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f Ca(V)1.2 exceeded that of Ca(V)1.3 and Ca(V)2.3 two-fold. Insulin secretion, which rose tenfold in response to 16.7 mM glucose, was completely abolished by 5 microM isradipine that blocks Ca(V)1.2 and Ca(V)1.3. Similarly, the increase in intracellular calcium in response to 15 mM glucose was decreased in the presence of 5 microM isradipine, and the frequency of calcium spikes was decreased to the level seen at 2.8 mM glucose. By contrast, inhibition of Ca(V)2.3 with 100 nM SNX-482 did not significantly affect insulin secretion or intracellular calcium. Using RNA interference, Ca(V)1.2 mRNA and protein levels were knocked down by approximately 65% and approximately 34% respectively, which reduced insulin secretion in response to 16.7 mM glucose by 50%. Similar reductions in calcium currents and cell capacitance were seen in standard whole-cell patch-clamp experiments. The remaining secretion of insulin could be reduced to the basal level by 5 microM isradipine. Calcium influx underlying this residual insulin secretion could result from persisting Ca(V)1.2 expression in transfected cells since knock-down of Ca(V)1.3 did not affect glucose-stimulated insulin secretion. In summary, our results suggest that Ca(V)1.2 is critical for insulin secretion in INS-1 832/13 cells.

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extinction, but not acquisition, of cocaine conditioned place preference (CPP) in male mice increased Cav1.2 L-type Ca2+ channel mRNA and protein in postsynaptic density (PSD) fractions of the hippocampus, a brain region involved in drug-context associations. Moreover, viral-mediated deletion of Cav1.2 in the dorsal hippocampus attenuated extinction of cocaine CPP. Molecular studies examining downstream Cav1.2 targets revealed that extinction recruited calcium/calmodulin (Ca2+/CaMK)-dependent protein kinase II (CaMKII) to the hippocampal PSD. This occurred in parallel with an increase in phosphorylation of the AMPA GluA1 receptor subunit at serine 831 (S831), a CaMKII site, along with an increase in total PSD GluA1. The necessity of S831 GluA1 was further demonstrated by the lack of extinction in S831A GluA1 phosphomutant mice. Of note hippocampal GluA1 levels remained unaltered at the PSD, but were reduced near the PSD and at perisynaptic sites of dendritic spines in extinction-resistant S831A mutant mice. Finally, conditional knock-out of Cav1.2 in dopamine D1 receptor (D1R)-expressing cells resulted in attenuation of cocaine CPP extinction and lack of extinction-dependent changes in hippocampal PSD CaMKII expression and S831 GluA1 phosphorylation. In summary, we demonstrate an essential role for the hippocampal Cav1.2/CaMKII/S831 GluA1 pathway in cocaine CPP extinction, with data supporting contribution of hippocampal D1R-expressing cells in this process. These findings demonstrate a novel role for Cav1.2 channels in extinction of contextual cocaine-associated memories.SIGNIFICANCE STATEMENT Continued drug-seeking behavior, a defining characteristic of cocaine addiction, can be precipitated by contextual cues, yet the molecular mechanisms required for extinction of these context-specific memories remain poorly understood. Here, we have uncovered a novel and selective role of the Cav1.2 L-type Ca2+ channel and its downstream signaling pathway in the hippocampus that mediate extinction of cocaine conditioned place preference (CPP). We additionally provide evidence that supports a role of Cav1.2 within dopamine D1 receptor-expressing cells of the hippocampus for extinction of cocaine CPP. Therefore, these findings reveal a previously unknown role of Cav1.2 channels within the hippocampus and in D1 receptor-expressing cells in extinction of cocaine-associated memories, providing a framework for further exploration of mechanisms underlying extinction of cocaine-seeking behavior.

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le for Cav1.3 and Cav1.4 as the primary Cav channels in IHCs and PRs, respectively. Because of the unique features of transmission at ribbon synapses, Cav1.3 and Cav1.4 exhibit unusual properties that are well-suited for their physiological roles. These properties may be intrinsic to the channel subunit(s) and/or may be conferred by regulatory interactions with synaptic signaling molecules. This review will cover advances in our understanding of the function of Cav1 channels at sensory ribbon synapses, and how dysregulation of these channels leads to disorders of vision and hearing.

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n in sample sets of 2,175 POAG cases and 2,064 controls from Sweden, the UK and Australia (combined OR = 1.18, P = 0.0015) and in 299 POAG cases and 580 unaffected controls from Hong Kong and Shantou, China (combined OR = 5.42, P = 0.0021). The risk variant identified here is located close to CAV1 and CAV2, both of which are expressed in the trabecular meshwork and retinal ganglion cells that are involved in the pathogenesis of POAG.

