ABSTRACT
Synthetic peptides of defined amino acid sequence are commonly used as unique antigens for production of antibodies to more complex target proteins. We previously showed that an affinity-purified, site-directed polyclonal antibody (CW90) raised against a peptide antigen (CNGRMPNIAKDVFTKM) anticipated to be specific to a T-type voltage-dependent Ca(2+) channel subunit identified recombinant rat alpha1I/Ca(V)3.3 and two endogenous mouse proteins distinct in their developmental expression and apparent molecular mass (neonatal form 260 kDa, mature form 190 kDa) [Yunker AM, Sharp AH, Sundarraj S, Ranganathan V, Copeland TD, McEnery MW (2003) Immunological characterization of T-type voltage-dependent calcium channel Ca(V)3.1 (alpha 1G) and Ca(V)3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development. Neuroscience 117:321-335]. In the present study, we further characterize the biochemical properties of the CW90 antigens. We show for the first time that recombinant alpha1I/Ca(V)3.3 is modified by N-glycosylation. Using peptide:N-glycosidase F (PNGase F), an enzyme that removes polysaccharides attached at Asn residues, and endoneuraminidase-N (Endo-N), which specifically removes polysialic acid modifications, we reveal that differential glycosylation fully accounts for the large difference in apparent molecular mass between neonatal and adult CW90 antigens and that the neonatal form is polysialylated. As very few proteins are substrates for Endo-N, we carried out extensive analyses and herein present evidence that CW90 reacts with recombinant alpha1I/Ca(V)3.3 as well as endogenous neural cell adhesion molecule-180 (NCAM-180). We demonstrate the basis for CW90 cross-reactivity is a five amino acid epitope (AKDVF) present in both alpha1I/Ca(V)3.3 and NCAM-180. To extend these findings, we introduce a novel polyclonal anti-peptide antibody (CW678) that uniquely recognizes NCAM-180 and a new antibody (CW109) against alpha1I/Ca(V)3.3. Western blot analyses obtained with CW678, CW109 and CW90 on a variety of samples confirm that the endogenous CW90 signals are fully attributed to the two developmental forms of NCAM-180. Using CW678, we present novel data on differentiation-dependent NCAM-180 expression in human neuroblastoma IMR32 cells. These results strongly suggest the need for careful analyses to validate anti-peptide antibodies when targeting membrane proteins of low abundance.
Subject(s)
Antibodies/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/physiology , Membrane Transport Proteins/physiology , Amino Acid Sequence , Animals , Animals, Newborn , Brain , Calcium Channels, T-Type/drug effects , Cell Line , Cross Reactions , Epitopes , Humans , Kidney , Membrane Transport Proteins/drug effects , Mice , Molecular Weight , Peptide Fragments/immunology , Wheat Germ AgglutininsABSTRACT
Spinal nucleus of bulbocavernosus and its target musculature, the bulbocavernosus and levator ani muscles, are sexually dimorphic, and their sexual differentiation depends on plasmatic levels of testosterone. Electrophysiological and immunocytochemical studies have demonstrated that at mammalian adult neuromuscular junctions only P/Q-type Ca2+ channels (Ca(v2.1)), mediate evoked transmitter release. Here we report that N-type Ca2+ channel (Ca(v2.2)) blocker omega-Conotoxin GVIA, as well as Ca(v2.1) blocker omega-Agatoxin IVA, significantly reduced quantal content of transmitter release by approximately 80% and approximately 70% respectively at levator ani muscle of the adult rats, indicating that neuromuscular transmission is jointly mediated by both types of channels. In these synapses, we also observed that castration and restitution of plasmatic testosterone in rats resulted in changes in the sensitivity to omega-Conotoxin GVIA. Castration induced, whereas testosterone treatment avoided, functional loss of Ca(v2.2), as mediators of transmitter release in these synapses. Strikingly, the expression and localization of alpha1B subunits, which form the pore of the Ca(v2.2) channel, were similar at control, gonadectomized and gonadectomized testosterone-treated rats, suggesting that testosterone may regulate the coupling mechanisms between Ca(v2.2) and transmitter release at the neuromuscular junctions of these sexually dimorphic motoneurons.
