Antigen retrieval pre-treatment causes a different expression pattern of Cav3.2 in rat and mouse spinal dorsal horn.

Cav3 channels consist of three isoforms, Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I), which produce low-threshold spikes that trigger burst firings in nociceptive neurons of the spinal dorsal horn (SDH) and dorsal root ganglion (DRG). Although Cav3.2 plays a crucial role in pathological pain, its distribution in SDH still remains controversial. One study showed that Cav3.2 is ubiquitously expressed in neurons, but another study implied that Cav3.2 is expressed restricted to astrocytes. To unravel these discrepancies, we used methods of immunohistochemistry either with or without antigen retrieval (AR) pre-treatment to detect Cav3 in SDH and DRG from both rats and mice. Moreover, Cav3.2 mRNA was detected in mice SDH using in situ hybridization. We found that the expression pattern of Cav3.2 but not Cav3.1 and Cav3.3 in SDH were largely different with or without AR pre-treatment, which showed a neuron-like and an astrocyte-like appearance, respectively. Double staining further demonstrated that Cav3.2 was mainly co-stained with the neuronal marker NeuN in the presence of AR but was with glial fibrillary acidic protein (GFAP, marker for astrocytes) in the absence of AR pre-treatment. Importantly, Cav3.2 mRNA was mainly co-localized with Cav3.2 but not GFAP. Together, our findings indicate that AR pre-treatment or not impacts the expression pattern of Cav3.2, which may make a significant contribution to the future study of Cav3.2 in SDH.

Inflammatory mediator bradykinin increases population of sensory neurons expressing functional T-type Ca(2+) channels.

T-type Ca(2+) channels are important regulators of peripheral sensory neuron excitability. Accordingly, T-type Ca(2+) currents are often increased in various pathological pain conditions, such as inflammation or nerve injury. Here we investigated effects of inflammation on functional expression of T-type Ca(2+) channels in small-diameter cultured dorsal root ganglion (DRG) neurons. We found that overnight treatment of DRG cultures with a cocktail of inflammatory mediators bradykinin (BK), adenosine triphosphate (ATP), norepinephrine (NE) and prostaglandin E2 (PGE2) strongly increased the population size of the small-diameter neurons displaying low-voltage activated (LVA, T-type) Ca(2+) currents while having no effect on the peak LVA current amplitude. When applied individually, BK and ATP also increased the population size of LVA-positive neurons while NE and PGE2 had no effect. The PLC inhibitor U-73122 and B2 receptor antagonist, Hoe-140, both abolished the increase of the population of LVA-positive DRG neurons. Inflammatory treatment did not affect CaV3.2 mRNA or protein levels in DRG cultures. Furthermore, an ubiquitination inhibitor, MG132, did not increase the population of LVA-positive neurons. Our data suggest that inflammatory mediators BK and ATP increase the abundance of LVA-positive DRG neurons in total neuronal population by stimulating the recruitment of a 'reserve pool' of CaV3.2 channels, particularly in neurons that do not display measurable LVA currents under control conditions.

Reactivity to the p305 Epitope of the α1G T-Type Calcium Channel and Autoimmune-Associated Congenital Heart Block.

BACKGROUND: Only 2% of mothers positive for anti-SSA/Ro (Ro) antibodies have children with congenital heart block (CHB). This study aimed to determine whether reactivity with p305, an epitope within the α1G T-type calcium channel, confers added risk over anti-Ro antibodies. METHODS AND RESULTS: Using sera from anti-Ro-exposed pregnancies resulting in offspring with CHB, no disease but CHB-sibling, and no disease and no CHB-sibling, as well as disease (lupus without anti-Ro) and healthy controls, reactivities were determined for binding to Ro60, p305, and an epitope within Ro60, p133-Ro60, which shares structural properties with p305, including key amino acids and an α-helical structure. Candidate peptides were further evaluated in an in vitro model that assessed the binding of maternal antibodies to apoptotic cells. In anti-Ro-positive mothers, anti-p305 autoantibodies (>3 SD above healthy controls) were detected in 3/59 (5%) CHB pregnancies, 4/30 (13%) unaffected pregnancies with a CHB-sibling, and 0/42 (0%) of unaffected pregnancies with no CHB-sibling. For umbilical bloods (61 CHB, 41 healthy with CHB sibling), no association of anti-p305 with outcome was detected; however, overall levels of anti-p305 were elevated compared to mothers during pregnancy in all groups studied. For anti-p133-Ro60, reactivity paralleled that of anti-p305. In the screen employing apoptotic cells, p133-Ro60, but not p305, significantly attenuated the binding of immunoglobulin G isolated from a mother whose child had CHB (42.1% reduced to 13.9%, absence/presence of p133-Ro60, respectively, P<0.05). CONCLUSIONS: These data suggest that anti-p305 is not a robust maternal marker for assessing increased risk of CHB during an anti-SSA/Ro pregnancy.

