintenance of ionic homeostasis in injured brain regions, enhance the release of neurotrophic agents to promote neuronal survival and differentiation, and/or enhance signaling in astrocytic networks in response to injury.

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase.

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIalpha subunit would reveal those tissues and signaling events that require anchored PKA. RIIalpha knockout mice appear normal and healthy. In adult skeletal muscle, RIalpha protein levels increased to partially compensate for the loss of RIIalpha. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIalpha knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA-AKAP interactions. The C subunit colocalized with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIalpha subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIalpha subunit. The potentiation of the L-type Ca2+ channel in RIIalpha knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIalpha is capable of physiologically relevant anchoring interactions.

Biochemical properties and subcellular distribution of the BI and rbA isoforms of alpha 1A subunits of brain calcium channels.

Biochemical properties and subcellular distribution of the class A calcium channel alpha 1 subunits (alpha 1A) from rat and rabbit brain were examined using site-directed anti-peptide antibodies specific for rat rbA (anti-CNA3) and for rabbit BI (anti-NBI-1 and anti-NBI-2) isoforms of alpha 1A. In immunoblotting experiments, anti-CNA3 specifically identifies multiple alpha 1A polypeptides with apparent molecular masses of 210, 190, and 160 kD, and anti-NBI-1 and anti-NBI-2 specifically recognize 190-kD alpha 1A polypeptides in rat brain membrane. In rabbit brain, anti-NBI-1 or anti-NBI-2 specifically detect alpha 1A polypeptides with apparent molecular masses of 220, 200, and 190 kD, while anti-CNA3 specifically recognizes 190-kD alpha 1A polypeptides. These polypeptides evidently represent multiple isoforms of alpha 1A present in both rat and rabbit brain. Anti-CNA3 specifically immunoprecipitates high affinity receptor sites for omega-conotoxin MVIIC (Kd approximately 100 pM), whereas anti-NBI-2 immunoprecipitates two distinct affinity receptor sites for omega-conotoxin MVIIC (Kd approximately 100 pM and approximately 1 microM). Coimmunoprecipitation experiments indicate that alpha 1A subunits recognized by anti-CNA3 and anti-NBI-2 are associated with syntaxin in a stable, SDS-resistant complex and with synaptotagmin. Immunofluorescence studies reveal that calcium channels recognized by anti-NBI-2 are localized predominantly in dendrites and nerve terminals forming synapses on them, while calcium channels recognized by anti-CNA3 are localized more prominently in cell bodies and in nerve terminals. The mossy fiber terminals in hippocampus and the terminals of climbing and parallel fibers in cerebellum are differentially stained by these isoform-specific antibodies. These results indicate that both rbA and BI isoforms of alpha 1A are expressed in rat and rabbit brain and form calcium channels having alpha 1A subunits with distinct molecular mass, pharmacology, and subcellular localization.

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.

Effects of expression of a mouse brain L-type calcium channel alpha 1 subunit on secretion from bovine adrenal chromaffin cells.

Regulated exocytosis from bovine chromaffin cells is stimulated by the influx of Ca2+ through plasma membrane ion channels that are opened by nicotinic stimulation and/or depolarization. Recently, we developed a novel method that enabled us to investigate the function of a cloned Ca2+ channel type C alpha 1 subunit in forming channels that stimulate exocytosis. In the present study, we demonstrate by immunocytochemistry that bovine chromaffin cells normally express an epitope specific for the type C alpha 1 subunit. We investigated the effects of expression of additional class C alpha 1 subunits (mouse brain clone) on various aspects of secretory function in bovine chromaffin cells by measuring secretion of cotransfected human growth hormone (GH, a reporter for the regulated secretory pathway in the transfected cells). New channels were activated in response to depolarization by both elevated K+ and nicotinic cholinergic agonist. The new channels had their greatest effects when secretion was stimulated suboptimally. Secretion was enhanced only after the first 30 sec of stimulation, and the enhancement extended beyond 5 min of continuous stimulation. In contrast to the endogenous L-type Ca2+ channels, the latency was not decreased by the dihydropyridine L-type Ca2+ channel agonist, Bay K 8644. The findings suggest that (i) the Ca(2+)-sensitive mechanism for triggering or maintaining exocytosis is capable of being saturated by high levels of Ca2+, (ii) secretion caused by nicotinic agonist stimulation can be significantly enhanced by activation of voltage-sensitive Ca2+ channels, and (iii) the effects on secretion of the L-type Ca2+ channels formed on expression of the mouse brain class C alpha 1 subunit are distinct from those of endogenous L-type Ca2+ channels.

Type II brain sodium channel expression in non-neuronal cells: embryonic rat osteoblasts.

Although voltage-sensitive sodium channels play a central role in electrogenesis in neurons, rat brain sodium channels are also present in some glial cells. To determine whether rat brain sodium channel alpha-subunit isotypes are expressed in other cell types, we examined osteoblasts within the embryonic day 17 (E17) vertebral column with in situ hybridization and immunocytochemical methods. For in situ hybridization studies, riboprobes hybridizing to isoform-specific sequences in the 3'-noncoding region of sodium channel mRNAs (NCI, NCII and NCIII) were utilized. Sodium channel mRNA I and III were not detectable in osteoblasts of the vertebra centrum or neural arches in E17 rats. In contrast, sodium channel mRNA II was moderately expressed by osteoblasts in the developing vertebral column of E17 rats. In immunocytochemical experiments, antipeptide antibodies directed against conserved and isotype-specific regions of the sodium channel alpha-subunit were used. Antibody SP20, which recognizes a conserved region of the sodium channel, intensely stains osteoblasts in both the vertebra centrum and neural arches. Antibody SP11-I, which recognizes sodium channel I, exhibited negligible-to-low levels of immunostaining in vertebral column osteoblasts. Osteoblasts reacted with antibody SP11-II, which recognizes sodium channel II, displayed moderate levels of immunostaining. Antibody SP32-III, which recognizes sodium channel III, displayed negligible levels of staining in osteoblasts within vertebra centrum and neural arches. These results demonstrate that osteoblasts in situ within E17 vertebral columns express sodium channel II mRNA and protein. Together with previous electrophysiological observations, the present results suggest that functional sodium channels are expressed in osteoblasts in vivo. These results extend the range of non-neuronal cells known to express rat brain sodium channels.

Structure and function of the beta 2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM motif.

Voltage-gated sodium channels in brain neurons are complexes of a pore-forming alpha subunit with smaller beta 1 and beta 2 subunits. cDNA cloning and sequencing showed that the beta 2 subunit is a 186 residue glycoprotein with an extracellular NH2-terminal domain containing an immunoglobulin-like fold with similarity to the neural cell adhesion molecule (CAM) contactin, a single transmembrane segment, and a small intracellular domain. Coexpression of beta 2 with alpha subunits in Xenopus oocytes increases functional expression, modulates gating, and causes up to a 4-fold increase in the capacitance of the oocyte, which results from an increase in the surface area of the plasma membrane microvilli. beta 2 subunits are unique among the auxiliary subunits of ion channels in combining channel modulation with a CAM motif and the ability to expand the cell membrane surface area. They may be important regulators of sodium channel expression and localization in neurons.