Subject(s)
Calcium Channels, L-Type/metabolism , Gene Expression Regulation/drug effects , Neuromuscular Junction/drug effects , Testosterone/pharmacology , Animals , Animals, Newborn , Calcium Channel Blockers/pharmacology , Calcium Channels, N-Type , Diaphragm/cytology , Diaphragm/drug effects , Drug Interactions , Evoked Potentials/drug effects , Evoked Potentials/physiology , Evoked Potentials/radiation effects , Immunohistochemistry/methods , Male , Orchiectomy/methods , Pelvic Floor , Radioimmunoassay/methods , Rats , Rats, Sprague-Dawley , Receptors, Cholinergic/metabolism , omega-Agatoxin IVA/pharmacology , omega-Conotoxin GVIA/pharmacologyABSTRACT
In the WAG/Rij rat, a model for human absence epilepsy, spike-wave discharges (SWD) and absence epileptic behavior develop after the age of 3 months. The rostral part of the reticular thalamic nucleus (rRTN) is involved in SWD. Ca(2+) channels play a central role in the initiation and maintenance of burst firing activity of thalamic cells. We hypothesize that a changed expression of alpha(1)-subunits of one or more high voltage-activated Ca(2+) channel types in the rRTN underlies the development of SWD. To test this hypothesis we compared 3- and 6-month-old WAG/Rij rats with nonepileptic, age-matched control rats. By immunocytochemistry, the expressions of alpha(1)1.3-, alpha(1)2.1-, alpha(1)2.2-, and alpha(1)2.3-subunits were shown in both strains, demonstrating the presence of Ca(v)1.3, Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 channels, respectively. Quantification of channel expression indicates that the development of SWD in WAG/Rij rats is concomitant with an increased expression of Ca(v)2.1 channels in the rRTN. These channels are mainly presynaptic, as revealed by double immunofluorescence involving the presynapse marker syntaxin. The mechanism by which this increase could be related to the occurrence of SWD has been discussed.
Subject(s)
Calcium Channels/biosynthesis , Epilepsy, Absence/metabolism , Gene Expression Regulation/physiology , Thalamic Nuclei/metabolism , Animals , Calcium Channels/genetics , Epilepsy, Absence/genetics , Male , Rats , Rats, Inbred ACIABSTRACT
Low voltage-activated calcium channels (LVAs; "T-type") modulate normal neuronal electrophysiological properties such as neuronal pacemaker activity and rebound burst firing, and may be important anti-epileptic targets. Proteomic analyses of available alpha 1G/Ca(V)3.1 and alpha 1I/Ca(V)3.3 sequences suggest numerous potential isoforms, with specific alpha 1G/Ca(V)3.1 or alpha 1I/Ca(V)3.3 domains postulated to be conserved among isoforms of each T-type channel subtype. This information was used to generate affinity-purified anti-peptide antibodies against sequences unique to alpha 1G/Ca(V)3.1 or alpha 1I/Ca(V)3.3, and these antibodies were used to compare and contrast alpha 1G/Ca(V)3.1 and alpha 1I/Ca(V)3.3 protein expression by western blotting and immunohistochemistry. Each antibody reacted with appropriately sized recombinant protein in HEK-293 cells. Regional and developmental differences in alpha 1G/Ca(V)3.1 and alpha 1I/Ca(V)3.3 protein expression were observed when the antibodies were used to probe regional brain dissections prepared from perinatal mice and adult rodents and humans. Mouse forebrain alpha 1G/Ca(V)3.1 (approximately 240 kDa) was smaller than cerebellar (approximately 260 kDa) alpha 1G/Ca(V)3.1, and expression of both proteins increased during perinatal development. In contrast, mouse midbrain and diencephalic tissues evidenced an alpha 1I/Ca(V)3.3 immunoreactive doublet (approximately 230 kDa and approximately 190 kDa), whereas other brain regions only expressed the small alpha 1I/Ca(V)3.3 isoform. A unique large alpha 1I/Ca(V)3.3 isoform (approximately 260 kDa) was expressed at birth and eventually decreased, concomitant with the appearance and gradual increase of the small alpha 1I/Ca(V)3.3 isoform. Immunohistochemistry supported the conclusion that LVAs are expressed in a regional manner, as cerebellum strongly expressed alpha 1G/Ca(V)3.1, and olfactory bulb and midbrain contained robust alpha 1I/Ca(V)3.3 immunoreactivity. Finally, strong alpha 1I/Ca(V)3.3, but not alpha 1G/Ca(V)3.1, immunoreactivity was observed in brain and spinal cord by embryonic day 14 in situ. Taken together, these data provide an anatomical and biochemical basis for interpreting LVA heterogeneity and offer evidence of developmental regulation of LVA isoform expression.
Subject(s)
Calcium Channels, T-Type/biosynthesis , Calcium Channels, T-Type/immunology , Animals , Brain/immunology , Brain/metabolism , Female , Gene Expression Regulation, Developmental/physiology , Humans , Membrane Transport Proteins , Mice , Pregnancy , Protein Isoforms/biosynthesis , Protein Isoforms/immunology , RatsABSTRACT
Phenotypes of several heritable disorders including forms of hearing loss, myelin diseases, hypomagnesemia, and cataracts are linked to missense mutations in single alleles encoding membrane proteins having four transmembrane spans. In some cases, the mutant proteins exhibit dominant negative or gain-of-function behavior whereby heterozygous coexpression of mutant and wild-type genes leads to more serious pathology than is the case for individuals in which only a single wild-type allele is expressed. An example is found in the relationship of peripheral myelin protein 22 (PMP22) to Charcot-Marie-Tooth disease (CMTD) type 1A. A number of disease-linked PMP22 mutants fail to undergo normal trafficking beyond the endoplasmic reticulum or intermediate compartment to reach the cell surface. Moreover, recent evidence suggests that pathology resulting from this mistrafficking-based loss of function may also be augmented by the ability of some mutants to disrupt normal trafficking of the product of the wild-type PMP22 allele. The basis for this phenomenon appears to be the heterodimerization of trafficking-incompetent mutants with wild-type PMP22, such that both the wild-type protein and the mutant forms are retained early in the secretory pathway. The full cellular and structural biological details of these observations remain to be elucidated. However, the model suggested by the existing data regarding the relationship of PMP22 to CMTD may be useful to explain phenotypes of several other diseases involving other tetraspan membrane proteins and to facilitate predictions regarding previously undetected disease-protein linkages.