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00;'>CAV1, which is reported to be overexpressed in tumors, with breast cancer within a Taiwanese population. PATIENTS AND METHODS: A total of 1232 patients with breast cancer and equal number of healthy controls in central Taiwan were genotyped via polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and six polymorphic variants of CAV1 were analyzed for their association with breast cancer susceptibility. RESULTS: The distribution of genotypes of CAV1 G14713A and T29107A were significantly different between breast cancer and control groups (p=5.6x10(-5) and 1.9x10(-4), respectively), while those for CAV1 C239A, G21985A, T28608A and G32124A were not significant (p>0.05). The percentages of AG genotype of G14713A and TT genotype of T29107A are higher in the cancer group than in the control group. The two single nucleotide polymorphisms were chosen for haplotype analysis and the data showed that compared with GG/TT haplotype of CAV1 G14713A/T29107A, the GG/AT and GG/AA groups have a lower risk of breast cancer (odds ratio, OR=0.69, 95% confidence interval, CI=0.57-0.92). On the contrary, the AG/TT haplotype confers a higher risk of breast cancer (OR=1.50, 95% CI=1.14-2.12). CONCLUSION: Our results provide evidence for CAV1 genotypes being involved in predisposition to breast cancer. The association of the potential risk haplotype agrees well with a role of CAV1 genotype in breast cancer risk and the association with tumor progression needs further investigation.

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ion is regulated by the entry of dietary Ca(2+) through Ca(v)1.3 in the apical membrane. When rat jejunum was perfused with 75 mM glucose, Ca(2+)-deplete conditions, or perfusion with the L-type antagonists nifedipine and verapamil strongly diminished the phloretin-sensitive apical GLUT2, but not the phloretin-insensitive SGLT1 component of glucose absorption. Western blotting showed that in each case there was a significant decrease in apical GLUT2 level, but no change in SGLT1 level. Inhibition of apical GLUT2 absorption coincided with inhibition of unidirectional (45)Ca(2+) entry by nifedipine and verapamil. At 10 mM luminal Ca(2+), (45)Ca(2+) absorption in the presence of 75 mM glucose was 2- to 3-fold that in the presence of 75 mM mannitol. The glucose-induced component was SGLT1-dependent and nifedipine-sensitive. RT-PCR revealed the presence of Ca(v)beta(3) in jejunal mucosa; Western blotting and immunocytochemistry localized Ca(v)beta(3) to the apical membrane, together with Ca(v)1.3. We conclude that in times of dietary sufficiency Ca(v)1.3 may mediate a significant pathway of glucose-stimulated Ca(2+) entry into the body and that luminal supply of Ca(2+) is necessary for GLUT2-mediated glucose absorption. The integration of glucose and Ca(2+) absorption represents a complex nutrient-sensing system, which allows both absorptive pathways to be regulated rapidly and precisely to match dietary intake.

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. We used real-time RT-PCR, Western blotting, and indirect immunostaining to characterize the developmental expression and localization of alpha1D Ca(2)(+) channel in rat hearts. Both protein and mRNA levels of alpha1D Ca(2)(+) channel decreased postnatally. Two forms of alpha1D Ca(2)(+) channel protein (250 and 190 kD) were observed, with the full-length (250 kD) channel protein being predominant in the prenatal stages. Both Western blots and confocal imaging demonstrated that alpha1D Ca(2)(+) channel protein was expressed in both atria and ventricles at fetal and neonatal stages but was absent in the adult ventricles. Interestingly, alpha1D Ca(2)(+) channel was also found at the nucleus/perinucleus of immature but not adult atrial cells. Furthermore, the nuclear staining was reproduced in adult atrial cell line, HL-1 cells, which possess immature properties. The data are first to show that alpha1D Ca(2)(+) channel has unique age-dependent expression profile and subcellular localization in the heart, suggesting a developmental stage-dependent specific function.

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lation of HVA channel gating. It has been suggested, however, that Ca(V)beta-mediated plasma membrane targeting could be uncoupled from Ca(V)beta-mediated modulation of channel gating. In addition to Ca(V)beta, Ca(V)alpha2delta and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca(V)alpha2delta caused a 5-fold stimulation of the whole cell currents measured with Ca(V)1.2 and Ca(V)beta3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca(V)1.2, extracellularly tagged Ca(V)1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca(V)1.2 with either Ca(V)alpha2delta, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca(V)1.2 subunits. In contrast, co-expression with Ca(V)beta3 stimulated plasma membrane expression of Ca(V)1.2 by a 10-fold factor. Mutations within the alpha interaction domain of Ca(V)1.2 or within the nucleotide kinase domain of Ca(V)beta3 disrupted the Ca(V)beta3-induced plasma membrane targeting of Ca(V)1.2. Altogether, these data support a model where high affinity binding of Ca(V)beta to the I-II linker of Ca(V)alpha1 largely accounts for Ca(V)beta-induced plasma membrane targeting of Ca(V)1.2.