Congenital heart block maternal sera autoantibodies target an extracellular epitope on the α1G T-type calcium channel in human fetal hearts.

BACKGROUND: Congenital heart block (CHB) is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV) block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit α1G may be a fetal target of maternal sera autoantibodies in CHB. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G (α1G gene) in the AV junction of human fetal hearts compared to the apex (18-22.6 weeks gestation). Using human fetal hearts (20-22 wks gestation), our immunoprecipitation (IP), Western blot analysis and immunofluorescence (IF) staining results, taken together, demonstrate accessibility of the α1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the α1G protein. By ELISA we demonstrated maternal sera reactivity to α1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305-319 of the extracellular loop linking transmembrane segments S5-S6 in α1G repeat I). Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved) of the α1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN) cells. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of α1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for α1H suggesting that autoantibodies can target multiple fetal targets.

Immunohistological demonstration of CaV3.2 T-type voltage-gated calcium channel expression in soma of dorsal root ganglion neurons and peripheral axons of rat and mouse.

Previous behavioral studies have revealed that CaV3.2 T-type calcium channels support peripheral nociceptive transmission and electrophysiological studies have established the presence of T-currents in putative nociceptive sensory neurons of dorsal root ganglion (DRG). To date, however, the localization pattern of this key nociceptive channel in the soma and peripheral axons of these cells has not been demonstrated due to lack of isoform-selective anti-CaV3.2 antibodies. In the present study a new polyclonal CaV3.2 antibody is used to localize CaV3.2 expression in rodent DRG neurons using different staining techniques including confocal and electron microscopy (EM). Confocal microscopy of both acutely dissociated cells and short-term cultures demonstrated strong immunofluorescence of anti-CaV3.2 antibody that was largely confined to smaller diameter DRG neurons where it co-localized with established immuno-markers of unmyelinated nociceptors, such as, CGRP, IB4 and peripherin. In contrast, a smaller proportion of these CaV3.2-labeled DRG cells also co-expressed neurofilament 200 (NF200), a marker of myelinated sensory neurons. In the rat sciatic nerve preparation, confocal microscopy demonstrated anti-CaV3.2 immunofluorescence which was co-localized with both peripherin and NF200. Further, EM revealed immuno-gold labeling of CaV3.2 preferentially in association with unmyelinated sensory fibers from mouse sciatic nerve. Finally, we demonstrated the expression of CaV3.2 channels in peripheral nerve endings of mouse hindpaw skin as shown by co-localization with Mrgpd-GFP-positive fibers. The CaV3.2 expression within the soma and peripheral axons of nociceptive sensory neurons further demonstrates the importance of this channel in peripheral pain transmission.

The antimicrobial peptide LL-37 alters human osteoblast Ca2+ handling and induces Ca2+-independent apoptosis.

The human antimicrobial peptide cathelicidin LL-37 has, besides its antimicrobial properties, also been shown to regulate apoptosis in a cell type-specific manner. Mechanisms involved in LL-37-regulated apoptotic signaling are not identified. Here, we show that LL-37 reduces the human osteoblast-like MG63 cell number and cell viability in the micromolar concentration range with an IC50 value of about 5 µM. Treatment with 4 µM LL-37 increased the number of annexin V-positive cells and stimulated activation of caspase 3 showing that LL-37 promotes apoptosis. Treatment with 4 µM LL-37 caused an acute and sustained rise in intracellular Ca(2+) concentration assessed by laser-scanning confocal microscopy of Fluo-4-AM-loaded MG63 cells. LL-37 increased Ca(2+) also in the presence of the respective L- and T-type voltage-sensitive Ca(2+) channel blockers nifedipine and NiCl2. LL-37 had no effect on Ca(2+) in cells incubated with Ca(2+)-free solution. LL-37 (4 and 8 µM) reduced the MG63 cell number both in the presence and absence of Ca(2+) in the medium. In conclusion, LL-37 reduces the osteoblast cell number by promoting apoptosis, and furthermore, LL-37 stimulates Ca(2+) inflow via a mechanism independent of voltage-sensitive Ca(2+) channels. Interestingly, LL-37-induced lowering of the cell number seems to be mediated via a mechanism independent of Ca(2+).