Subject(s)
Charcot-Marie-Tooth Disease/genetics , Myelin Proteins/genetics , Myelin Proteins/metabolism , Protein Structure, Tertiary , Protein Transport/physiology , Amino Acid Sequence , Charcot-Marie-Tooth Disease/metabolism , Humans , Molecular Sequence Data , Mutation , Myelin Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Sequence AlignmentABSTRACT
T-type Ca(2+) currents have been proposed to be involved in the genesis of spike-and-wave discharges, a sign of absence seizures, but direct evidence in vivo to support this hypothesis has been lacking. To address this question, we generated a null mutation of the alpha(1G) subunit of T-type Ca(2+) channels. The thalamocortical relay neurons of the alpha(1G)-deficient mice lacked the burst mode firing of action potentials, whereas they showed the normal pattern of tonic mode firing. The alpha(1G)-deficient thalamus was specifically resistant to the generation of spike-and-wave discharges in response to GABA(B) receptor activation. Thus, the modulation of the intrinsic firing pattern mediated by alpha(1G) T-type Ca(2+) channels plays a critical role in the genesis of absence seizures in the thalamocortical pathway.
Subject(s)
Calcium Channels, T-Type/physiology , Cerebral Cortex/physiology , Epilepsy, Absence/physiopathology , Neurons/physiology , Receptors, GABA-B/physiology , Seizures/physiopathology , Thalamus/physiology , 4-Butyrolactone/pharmacology , Animals , Baclofen/pharmacology , Calcium Channels, T-Type/deficiency , Calcium Channels, T-Type/genetics , Cerebral Cortex/physiopathology , Electroencephalography , Epilepsy, Absence/genetics , Immunity, Innate/genetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/drug effects , Protein Subunits , Seizures/genetics , Thalamic Nuclei/physiology , Thalamic Nuclei/physiopathology , Thalamus/physiopathologyABSTRACT
Inherited forms of ataxia and absence seizures in mice have been linked to defects in voltage-dependent calcium channel subunits. However, a correlation between the sites of neuronal dysfunction and the impact of the primary lesion upon calcium channel subunit expression or function has not been clearly established. For example, the mutation in stargazer mice has pleiotropic consequences including synaptic alterations in cerebellar granule cells, hippocampal CA3/mossy fibers, and cortical neurons in layer V that, presumably, lead to ataxia and seizures. Genetic analysis of stargazer mice determined that the defective gene encodes a protein expressed in brain (gamma2) with limited homology to the skeletal muscle L-type calcium channel gamma1 subunit. Although additional gamma isoforms have been subsequently identified primarily in neural tissue, little was known about the proteins they encode. Therefore, this study explored the distribution and biochemical properties of gamma2 and other gamma isoforms in wild-type and stargazer brain. We cloned human gamma2, gamma3, and gamma4 isoforms, produced specific anti-peptide antibodies to gamma isoforms and characterized both heterologously expressed and endogenous gamma. We identified regional specificity in the expression of gamma isoforms by western analysis and immunohistochemistry. We report for the first time that the mutation in the stargazer gene resulted in the loss of gamma2 protein. Furthermore, no compensatory changes in the expression of gamma3 or gamma4 protein were evident in stargazer brain. In contrast to other voltage-dependent calcium channel subunits, gamma immunostaining was striking in that it was primarily detected in regions highly enriched in excitatory glutamatergic synapses and faintly detected in cell bodies, suggesting a role for gamma in synaptic functions. Sites of known synaptic dysfunction in stargazer (the hippocampal CA3 region, dentate gyrus, and cerebellar molecular layer) were revealed as relying primarily upon gamma2, as total gamma isoform expression was dramatically decreased in these regions. Electron microscopy localized anti-gamma antibody immunostaining to dendritic structures of hippocampal mossy fiber synapses, with enrichment at postsynaptic densities. To assess the association of native gamma with voltage-dependent calcium channel or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits, gamma isoforms (gamma2, gamma3 and gamma4) were detergent solubilized from mouse forebrain. Antibodies against a highly conserved C-terminal epitope present in gamma2, gamma3 and gamma4 immunoprecipitated voltage-dependent calcium channel subunits (alpha1B), providing the first in vivo evidence that gamma and voltage-dependent calcium channels form stable complexes. Furthermore, both anti-gamma2 antibodies and anti-alpha1B antibodies independently immunoprecipitated the AMPA receptor subunit, GluR1, from mouse forebrain homogenates. In summary, loss of gamma2 immunoreactivity in stargazer is precisely localized so as to contribute to previously characterized synaptic defects. The data in this paper provide compelling evidence that gamma isoforms form complexes in vivo with voltage-dependent calcium channels as well as AMPA receptors, are selectively and differentially expressed in neuronal processes, and localize primarily to dendritic structures in the hippocampal mossy fiber region.