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tumor suppressor versus oncogene paradox of CAV1 during tumor development tempted us to investigate the role for the epigenetic drift of CAV1 gene regulation. METHODS: We have analyzed CAV1 gene expression and associated epigenetic marks (DNA methylation and histone 3 modifications) in the CAV1 promoter in two colon cancer cell lines, under treatment with well established epigenetic modulators, AZA, SAM, TSA and SFN at varying concentrations. CAV1 gene promoter DNA methylation and histone modifications were analyzed by DNA methylation specific PCR, bisulphite modification of DNA and ChIP analyses following PCR respectively. RESULTS: Ectopic expression of CAV1 by epigenetic modulators inhibits colon cancer cell growth. CAV1 promoter DNA remains unmethylated before and after treatment with epigenetic modulators, which confirmed that DNA methylation is not the regulator of CAV1 expression in colon cancer. There was enrichment of H3K4me3 and H3K9AcS10P and depletion of H3K9me3 modifications around the CAV1 promoter. CONCLUSIONS: Our data provides novel insight into the regulation of CAV1 gene by histone H3 modifications and enhance the amplitude of the cancer epigenome.

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nd Cav1.3 with estrogen in female sexual dysfunction.
METHODS: Forty female SD rats of 8 weeks were randomly divided into Groups A (2-week sham operation), B (4-week sham operation), C (2-week castration) and D (4-week castration). Two and 4 weeks after surgery, the serum estradiol level was determined with the automated immunochemiluminescence system and the expressions of RyR1 and Cav1.3 in the vaginal smooth muscle were detected by immunohistochemistry and RT-PCR. Gray scale ratio was used to represent the mRNA expression levels of RyR1 and Car1.3, and the optical density value to denote their protein expression levels.
RESULTS: Serum estradiol was significantly decreased in Group C ([0.210 +/- 0.026] nmol/L) as compared with A ([0.505 +/- 0.053] nmol/L) (P < 0.01), and so was it in Group D ([0.130 +/- 0.031] nmol/L) in comparison with B ([0.476 +/- 0.058] nmol/L) (P < 0.01). RyR1 and Cav1.3 were expressed in all groups. The mRNA expressions of RyR1 and Cav1.3 were significantly reduced in Group C (0. 680 +/- 0.073 and 0.580 +/- 0.043) as compared with A (0.950 +/- 0.064 and 0.870 +/- 0.019) (P < 0.01), as well as in Group D (0.220 +/- 0.032 and 0.190 +/- 0.020) in comparison with B (0.890 +/- 0.072 and 0.820 +/- 0.021) (P < 0.01). The protein expressions of RyR1 and Cav1.3 were significantly down-regulated in Group C (96.67 +/- 7.75 and 87.97 +/- 6.96) as compared with A (123.69 +/- 10.66 and 106.46 +/- 8.04) (P < 0.01), and so were they in D (86.45 +/- 8.16 and 69.43 +/- 8.30) in comparison with B (109.31 +/- 9.87 and 97.38 +/- 7.56) (P < 0.01).
CONCLUSION: Both RyR1 and Cav1.3 were expressed in the vaginal smooth muscle cells of the rats, and estrogen might be involved in the regulation of female sexual reaction by acting on the expressions of RyR1 and Cav1.3.

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cing factor Rbfox2 is known to regulate the CaV1.2 pre-mRNA alternative splicing events during neuronal development. However, Rbfox2's roles in modulating the key function of vascular CaV1.2 channel and in the pathogenesis of hypertension remain elusive. Here, we report that the proportion of CaV1.2 channels with alternative exon 9* is increased by 10.3%, whereas that with alternative exon 33 is decreased by 10.5% in hypertensive arteries. Surprisingly, the expression level of Rbfox2 is increased ≈3-folds, presumably because of the upregulation of a dominant-negative isoform of Rbfox2. In vascular smooth muscle cells, we find that knockdown of Rbfox2 dynamically increases alternative exon 9*, whereas decreases exon 33 inclusion of CaV1.2 channels. By patch-clamp studies, we show that diminished Rbfox2-induced alternative splicing shifts the steady-state activation and inactivation curves of vascular CaV1.2 calcium channel to hyperpolarization, which makes the window current potential to more negative. Moreover, siRNA-mediated knockdown of Rbfox2 increases the pressure-induced vascular myogenic tone of rat mesenteric artery. Taken together, our data indicate that Rbfox2 modulates the functions of vascular CaV1.2 calcium channel by dynamically regulating the expressions of alternative exons 9* and 33, which in turn affects the vascular myogenic tone. Therefore, our work suggests a key role for Rbfox2 in hypertension, which provides a rational basis for designing antihypertensive therapies.