CACNA1H antibodies associated with headache with neurological deficits and cerebrospinal fluid lymphocytosis (HaNDL).

BACKGROUND: Patients with the syndrome of headache with neurological deficits and lymphocytosis (HaNDL) typically present with recurrent and temporary attacks of neurological symptoms and cerebrospinal fluid lymphocytosis. AIM AND METHODS: To identify potential HaNDL-associated antibodies directed against neuronal surface and/or synapse antigens, sera of four HaNDL patients and controls were screened with indirect immunohistochemistry, immunofluorescence, cell-based assay, radioimmunoassay, protein macroarray and enzyme-linked immunosorbent assay (ELISA). RESULTS: Although HaNDL sera did not yield antibodies to any of the well-characterized neuronal surface or synapse antigens, protein macroarray and ELISA studies showed high-titer antibodies to a subunit of the T-type voltage-gated calcium channel (VGCC), CACNA1H, in sera of two HaNDL patients. CONCLUSION: Our results support the notion that ion channel autoimmunity might at least partially contribute to HaNDL pathogenesis and occurrence of neurological symptoms.

Transfer of beta subunit regulation from high to low voltage-gated Ca2+ channels.

High voltage-activated Ca(2+) channel expression and gating is controlled by their beta subunits. Although the sites of interaction are known at the atomic level, how beta modulates gating remains to be determined. Using a chimeric approach, beta subunit regulation was conferred to a low voltage-activated channel. Regulation was dependent on a rigid linker connecting the alpha(1) interaction domain to IS6. Chimeric channels also revealed a role for IS6 in channel gating. Taken together, these results support a direct coupling model where beta subunits alter movements in IS6 that occur as the channel transits between closed, open, and inactivated states.

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.

Direct inhibition of expressed cardiac l- and t-type calcium channels by igg from mothers whose children have congenital heart block.

BACKGROUND: Congenital heart block (CHB) is a disease that affects the offspring of mothers with autoimmune diseases. We recently reported that maternal sera containing antibodies against SSA/Ro and SSB/La ribonucleoproteins (positive IgG) inhibited L-type Ca current in isolated cardiac myocytes and induced sinus bradycardia in a murine model of CHB. The direct interaction of positive IgG with L-type Ca channel proteins and the possible inhibition of T-type Ca current that could account for the sinus bradycardia remain unknown. METHODS AND RESULTS: The 2-electrode voltage-clamp technique was used to record currents via L-type (I(Ba)-alpha(1C) or I(Ba)-alpha(1C)+beta(2a)+alpha(2)/delta) and T-type (I(Ba)-alpha(1H)) Ca channels, Na channels (I(Na)-hH1), and K channels (I(Ks)-minK+KvLQT1) expressed in Xenopus oocytes. Positive IgG (350 microgram/mL) inhibited I(Ba)-alpha(1C) by 50.6+/-4.7% (P<0.01) and I(Ba)-alpha(1C)+beta(2a)+alpha(2)/delta by 50.9+/-4.2% (P<0.01); I(Ba)-alpha(1H) was reduced by 18.9+/-1.0% (P<0.01). Immunoblot data show cross-reactivity of positive IgG with alpha(1C) subunit. Pretreatment of oocytes with atropine (1 micromol/L) or acetylcholine (10 micromol/L) did not affect the inhibitory effect of IgG on I(Ba)-alpha(1C) and I(Ba)-alpha(1C)+beta(2a)+alpha(2)/delta (P<0.05). Positive IgG had no effect, however, on either I(Na)-hH1 or I(Ks)-minK+KvLQT1. CONCLUSIONS: Positive IgG inhibited expressed L-type I:(Ba) and cross-reacted with the alpha(1C) subunit in Xenopus oocytes, providing strong evidence that maternal antibodies interact directly with the pore-forming alpha(1)-subunit of Ca channels. In addition, we show for the first time that positive IgG also inhibited T-type I(Ba) but not I(Na)-hH1 or I(Ks)-minK+KvLQT1. This could provide, in part, the ionic basis of sinus bradycardia reported in animal models of CHB and clinically in humans.

Immunodetection of alpha1E voltage-gated Ca(2+) channel in chromogranin-positive muscle cells of rat heart, and in distal tubules of human kidney.

The calcium channel alpha1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of alpha1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac alpha1E was determined by SDS-PAGE and immunoblotting (218 +/- 6 kD; n = 3). Compared to alpha1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of alpha1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of alpha1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of alpha1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of alpha1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca(2+)-dependent secretion of peptide hormones such as ANP and urodilatin.