Subject(s)
Ataxia/metabolism , Brain/metabolism , Calcium Channels, L-Type/genetics , Epilepsy/metabolism , Mice, Neurologic Mutants/metabolism , Synapses/metabolism , Animals , Antibody Specificity , Ataxia/genetics , Ataxia/physiopathology , Brain/physiopathology , Brain/ultrastructure , Calcium Channels, L-Type/metabolism , Calcium Channels, N-Type/genetics , Calcium Channels, N-Type/metabolism , Calcium Signaling/genetics , Dendrites/metabolism , Dendrites/ultrastructure , Epilepsy/genetics , Epilepsy/physiopathology , Gene Expression/physiology , Hippocampus/metabolism , Hippocampus/ultrastructure , Immunohistochemistry/methods , Mice , Mice, Neurologic Mutants/abnormalities , Microscopy, Electron , Molecular Sequence Data , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Sequence Homology, Amino Acid , Synapses/ultrastructureABSTRACT
Calcium influx through N-type calcium channels mediates synaptic transmission at numerous central synapses and transduces nociceptive information in the spinal dorsal horn. However, the precise role of N-type calcium channels in pain perception is not fully elucidated. To address this issue, we generated and analyzed knockout mice for alpha(1B,) the pore-forming subunit of the N-type calcium channel. Homozygous mutants are viable, fertile, and show normal motor coordination. In small-diameter dorsal root ganglion neurons from mutants the density of calcium channel currents is significantly reduced, which can be accounted for by the abolition of N-type currents. We performed several pain-related behavioral tests using the mutant mice. alpha(1B)-Deficient mice show reduced response to mechanical stimuli in the von Frey test and increased tail flick latency in response to radiant heat, indicating altered spinal reflexes. However, pain response in the hot plate test is normal. In the formalin paw test, the mutant mice exhibit significantly attenuated response in Phase 2, but normal pain behaviors in Phase 1. The response to visceral inflammatory pain caused by acetic acid is also reduced in alpha(1B) knockout mice. These results suggest that the alpha(1B) subunit of N-type calcium channel plays a major role in pain perception by acting at the spinal level, but not at the supraspinal level.
Subject(s)
Calcium Channels, N-Type/deficiency , Ganglia, Spinal/metabolism , Mice, Knockout/metabolism , Neurons, Afferent/metabolism , Nociceptors/metabolism , Pain/metabolism , Animals , Behavior, Animal/physiology , Calcium Channel Blockers/pharmacology , Calcium Channels, N-Type/genetics , Electric Stimulation , Ganglia, Spinal/cytology , Ganglia, Spinal/growth & development , Hyperalgesia/genetics , Hyperalgesia/metabolism , Hyperalgesia/physiopathology , Inflammation/chemically induced , Inflammation/genetics , Inflammation/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Neurons, Afferent/cytology , Nociceptors/cytology , Nociceptors/growth & development , Pain/genetics , Pain/physiopathology , Pain Measurement , Pain Threshold/physiology , Physical Stimulation , Reaction Time/genetics , Spinal Cord/cytology , Spinal Cord/growth & development , Spinal Cord/metabolismSubject(s)
Calcium Channels/metabolism , Cerebellar Ataxia/physiopathology , Cerebellum/pathology , Gene Targeting , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels/genetics , Calcium Channels, N-Type , Calcium Channels, P-Type , Calcium Channels, Q-Type , Cells, Cultured , Cerebellar Ataxia/genetics , Cerebellum/cytology , Cerebellum/drug effects , Cerebellum/physiopathology , Humans , Mice , Mice, Transgenic , Nimodipine/pharmacology , Patch-Clamp Techniques , omega-Conotoxin GVIA/pharmacology , omega-Conotoxins/pharmacologyABSTRACT
Stargazin or [gamma]2, the product of the gene mutated in the stargazer mouse, is a homologue of the [gamma]1 protein, an accessory subunit of the skeletal muscle L-type Ca2+ channel. [gamma]2 is selectively expressed in the brain, and considered to be a putative neuronal Ca2+ channel subunit based mainly on homology to [gamma]1. Two new members of the [gamma] family expressed in the brain have recently been identified: [gamma]3 and [gamma]4. We have co-expressed, in Xenopus oocytes, the human [gamma]2, [gamma]3 and [gamma]4 subunits with the P/Q-type (Ca(V)2.1) Ca2+ channel and different regulatory subunits ([alpha]2-[delta]; [beta]1, [beta]2, [beta]3 or [beta]4). Subcellular distribution of the [gamma] subunits confirmed their membrane localization. Ba2+ currents, recorded using two-electrode voltage clamp, showed that the effects of the [gamma] subunits on the electrophysiological properties of the channel are, most of the time, minor. However, a fraction of the oocytes expressing [beta] subunits displayed an unusual slow-inactivating Ba2+ current. Expression of both [beta] and [gamma] subunits increased the appearance of the slow-inactivating current. Our data support a role for the [gamma] subunit as a brain Ca2+ channel modulatory subunit and suggest that [beta] and [gamma] subunits are involved in a switch between two regulatory modes of the P/Q-type channel inactivation.