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eived three daily doses of ethanol every 8 hours for 4 consecutive days; control rats received vehicle. The IC was dissected at various time intervals following alcohol withdrawal, and the mRNA and protein levels of the CaV1.3 and CaV1.2 α1 subunits were measured. In separate experiments, rats were tested for their susceptibility to alcohol withdrawal-induced seizures (AWS) 3, 24, and 48 hours after alcohol withdrawal.
RESULTS: In the alcohol-treated group, AWS were observed 24 hours after withdrawal; no seizures were observed at 3 or 48 hours. No seizures were observed at any time in the control-treated rats. Compared to control-treated rats, the mRNA level of the CaV1.3 α1 subunit was increased 1.4-fold, 1.9-fold, and 1.3-fold at 3, 24, and 48 hours, respectively. In contrast, the mRNA level of the CaV1.2 α1 subunit increased 1.5-fold and 1.4-fold at 24 and 48 hours, respectively. At 24 hours, Western blot analyses revealed that the levels of the CaV1.3 and CaV1.2 α1 subunits increased by 52% and 32%, respectively, 24 hours after alcohol withdrawal. In contrast, the CaV1.2 and CaV1.3 α1 subunits were not altered at either 3 or 48 hours during alcohol withdrawal.
CONCLUSIONS: Expression of the CaV1.3 α1 subunit increased in parallel with AWS development, suggesting that altered L-type CaV1.3 channel expression is an important feature of AWS pathogenesis.

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ocytes, it is unclear whether S100A1 exerts modulatory effects on other tissues in proximity with cardiac cells. Therefore, we sought to investigate the effects of exogenous S100A1 on Ca2+ signals and electrical properties of superior cervical ganglion (SCG) neurons. Immunostaining and Western blot assays indicated no endogenous S100A1 in SCG neurons. Cultured SCG neurons took up S100A1 when it was present in the extracellular medium. Inside the cell exogenous S100A1 localized in a punctate pattern throughout the cytoplasm but was excluded from the nuclei. S100A1 partially colocalized with markers for both receptor- and non-receptor-mediated endocytosis, indicating that in SCG neurons multiple endocytotic pathways are involved in S100A1 internalization. In compartmentalized SCG cultures, axonal projections were capable of uptake and transport of S100A1 toward the neuronal somas. Exogenous S100A1 applied either extra- or intracellularly enhanced Cav1 channel currents in a PKA-dependent manner, prolonged action potentials, and amplified action potential-induced Ca2+ transients. NMR chemical shift perturbation of Ca2+-S100A1 in the presence of a peptide from the regulatory subunit of PKA verifies that S100A1 directly interacts with PKA, and that this interaction likely occurs in the hydrophobic binding pocket of Ca2+-S100A1. Our results suggest the hypothesis that in sympathetic neurons exogenous S100A1 may lead to an increase of sympathetic output.

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pha(1)1.2 subunit of Ca(v)1.2. PKA is bound to Ca(v)1.2 within a macromolecular signaling complex consisting of the beta(2) adrenergic receptor, trimeric G(s) protein, and adenylyl cyclase for fast, localized, and hence specific signaling [Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Buret, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) Science 293, 98-101]. Protein phosphatase 2A (PP2A) serves to effectively balance serine 1928 phosphorylation by PKA through its association with the Ca(v)1.2 complex [Davare, M. A., Horne, M. C., and Hell, J. W. (2000) J. Biol. Chem. 275, 39710-39717]. We now show that native PP2A holoenzymes, as well as the catalytic subunit itself, bind to alpha(1)1.2 immediately downstream of serine 1928. Of those holoenzymes, only heterotrimeric PP2A containing B' and B' ' subunits copurify with alpha(1)1.2. Preventing the binding of PP2A by truncating alpha(1)1.2 28 residues downstream of serine 1928 hampers its dephosphorylation in intact cells. Our results demonstrate for the first time that a stable interaction of PP2A with Ca(v)1.2 is required for effective reversal of PKA-mediated channel phosphorylation. Accordingly, PKA as well as PP2A are constitutively associated with Ca(v)1.2 for its proper regulation by phosphorylation and dephosphorylation of serine 1928.

