Resorbable Nanocomposites with Bone-Like Strength and Enhanced Cellular Activity.

Bone cements for treatment of fractures at weight-bearing sites are subjected to dynamic physiological loading from daily activities. An ideal bone cement rapidly sets after injection, exhibits bone-like strength, stimulates osteogenic differentiation of endogenous cells, and resorbs at a rate aligned with patient biology. However, currently available materials fall short of these targeted properties. Nanocrystalline hydroxyapatite (nHA) enhances osteogenic differentiation, new bone formation, and osteoclast differentiation activity compared to amorphous or micron-scale crystalline hydroxyapatite. However, the brittle mechanical properties of nHA precludes its use in treatment of weight-bearing bone defects. In this study, we report settable nHA-poly(ester urethane) (PEUR) nanocomposites synthesized from nHA, lysine triisocyanate (LTI), and poly(caprolactone) triol via a solvent-free process. The nanocomposites are easily mixed and injected using a double-barrel syringe, exhibit mechanical properties exceeding those of conventional bone cements, enhance mineralization of osteoprogenitor cells in vitro, and undergo osteoclast-mediated degradation in vitro. This combination of properties cannot be achieved using other technologies, which underscores the potential of nHA-PEUR nanocomposites as a new approach for promoting bone healing at weight-bearing sites.

Site-directed antibodies to low-voltage-activated calcium channel CaV3.3 (alpha1I) subunit also target neural cell adhesion molecule-180.

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.

Testosterone modulates Ca(v2.2) calcium channels' functional expression at rat levator ani neuromuscular junction.

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.

Differential expression of high voltage-activated Ca2+ channel types in the rostral reticular thalamic nucleus of the absence epileptic WAG/Rij rat.

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.

Differential expression of alpha 1 and beta subunits of voltage dependent Ca2+ channel at the neuromuscular junction of normal and P/Q Ca2+ channel knockout mouse.

Voltage-dependent calcium channels (VDCC) have a key role in neuronal function transforming the voltage signals into intracellular calcium signals. They are composed of the pore-forming alpha(1) and the regulatory alpha(2)delta, gamma and beta subunits. Molecular and functional studies have revealed which alpha(1) subunit gene product is the molecular constituent of each class of native calcium channel (L, N, P/Q, R and T type). Electrophysiological and immunocytochemical studies have suggested that at adult mouse motor nerve terminal (MNT) only P/Q type channels, formed by alpha(1A) subunit, mediate evoked transmitter release. The generation of alpha(1A)-null mutant mice offers an opportunity to study the expression and localization of calcium channels at a synapse with complete loss of P/Q calcium channel. We have investigated the expression and localization of VDCCs alpha(1) and beta subunits at the wild type (WT) and knockout (KO) mouse neuromuscular junction (NMJ) using fluorescence immunocytochemistry. The alpha(1A) subunit was observed only at WT NMJ and was absent at denervated muscles and at KO NMJ. The subunits alpha(1B), alpha(1D) and alpha(1E) were also present at WT NMJ and they were over- expressed at KO NMJ suggesting a compensatory expression due to the lack of the alpha(1A). On the other hand, the beta(1b), beta(2a) and beta(4) were present at the same levels in both genotypes. The presence of other types of VDCC at WT NMJ indicate that they may play other roles in the signaling process which have not been elucidated and also shows that other types of VDCC are able to substitute the alpha(1A) subunit, P/Q channel under certain pathological conditions.

Immunological characterization of T-type voltage-dependent calcium channel CaV3.1 (alpha 1G) and CaV3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development.

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.

Mutations of peripheral myelin protein 22 result in defective trafficking through mechanisms which may be common to diseases involving tetraspan membrane proteins.

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.

Ion chromatography on-chip.

On-chip separation of inorganic anions by ion-exchange chromatography was realized. Micro separation channels were fabricated on a silicon wafer and sealed with a Pyrex cover plate using standard photolithography, wet and dry chemical etching, and anodic bonding techniques. Quaternary ammonium latex particles were employed for the first time to coat the separation channels on-chip. Owing to the narrow depths of the channels on the chip, 0.5-10 microm, there were more interactions of the analytes with the stationary phase on the chip than in a 50-microm I.D. capillary. With off-chip injection (20 nl) and UV detection, NO2-, NO3-, I-, and thiourea were separated using 1 mM KCl as the eluent. The linear ranges for NO2- and NO3- are from 5 to 1000 microM with the detection limits of 0.5 microM.

On-chip definition of picolitre sample injection plugs for miniaturised liquid chromatography.

The fabrication of components for a miniaturised liquid chromatography system on silicon has recently been reported by our research group [J. Cap. Electrophoresis Microchip Technol. 6 (1999) 33; Analyst 125 (2000) 25]. To date, inlet and outlet connection ports, separation micro-channels (20-200 microm in width, 0.5-10 microm in depth, 15-60 cm in length), and an intersection for picolitre injection have been etched on a silicon wafer and then sealed with a Pyrex cover plate on which platinum electrodes for on-chip detection have been patterned. The platinum electrodes have been used for the amperometric detection of phenol, using 20 nl off-chip injection. In this work we present our latest results obtained with on-chip pressure driven picolitre injection, designed to realize the full capabilities of this micro-LC system. The injection volume is dependent on the micro-channel depth, width, and also on the intersection length, allowing injection in the low nanolitre to picolitre range.

Enantiomeric separation of R,S-naproxen by conventional and nano-liquid chromatography with methyl-beta-cyclodextrin as a mobile phase additive.

Chiral separations of R,S-naproxen mixtures were obtained on an achiral column (ODS) with methyl-beta-cyclodextrin as a mobile phase additive using conventional and nano-LC. The optimised mobile phase composition was 20 mmol l(-1) methyl-beta-cyclodextrin, 20% (v/v) acetonitrile, and 50 mmol l(-1) sodium acetate buffer at pH 3 using hydrochloric acid for pH adjustment. In addition to UV detection at 232 nm, amperometric detection was also investigated. Without using any internal standard, the reproducibility of amperometric detection (+1.05 V vs. Ag/AgCl) over a long analysis cycle in LC was greatly improved by choosing the peak area ratio between R- and S-naproxen as the analytical readout (the relative standard deviation was 2.11%) and enantiomeric purity could be assessed directly. This method was successfully employed for enantiomeric purity assessment in commercial naproxen tablets. Finally, successful transfer from conventional LC to nano-LC was realised, resulting in over 1000-fold reduction in reagent consumption.

Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking alpha(1G) T-type Ca(2+) channels.

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.

Altered nociceptive response in mice deficient in the alpha(1B) subunit of the voltage-dependent calcium channel.

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.

Biochemical and anatomical evidence for specialized voltage-dependent calcium channel gamma isoform expression in the epileptic and ataxic mouse, stargazer.

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.

Functional roles of gamma2, gamma3 and gamma4, three new Ca2+ channel subunits, in P/Q-type Ca2+ channel expressed in Xenopus oocytes.

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.

Expression of Ca(2+) channel subunits during cardiac ontogeny in mice and rats: identification of fetal alpha(1C) and beta subunit isoforms.

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.

Calcium channel activation stabilizes a neuronal calcium channel mRNA.

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.

N-type calcium channel/syntaxin/SNAP-25 complex probed by antibodies to II-III intracellular loop of the alpha1B subunit.

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.

Metabolism and trafficking of N-type voltage-operated calcium channels in neurosecretory cells.

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.