Subject(s)
Calcium Channels, N-Type , Ion Channel Gating/physiology , Animals , Barium/pharmacokinetics , Calcium/metabolism , Calcium Channels, N-Type/chemistry , Calcium Channels, N-Type/genetics , Calcium Channels, N-Type/metabolism , Cell Line , Female , Gene Expression/physiology , Humans , Kidney/cytology , Kinetics , Oocytes/physiology , Patch-Clamp Techniques , Protein Structure, Tertiary , Transfection , Xenopus laevisABSTRACT
Functional cardiac L-type calcium channels are composed of the pore-forming alpha(1C) subunit and the regulatory beta(2) and alpha(2)/delta subunits. To investigate possible developmental changes in calcium channel composition, we examined the temporal expression pattern of alpha(1C) and beta(2) subunits during cardiac ontogeny in mice and rats, using sequence-specific antibodies. Fetal and neonatal hearts showed two size forms of alpha(1C) with 250 and 220 kDa. Quantitative immunoblotting revealed that the rat cardiac 250-kDa alpha(1C) subunit increased about 10-fold from fetal days 12-20 and declined during postnatal maturation, while the 220-kDa alpha(1C) decreased to undetectable levels. The expression profile of the 85-kDa beta(2) subunit was completely different: beta(2) was not detected at fetal day 12, rose in the neonatal stage, and persisted during maturation. Additional beta(2)-stained bands of 100 and 90 kDa were detected in fetal and newborn hearts, suggesting the transient expression of beta(2) subunit variants. Furthermore, two fetal proteins with beta(4) immunoreactivity were identified in rat hearts that declined during prenatal development. In the fetal rat heart, beta(4) gene expression was confirmed by RT-PCR. Cardiac and brain beta(4) mRNA shared the 3 prime region, predicting identical primary sequences between amino acid residues 62-519, diverging however, at the 5 prime portion. The data indicate differential developmental changes in the expression of Ca(2+) channel subunits and suggest a role of fetal alpha(1C) and beta isoforms in the assembly of Ca(2+) channels in immature cardiomyocytes.
Subject(s)
Calcium Channels, L-Type/metabolism , Gene Expression Regulation, Developmental/genetics , Heart/embryology , Amino Acid Sequence , Animals , Animals, Newborn , Calcium Channels, L-Type/genetics , Embryonic and Fetal Development , Immunoblotting , Mice , Molecular Sequence Data , Myocardium/metabolism , Peptide Fragments/immunology , RNA, Messenger/metabolism , Rats , Sequence Alignment , Time FactorsABSTRACT
We have identified a calcium-dependent pathway in neurons that regulates expression levels of the alpha1B subunit and N channel current. When neurons are depolarized and voltage-gated calcium channels activated, the half-life of cellular N channel alpha1B mRNA is prolonged. This stabilizing effect of depolarization is mediated through the 3' untranslated region of a long form of the alpha1B mRNA and may represent a form of modulation of N-channel levels that does not require changes in gene transcription. Increases in N channel expression would affect several key neuronal functions controlled by calcium, including transmitter release and neurite outgrowth.
Subject(s)
Calcium Channels/genetics , Calcium Channels/physiology , Gene Expression Regulation , Neurons/physiology , RNA, Messenger/metabolism , Superior Cervical Ganglion/physiology , 3' Untranslated Regions/genetics , Action Potentials/physiology , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type , Cells, Cultured , Membrane Potentials , Nerve Growth Factors/pharmacology , Neurons/drug effects , Nimodipine/pharmacology , Peptides/pharmacology , RNA, Messenger/genetics , Rats , Transcription, Genetic , omega-Conotoxin GVIAABSTRACT
Neuronal voltage-dependent calcium channels are integral components of cellular excitation and neurosecretion. In addition to mediating the entry of calcium across the plasma membrane, both N-type and P/Q-type voltage-dependent calcium channels have been shown to form stable complexes with synaptic vesicle and presynaptic membrane proteins, indicating a structural role for the voltage-dependent calcium channels in secretion. Recently, detailed structural analyses of N-type calcium channels have identified residues amino acids 718-963 as the site in the rat alpha1B subunit that mediates binding to syntaxin, synaptosome-associated protein of 25,000 mol. wt and synaptotagmin [Sheng et al. (1996) Nature 379, 451-454]. The purpose of this study was to employ site-directed antibodies to target domains within and outside of the interaction site on the rat alpha1B to probe potential binding sites for syntaxin/SNAP-25/synaptotagmin. Our results demonstrate that both antibodies employed in this study have access to their epitopes on the alpha1B as evidenced by equivalent immunoprecipitation of native [125I]omega-conotoxin GVIA-labeled alpha1B protein from CHAPS-solubilized preparations. The N-type voltage-dependent calcium channel immunoprecipitated by Ab CW14, the antibody directed to a domain outside of the synprint site, is associated with syntaxin and SNAP-25 with the recovery of these proteins, increasing in parallel to the recovery of alpha1B. However, when we used the antibody raised to an epitope within the synprint site (Ab CW8) to immunoprecipitate N-type calcium channels, the alpha1B was depleted of more than 65% of syntaxin and 80% of SNAP-25 when compared to the recovery of these proteins using Ab CW14. This is the first report of a defined epitope on the alpha1B subunit II-III loop (amino acids 863-875) whose perturbation by a site-directed antibody influences the dissociation of SNAP-25 and syntaxin.