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n the regulation of Ca2+ influx through Ca(v)1.2 (L-type) Ca2+ channels. CaBP1 interacts directly with the alpha1 subunit of Ca(v)1.2 at sites that also bind CaM. CaBP1 binding to one of these sites, the IQ domain, is Ca2+ dependent and competitive with CaM binding. The physiological significance of this interaction is supported by the association of Ca(v)1.2 and CaBP1 in postsynaptic density fractions purified from rat brain. Moreover, in double-label immunofluorescence experiments, CaBP1 and Ca(v)1.2 colocalize in numerous cell bodies and dendrites of neurons, particularly in pyramidal cells in the CA3 region of the hippocampus and in the dorsal cortex. In electrophysiological recordings of cells transfected with Ca(v)1.2, CaBP1 greatly prolonged Ca2+ currents, prevented Ca2+-dependent inactivation, and caused Ca2+-dependent facilitation of currents evoked by step depolarizations and repetitive stimuli. These effects contrast with those of CaM, which promoted strong Ca2+-dependent inactivation of Ca(v)1.2 with these same voltage protocols. Our findings reveal how Ca2+-binding proteins, such as CaM and CaBP1, differentially adjust Ca2+ influx through Ca(v)1.2 channels, which may specify diverse modes of Ca2+ signaling in neurons.

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ll carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

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er axons and regulate axon pathfinding. To elucidate which channel molecules are present in pioneer axons, immunohistochemical analysis was performed to examine 14 voltage-gated ion channels (Kv1.1-Kv1.3, Kv3.1-Kv3.4, Kv4.3, Cav1.2, Cav1.3, Cav2.2, Nav1.2, Nav1.6, and Nav1.9) in nine axonal tracts in the developing rat forebrain, including the optic nerve, corpus callosum, corticofugal fibers, thalamocortical axons, lateral olfactory tract, hippocamposeptal projection, anterior commissure, hippocampal commissure, and medial longitudinal fasciculus. We found A-type K(+) channel Kv3.4 in both pioneer axons and early follower axons and L-type Ca(2)(+) channel Cav1.2 in pioneer axons and early and late follower axons. Spatially, Kv3.4 and Cav1.2 were colocalized with markers of pioneer neurons and pioneer axons, such as deleted in colorectal cancer (DCC), in most fiber tracts examined. Temporally, Kv3.4 and Cav1.2 were expressed abundantly in most fiber tracts during axon pathfinding but were downregulated beginning in synaptogenesis. By contrast, delayed rectifier Kv channels (e.g., Kv1.1) and Nav channels (e.g., Nav1.2) were absent from these fiber tracts (except for the corpus callosum) during pathfinding of pioneer axons. These data suggest that Kv3.4 and Cav1.2, two high-voltage-activated ion channels, may act together to control Ca(2)(+) -dependent electrical activity of pioneer axons and play important roles during axon pathfinding.

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toreceptor function and will thus lead to retinal degeneration. However, the RPE function which is influenced by bestrophin-1 is so far not identified. Previously we showed that bestrophin-1 interacts with L-type Ca(2)(+) channels of the CaV1.3 subtype and that the endogenously expressed bestrophin-1 is required for intracellular Ca(2)(+) regulation. A hallmark of Best's disease is the fast lipofuscin accumulation occurring already at young ages. Therefore, we addressed the hypothesis that bestrophin-1 might influence phagocytosis of photoreceptor outer segments (POS) by the RPE. Here, siRNA knock-down of bestrophin-1 expression as well as inhibition of L-type Ca(2)(+) channel activity modulated the POS phagocytosis in vitro. In vivo CaV1.3 expression appeared to be diurnal regulated with a higher expression rate in the afternoon. Compared to wild-type littermates, Ca V 1.3 (-/-) mice showed a shift in the circadian POS phagocytosis with an increased activity in the afternoon. Thus we suggest that mutant bestrophin-1 leads to an impaired regulation of the POS phagocytosis by the RPE which would explain the fast lipofuscin accumulation in Best patients.

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ory brainstem response and the expressions of Ca(v)1.3.3 calcium channels at the protein and mRNA levels were detected by immunohistochemistry, western blot, and real-time RT-PCR. Associated with the auditory brainstem response threshold increased with age, the Ca(v)1.3.3 expression was gradually decreased. In comparison with 4-week-old mice, Ca(v)1.3.3 expressions in the cochlea at 14, 24, and 48 weeks of age were significantly and gradually decreased at both the protein and the mRNA levels. Immunohistochemistry showed that the expression of Ca(v)1.3.3 was apparently reduced at the inner hair cells, outer hair cells, and stria vascularis in the cochlear lateral wall in the aged mice. Our findings indicate that Ca(v)1.3.3 calcium channel expression in the cochlea is reduced in the ARHL mice and is associated with ARHL. The data also support a view that Ca(v)1.3.3 calcium channel is a good target for prevention and therapy of ARHL.