Subject(s)
Calcium Channels/metabolism , Calcium-Binding Proteins , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Amino Acid Sequence , Animals , Antibodies , Antibody Specificity , Binding Sites , Calcium/metabolism , Calcium Channels/chemistry , Calcium Channels/isolation & purification , Cell Membrane/metabolism , Humans , Macromolecular Substances , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/isolation & purification , Membrane Proteins/chemistry , Membrane Proteins/isolation & purification , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/isolation & purification , Peptide Fragments/chemistry , Peptide Fragments/immunology , Protein Structure, Secondary , Qa-SNARE Proteins , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Synaptosomal-Associated Protein 25 , SynaptotagminsABSTRACT
The N-type voltage-operated calcium channel has been characterized over the years as a high-threshold channel, with variable inactivation kinetics, and a unique ability to bind with high affinity and specificity omega-conotoxin GVIA and related toxins. This channel is particularly expressed in some neurons and endocrine cells, where it participates in several calcium-dependent processes, including secretion. Omega-conotoxin GVIA was instrumental not only for the biophysical and pharmacological characterization of N-type channels but also for the development of in vitro assays for studying N-type VOCC subcellular localization, biosynthesis, turnover, as well as short-and long-term regulation of its expression. We here summarize our studies on N-type VOCC expression in neurosecretory cells, with a major emphasis on recent data demonstrating the presence of N-type channels in intracellular secretory organelles and their recruitment to the cell surface during regulated exocytosis.
Subject(s)
Calcium Channels, N-Type , Calcium Channels/metabolism , Nerve Tissue Proteins/metabolism , Neurosecretory Systems/metabolism , Peptides/pharmacology , Animals , Biological Transport , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Calcium Signaling/drug effects , Cell Membrane/metabolism , Down-Regulation , Humans , Membrane Potentials , Nerve Tissue Proteins/drug effects , Neurosecretory Systems/drug effects , PC12 Cells/metabolism , Peptides/metabolism , Rats , Up-Regulation , omega-Conotoxin GVIAABSTRACT
Voltage-gated calcium channels (VDCC) are essential to neuronal maturation and differentiation. It is believed that important signaling information is encoded by VDCC-mediated calcium influx that has both spatial and temporal components. VDCC are multimeric complexes comprised of a pore-forming alpha1 subunit and auxiliary beta and alpha2/delta subunits. Changes in the fractional contribution of distinct calcium conductances to the total calcium current have been noted in developing and differentiating neurons. These changes are anticipated to reflect the differential expression and localization of the pore-forming alpha1 subunits. However, as in vitro studies have established that beta regulates the channel properties and targeting of alpha1, attention has been directed toward the developmental expression and assembly of beta isoforms. Recently, changes in the beta component of the omega-conotoxin GVIA (CTX)-sensitive N-type VDCC have indicated differential assembly of alpha1B with beta in postnatal rat brain. In addition, unique properties of beta4 have been noted with respect to its temporal pattern of expression and incorporation into N-type VDCC complexes. Therefore, the expression and assembly of specific alpha1/beta complexes may reflect an elaborate cellular strategy for regulating VDCC diversity. The importance of these developmental findings is bolstered by a recent study which identified mutations in the beta4 as the molecular defect in the mutant epileptic mouse (lethargic; lh/lh). As beta4 is normally expressed in both forebrain and cerebellum, one may consider the impact of the loss of beta4 upon VDCC assembly and activity. The importance of the beta1b and beta4 isoforms to calcium channel maturation and assembly is discussed.
Subject(s)
Calcium Channels/physiology , Nerve Tissue Proteins/physiology , Animals , Brain/embryology , Brain/growth & development , Brain/metabolism , Calcium/metabolism , Calcium Channels/biosynthesis , Calcium Channels/chemistry , Calcium Channels/deficiency , Calcium Channels/genetics , Calcium Signaling , Cell Differentiation , Cell Line , Epilepsy/genetics , Epilepsy/metabolism , Gene Expression Regulation, Developmental , Humans , Ion Channel Gating , Mice , Mice, Mutant Strains , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Neurons/metabolism , PC12 Cells/metabolism , Protein Conformation , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Rabbits , RatsABSTRACT
Voltage-dependent calcium channels (VDCC) are multisubunit complexes whose expression and targeting require the assembly of the pore-forming alpha1 with auxiliary beta and alpha2/delta subunits. The developmentally regulated expression and differential assembly of beta isoforms with the alpha1B subunit to form N-type VDCC suggested a unique role for the beta4 isoform in VDCC maturation (Vance, C. L., Begg, C. M., Lee, W.-L., Haase, H., Copeland, T. D., and McEnery, M. W. (1998) J. Biol. Chem. 273, 14495-14502). The focus of this study is the expression and assembly of alpha1B and beta isoforms in the epileptic mouse, lethargic (lh/lh), a mutant anticipated to produce a truncated beta4 subunit (Burgess, D. L., Jones, J. M., Meisler, M. H., and Noebels, J. L. (1997) Cell 88, 385-392). In this report, we demonstrate that neither full-length nor truncated beta4 protein is expressed in lh/lh mice. The absence of beta4 in lh/lh mice is associated with decreased expression of N-type VDCC in forebrain and cerebellum. The most surprising characteristic of the lh/lh mouse is increased expression of beta1b protein. This result suggests a previously unidentified cellular mechanism wherein expression of the total pool of available beta subunits is under tight metabolic regulation. As a consequence of increased beta1b expression, the beta1b is increased in its incorporation into alpha1B/beta complexes relative to wild type. Thus, in striking similarity to the population of N-type VDCC present in immature rat brain, the population of N-type VDCC present in adult lh/lh mice is characterized by the absence of beta4 with increased beta1b expression and assembly into N-type VDCC. It is intriguing to speculate that the increased excitability and susceptibility to seizures observed in the lh/lh mouse arises from the inappropriate expression of an immature population of N-type VDCC throughout neuronal development.
Subject(s)
Calcium Channels/biosynthesis , Epilepsy/metabolism , Animals , Brain/drug effects , Brain/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels/genetics , Mice , Mice, Neurologic Mutants , Neurons/drug effects , Neurons/metabolism , Peptides/pharmacology , Protein Conformation , Rats , omega-Conotoxin GVIAABSTRACT
Calcium functions as an essential second messenger during neuronal development and synapse acquisition. Voltage-dependent calcium channels (VDCC), which are critical to these processes, are heteromultimeric complexes composed of alpha1, alpha2/delta, and beta subunits. beta subunits function to direct the VDCC complex to the plasma membrane as well as regulate its channel properties. The importance of beta to neuronal functioning was recently underscored by the identification of a truncated beta4 isoform in the epileptic mouse lethargic (lh) (Burgess, D. L., Jones, J. M., Meisler, M. H., and Noebels, J. L. (1997) Cell 88, 385-392). The goal of our study was to investigate the role of individual beta isoforms (beta1b, beta2, beta3, and beta4) in the assembly of N-type VDCC during rat brain development. By using quantitative Western blot analysis with anti-alpha1B-directed antibodies and [125I-Tyr22]omega-conotoxin GVIA (125I-CTX) radioligand binding assays, we observed that only a small fraction of the total alpha1B protein present in embryonic and early postnatal brain expressed high affinity 125I-CTX-binding sites. These results suggested that subsequent maturation of alpha1B or its assembly with auxiliary subunits was required to exhibit high affinity 125I-CTX binding. The temporal pattern of expression of beta subunits and their assembly with alpha1B indicated a developmental pattern of expression of beta isoforms: beta1b increased 3-fold from P0 to adult, beta4 increased 10-fold, and both beta2 and beta3 expression remained unchanged. As the beta component of N-type VDCC changed during postnatal development, we were able to identify both immature and mature forms of N-type VDCC. At P2, the relative contribution of beta is beta1b > beta3 >> beta2, whereas at P14 and adult the distribution is beta3 > beta1b = beta4. Although we observed no beta4 associated with the alpha1B at P2, beta4 accounted for 14 and 25% of total alpha1B/beta subunit complexes in P14 and adult, respectively. Thus, of the beta isoforms analyzed, only the beta4 was assembled with the rat alpha1B to form N-type VDCC with a time course that paralleled its level of expression during rat brain development. These results suggest a role for the beta4 isoform in the assembly and maturation of the N-type VDCC.
Subject(s)
Brain/growth & development , Calcium Channels/chemistry , Gene Expression Regulation, Developmental/genetics , Age Factors , Amino Acid Sequence , Animals , Binding Sites , Calcium Channels/classification , Cell Line , Charybdotoxin/metabolism , Epitopes/immunology , Humans , Iodine Radioisotopes/metabolism , Molecular Sequence Data , Peptide Fragments/immunology , Protein Binding , RatsABSTRACT
While emphasis has been placed upon those proteins which either mediate or respond to the rapid influx of calcium following depolarization, there has been little emphasis upon those proteins which aid in the reequilibration of the membrane potential. In an effort to identify presynaptic membrane proteins implicated in neurosecretion, monoclonal antibodies were screened against proteins which cosegregated with neuronal voltage-dependent calcium channels (VDCC) following immunoprecipitation. One monoclonal antibody (mAb 9A7) identified a 110-kDa protein. Micropeptide sequencing of (i) the mAb 9A7 immunoaffinity purified antigen and (ii) the 110-kDa protein present in the neuronal (N-type) VDCC preparation (McEnery et al., 1991, Proc. Natl. Acad. Sci. 88, 11095-11099) indicated identity with the alpha subunit(s) of the Na,K-ATPase. Further characterization by Western blotting, immunochemical localization, and immunoaffinity purification indicated that mAb 9A7 not only recognized the alpha3 isoform which is predominant in neuronal tissues but also identified the alpha1 and alpha2 isoforms. mAb 9A7 exhibited a wide cross-species reactivity and recognized human, rat, and mouse alpha subunit isoforms at an internal epitope. The pan-specificity of mAb 9A7 and the differential mobility of the alpha1 isoform relative to the alpha2 and alpha3 permitted parallel detection of multiple alpha isoforms. Western blot analysis of undifferentiated rat pheochromocytoma cell line (PC12) and human neuroblastoma (IMR32) cells indicated coexpression of the alpha1 and alpha3 isozymes. Upon differentiation of IMR32 cells by dibutrylyl-cAMP, a substantial increase in the alpha3 relative to the alpha1 isoform was observed. While the enrichment of total Na,K-ATPase may reflect the increased demand for ATP-dependent ion transport as IMR32 cells become more excitable, the specific increase in the alpha3 isoform suggests a unique role of this isoform during IMR32 cell differentiation.