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otide polymorphism (SNP) in caveolin-1 (CAV1 rs4730751) and 2 coding SNPs in transforming growth factor-beta 1 (TGFB1 rs1800471, codon 25; and rs1800470, codon 10) were associated with the NSF phenotype. RESULTS: Forty-one patients with a history of chronic kidney disease and GCCA administration were studied, including NSF cases (n = 17) and control subjects (n = 24) without clinical or histological evidence of NSF. No significant differences in the genotype frequencies at these SNPs in TGFB1 and CAV1 were found between patients with NSF and subjects without NSF. CONCLUSIONS: We conclude that polymorphisms in the genes encoding TGFB1 and CAV1 previously associated with the development and progression of fibrosis in several organ systems are not associated with development of NSF in this cohort of patients with renal impairment after GCCA exposure.

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;'>CAV1 is linked to CAD and MI in humans. In this study we analyzed a tagSNP for CAV1 in intron 2, rs3807989, for potential association with CAD.
METHODS AND RESULTS: We performed case-control association studies in three independent Chinese Han populations from GeneID, including 1249 CAD cases and 841 controls in Population I, 1260 cases and 833 controls in Population II and 790 cases and 1212 controls in Population III (a total of 3299 cases and 2886 controls). We identified significant association between rs3807989 and CAD in three independent populations and in the combined population (Padj = 2.18 × 10(-5), OR = 1.19 for minor allele A). We also detected significant association between rs3807989 and MI (Padj = 5.43 × 10(-5), OR = 1.23 for allele A). Allele A of SNP rs3807989 was also associated with a decreased level of LDL cholesterol. Although rs3807989 is a tagSNP for both CAV1 and nearby CAV2, allele A of SNP rs3807989 was associated with an increased expression level of CAV1 (both mRNA and protein), but not CAV2.
CONCLUSIONS: The data in this study demonstrated that rs3807989 at the CAV1/CAV2 locus was associated with significant risk of CAD and MI by increasing expression of CAV1 (but not CAV2). Thus, CAV1 becomes a strong candidate susceptibility gene for CAD/MI in humans.

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e, we used DNA sequence analysis to screen the coding regions of human CAV1 (MIM 601047).
RESULTS: We found a heterozygous frameshift mutation in CAV1, designated I134fsdelA-X137, in a female patient who had atypical partial lipodystrophy, with subcutaneous fat loss affecting the upper part of her body and face, but sparing her legs, gluteal region and visceral fat stores. She had severe type 5 hyperlipoproteinemia, with recurrent pancreatitis. In addition, she had some atypical features, including congenital cataracts and neurological findings. Her father was also heterozygous for this mutation, and had a similar pattern of fat redistribution, hypertriglyceridemia and congenital cataracts, with milder neurological involvement. An unrelated patient had a different heterozygous frameshift mutation in the CAV1 gene, designated -88delC. He also had a partial lipodystrophy phenotype, with subcutaneous fat loss affecting the arms, legs and gluteal region, but sparing his face, neck and visceral fat stores. He also had severe type 5 hyperlipoproteinemia, with recurrent pancreatitis; however he had no clinically apparent neurological manifestations. The mutations were absent from the genomes of 1063 healthy individuals.
CONCLUSION: Thus, very rare CAV1 frameshift mutations appear to be associated with atypical lipodystrophy and hypertriglyceridemia.

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CaV1.3 channels are modulated by the insulin-like growth factor-1/receptor tyrosine kinase (IGF-1/RTK) through a signaling pathway that involves phospholipase C, calcium release from IP3-sensitive internal stores, and calcium/calmodulin kinase II. In addition, we find that the IGF-1-induced modulation requires phosphorylation of a specific serine residue, S1486, in the EF hand motif of the CaV1.3 subunit. This modulation alters CaV1.3 activity, causing a left shift in the current-voltage relationship and strongly potentiating peak currents at hyperpolarized membrane potentials. We also find that CaV1.3 channels and their RTK-dependent potentiation contribute to the regulation of the survival-promoting transcription factor cAMP response element-binding protein (CREB): in both cortical and hippocampal neurons, depolarization and IGF-1 rapidly increase phospho-CREB levels in a manner that requires CaV1.3 activity and the S1486 phosphorylation site to achieve a full effect. Although the full effects of CaV1.3 channels remain to be determined, their preferential localization to dendritic shafts and spine heads coupled with their ability to activate at relatively hyperpolarized and even subthreshold potentials suggests that CaV1.3 activity may subserve different cellular functions from CaV1.2 and, in particular, may be important in transducing signals initiated by excitatory neurotransmission.

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vestigate the impact of the cell shape changes, due to abnormal formation of extensive square array junctions, on the lens opacification in the caveolin-1 knockout mice. The cav1-KO and wild-type mice at age 1-22 months were used. By light microscopy examinations, cav1-KO lenses at age 1-18 months were transparent in both cortical and nuclear regions, whereas some lenses older than 18 months old exhibited nuclear cataracts. Scanning EM consistently observed the massive formation of ridge-and-valley membrane surfaces in young fibers at approximately 150 mum deep in all cav1-KO lenses studied. In contrast, the typical ridge-and-valleys were only seen in mature fibers deeper than 400 mum in wild-type lenses. The resulting extensive ridge-and-valleys dramatically altered the overall cell shape in cav1-KO lenses. Remarkably, despite dramatic shape changes, these deformed fiber cells remained intact and made close contact with their neighboring cells. By freeze-fracture TEM, ridge-and-valleys exhibited the typical orthogonal arrangement of 6.6 nm square array intramembrane particles and displayed the narrowed extracellular spaces. Immunofluorescence analysis showed that AQP0 C-terminus labeling was significantly decreased in outer cortical fibers in cav1-KO lenses. However, freeze-fracture immunogold labeling showed that the AQP0 C-terminus antibody was sparsely distributed on the wavy square array junctions, suggesting that the cleavage of AQP0 C-termini might not yet be complete. The cav1-KO lenses with nuclear cataracts showed complete cellular breakdown and large globule formation in the lens nucleus. This study suggests that despite dramatic cell shape changes, the massive formation of wavy square array junctions in intact fibers may provide additional adhesive support for maintaining the narrowed extracellular spaces that are crucial for the transparency of cav1-KO lenses.

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o discover the effects of NPW-GPR7 on the function of CaV1.2 calcium channels in the vascular smooth muscle cells (VSMCs) and vasotone of arterial vessels. METHODS: By whole-cell patch clamp, we studied the effects of NPW-23, the active form of NPW, on the CaV1.2 channels in the heterologously transfected human embryonic kidney 293 cells and VSMCs isolated from rat. Living system was used to explore the physiological function of NPW-23 in arterial myogenic tone. To investigate the pathological relevance, NPW mRNA level of mesenteric arteries was measured in the hypertensive and normotensive rats. RESULTS: NPW's receptor GPR7 was coexpressed with CaV1.2 channels in arterial smooth muscle. NPW-23 increased the ICa,L in transfected human embryonic kidney 293 cells and VSMCs via GPR7, which could be abrogated by phospholipase C (PLC)/protein kinase C (PKC) inhibitors, not protein kinase A or protein kinase G inhibitor. After NPW-23 application, the expression of pan phospho-PKC was increased; moreover, intracellular diacylglycerol level, the second messenger catalyzed by PLC, was increased 1.5-2-fold. Application with NPW-23 increased pressure-induced vasotone of the rat mesenteric arteries. Importantly, the expression of NPW was decreased in the hypertensive rats. CONCLUSION: NPW-23 regulates ICa,L via GPR7, which is mediated by PLC/PKC signaling, and such a mechanism plays a role in modulating vascular myogenic tone, which may involve in the development of vascular hypertension.

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span style='font-weight:700;'>Cav1.2 residues 1923-1942 is required for beta-adrenergic regulation of Cav1.2. Despite the prominence of PKA-mediated phosphorylation of Cav1.2 S1928 within the newly identified beta2AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the beta2AR from Cav1.2 upon beta-adrenergic stimulation rendering Cav1.2 refractory for several minutes from further beta-adrenergic stimulation. This effect is lost in S1928A knock-in mice. Although AMPARs are clustered at postsynaptic sites like Cav1.2, beta2AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the beta2AR from Cav1.2 is a uniquely specific desensitization mechanism of Cav1.2 regulation by highly localized beta2AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT-LTP) depends on Cav1.2 and its regulation by channel-associated beta2AR.

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tion of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.

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he robustness of L-type calcium current expression, the response to changes in Ca(2)(+) channel Cav1.2 gene dosage was studied in adult mice. Using a cardiac-specific inducible Cre recombinase system, Cav1.2 mRNA was reduced to 11 +/- 1% of control values in homozygous floxed mice and the mice died rapidly (11.9 +/- 3 days) after induction of gene deletion. In these homozygous knockout mice, echocardiographic analysis showed that myocardial contractility was reduced to 14 +/- 1% of control values shortly before death. For these mice, no effective compensatory changes in ion channel gene expression were triggered following deletion of both Cav1.2 alleles, despite the dramatic decay in cardiac function. In contrast to the homozygote knockout mice, following knockout of only one Cav1.2 allele, cardiac function remained unchanged, as did survival.Cav1.2mRNAexpression in the left ventricle of heterozygous knockout mice was reduced to 58 +/- 3% of control values and there was a 21 +/- 2% reduction in Cav1.2 protein expression. There was no significant reduction in L-type Ca(2)(+) current density in these mice. The results are consistent with a model of L-type calcium channel biosynthesis in which there are one or more saturated steps, which act to buffer changes in both total Cav1.2 protein and L-type current expression.