Subject(s)
Antibodies, Monoclonal/immunology , Neurons/enzymology , Sodium-Potassium-Exchanging ATPase/chemistry , Animals , Antibodies, Monoclonal/metabolism , Antibody Specificity , Blotting, Western , Brain/enzymology , Calcium Channels/chemistry , Calcium Channels/immunology , Cell Membrane/chemistry , Cell Membrane/enzymology , Cross Reactions , Epitopes , Humans , Immunohistochemistry , Isoenzymes/analysis , Isoenzymes/chemistry , Isoenzymes/metabolism , PC12 Cells , Precipitin Tests , Protein Conformation , Rats , Sequence Analysis , Sodium-Potassium-Exchanging ATPase/analysis , Sodium-Potassium-Exchanging ATPase/immunology , Sodium-Potassium-Exchanging ATPase/isolation & purification , Tumor Cells, CulturedABSTRACT
Human neuroblastoma cells (IMR32) respond to treatment with either dibutyryl-cAMP or nerve factor by acquiring a neuronal phenotype which is accompanied by a marked increase in the density of neuronal (N-type) VDCC currents. Using IMR32 cells as a model for neuronal differentiation, we were interested in examining possible changes in the level of expression of the alpha1B subunit of N-type calcium channels as well as beta subunit isoforms. Upon differentiation with dibutyryl-cAMP and 5-bromo-2-deoxyuridine for 16 days, we observed a dramatic increase in alpha1B protein which initiated between day 8 and 10. Day 10 evidenced maximal expression of alpha1B protein, which was followed by an interval of relatively constant expression of alpha1B (day 12 to day 16). Monitoring beta subunit expression using a pan specific anti-beta antibody (Ab CW20), we observed an increase in expression of a single 82 kDa beta subunit. The predominant 82 kDa beta subunit expressed throughout the course of differentiation was identified as the beta1b isoform using a panel of beta subunit specific antibodies. Of significance, neither the beta2 nor beta3 isoforms were detected in full differentiated IMR32 cells. Contrary to a previous report on the absence of neurotypic expression of VDCC beta subunits in a second model for in vitro differentiation, NGF-treated rat pheochromocytoma cells (PC12 cells) [1], we report the regulated expression of the beta1b protein in differentiated IMR32 cells suggesting a cell specific function for this beta subunit which parallels the acquisition of the neuronal phenotype. The restrictive expression of the beta1b in IMR32 cells may reflect a cell-type specific function that extends beyond its role as an auxiliary subunit of VDCC complexes.
Subject(s)
Calcium Channels/biosynthesis , Neurons/cytology , Neurons/metabolism , Amino Acid Sequence , Animals , Antibody Specificity , Bromodeoxyuridine/pharmacology , Bucladesine/pharmacology , Calcium Channels/chemistry , Cell Differentiation , Humans , Molecular Sequence Data , Molecular Weight , Neuroblastoma , Prosencephalon/metabolism , Rats , Tumor Cells, CulturedABSTRACT
Modulation of the neuronal omega-conotoxin GVIA-sensitive N-type voltage-dependent calcium channel (VDCC) by neurotransmitters and guanine nucleotides suggests a dynamic interaction between activated G-protein alpha subunits and the N-type VDCC. Our previous report on the purification of the N-type VDCC (McEnery, M. W., Snowman, A. M., Sharp, A. H., Adams, M. E., and Snyder, S. H. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11095-11099), has led us to investigate a possible association of CTXR with an endogenous G alpha subunit. The addition of the G-protein activator AIF4- modulated the 125I-CTX binding characteristics of the solubilized CTXR. Further immunological analyses employing G alpha subunit-specific antibodies to monitor the cofractionation of G alpha with 125I-CTX binding activity throughout the purification procedure indicate the selective recovery of Go alpha in the purified CTXR preparation, as neither Gs alpha, Gi alpha, nor G beta gamma could be detected. Furthermore, Go alpha associated with CTXR acted as a substrate for pertussis toxin-dependent ADP-ribosylation only upon the addition of exogenous G beta gamma subunits. These results strongly indicate a high affinity complex between an activated Go alpha and CTXR maintained throughout biochemical purification of the 125I-CTX receptor.