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ns by Western blot analysis and the co-localization of p-CaMKII/CaV1.2 by double-labeling immunofluorescence in the hippocampus of APP/PS1 mice and VD gerbils. Additionally, expression of these proteins and intracellular calcium levels were examined in cultured neurons treated with Aß1-42. The expression of CaV1.2 protein was increased in VD gerbils and in cultured neurons but decreased in APP/PS1 mice; the expression of calmodulin protein was increased in APP/PS1 mice and VD gerbils; levels of p-CaMKII, p-CREB and BDNF proteins were decreased in AD and VD models. The number of neurons in which p-CaMKII and CaV1.2 were co-localized, was decreased in the CA1 and CA3 regions in two models. Intracellular calcium was increased in the cultured neurons treated with Aß1-42. Collectively, our results suggest that the alterations in CaV1.2, calmodulin, p-CaMKII, p-CREB and BDNF can be reflective of an involvement in the impairment in memory and cognition in AD and VD models.

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rtant and its association with inherited diabetes-related phenotypes.
METHODS: Beta cells of human islets from cadaver donors were enriched using FACS to study the expression of the genes encoding voltage-gated calcium channel (Cav)1.2 and Cav1.3 by absolute quantitative PCR in whole human and rat islets, as well as in clonal cells. Single-cell exocytosis was monitored as increases in cell capacitance after treatment with small interfering (si)RNA against CACNA1D (which encodes Cav1.3). Three single nucleotide polymorphisms (SNPs) were genotyped in 8,987 non-diabetic and 2,830 type 2 diabetic individuals from Finland and Sweden and analysed for associations with type 2 diabetes and insulin phenotypes.
RESULTS: In FACS-enriched human beta cells, CACNA1D mRNA expression exceeded that of CACNA1C (which encodes Cav1.2) by approximately 60-fold and was decreased in islets from type 2 diabetes patients. The latter coincided with diminished secretion of insulin in vitro. CACNA1D siRNA reduced glucose-stimulated insulin release in INS-1 832/13 cells and exocytosis in human beta cells. Phenotype/genotype associations of three SNPs in the CACNA1D gene revealed an association between the C allele of the SNP rs312480 and reduced mRNA expression, as well as decreased insulin secretion in vivo, whereas both rs312486/G and rs9841978/G were associated with type 2 diabetes.
CONCLUSION/INTERPRETATION: We conclude that the L-type calcium channel Cav1.3 is important in human glucose-induced insulin secretion, and common variants in CACNA1D might contribute to type 2 diabetes.

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of the TS mutation on other L-channel gating mechanisms has yet to be investigated. We compared kinetic properties of exogenously expressed (HEK293 cells) rabbit cardiac L-channels with (G436R; corresponding to position 406 in human clone) and without (wild-type) the TS mutation. Our results surprisingly show that the TS mutation did not affect close-state voltage-dependent inactivation, which suggests different gating mechanisms underlie these two types of voltage-dependent inactivation. The TS mutation also significantly slowed activation at voltages less than 10 mV, and significantly slowed deactivation across all test voltages. Deactivation was slowed in the double mutant G436R/S439A, which suggests that phosphorylation of S439 was not involved. The L-channel agonist Bay K8644 increased the magnitude of both step and tail currents, but surprisingly failed to slow deactivation of TS channels. Our mathematical model showed that slowed deactivation plus impaired OSvdI combine to synergistically increase cardiac action potential duration that is a likely cause of arrhythmias in TS patients. Roscovitine, a tri-substituted purine that enhances L-channel OSvdI, restored TS-impaired OSvdI. Thus, inactivation-enhancing drugs are likely to improve cardiac arrhythmias and other pathologies afflicting TS patients.

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ndrites of hippocampal pyramidal neurons, but electrophysiological data suggest a broader distribution of these channels. To define the subcellular substrates underlying Ca(v)1.2 Ca(2+) signals, we analyzed the localization of Ca(v)1.2 in the hippocampal formation by using antibodies against the pore-forming alpha(1)-subunit of Ca(v)1.2 (alpha(1)1.2). By light microscopy, alpha(1)1.2-like immunoreactivity (alpha(1)1.2-IR) was detected in pyramidal cell soma and dendritic fields of areas CA1-CA3 and in granule cell soma and fibers in the dentate gyrus. At the electron microscopic level, alpha(1)1.2-IR was localized in dendrites, but also in axons, axon terminals, and glial processes in all hippocampal subfields. Plasmalemmal immunogold particles representing alpha(1)1.2-IR were more significant for small- than large-caliber dendrites and were largely associated with extrasynaptic regions in dendritic spines and axon terminals. These findings provide the first detailed ultrastructural analysis of Ca(v)1.2 localization in the brain and support functionally diverse roles of these channels in the hippocampal formation.