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P-type (CaV+2.1) Ca2+ channels are a central conduit of neuronal Ca2+ entry, so their Ca2+ feedback regulation promises widespread neurobiological impact. Heterologous expression of recombinant CaV2.1 channels demonstrates that the Ca2+ sensor calmodulin can trigger Ca2+-dependent facilitation (CDF) of channel opening. This facilitation occurs when local Ca2+ influx through individual channels selectively activates the C-terminal lobe of calmodulin. In neurons, however, such calmodulin-mediated processes have yet to be detected, and CDF of native P-type current has thus far appeared different, arguably triggered by other Ca2+ sensing molecules. Here, in cerebellar Purkinje somata abundant with prototypic P-type channels, we find that the C-terminal lobe of calmodulin does produce CDF, and such facilitation augments Ca2+ entry during stimulation by repetitive action-potential and complex-spike waveforms. Beyond recapitulating key features of recombinant channels, these neurons exhibit an additional modulatory dimension: developmental upregulation of CDF during postnatal week 2. This phenomenon reflects increasing somatic expression of CaV2.1 splice variants that manifest CDF and progressive dendritic targeting of variants lacking CDF. Calmodulin-triggered facilitation is thus fundamental to native CaV+2.1 and rapidly enhanced during early development. [ABSTRACT FROM AUTHOR]

Alternative splicing of the P/Q-type channel (Cav2.1) promises customization of the computational repertoire of neurons. Here we report that concerted splicing of its main α1A subunit, at both an EF-hand-like domain and the channel C terminus, controls the form of Ca2+-dependent facilitation (CDF), an activity-dependent enhancement of channel opening that is triggered by calmodulin. In recombinant channels, such alternative splicing switches CDF among three modes: (1) completely "ON" and driven by local Ca2+ influx through individual channels, (2) completely "OFF," and (3) partially OFF but inducible by elevated global Ca2+ influx. Conversion from modes 1 to 3 represents an unprecedented dimension of control. The physiological function of these variants is likely important, because we find that the distribution of EF-hand splice variants is strikingly heterogeneous in the human brain, varying both across regions and during development. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a progressive neurodegenerative disease with no known cure and affects approximately 1% of the elderly population. The major question in PD relates to the selective loss of dopaminergic neurons in patients. The underlying mechanism of genetic dysfunction and environmental toxins in contributing to the pathogenesis of PD may be oxidative stress. The interactions of genetic and environmental factors in PD may provide some answers to the longstanding question. In particular, the possibility that iron may provide selectivity to genetic susceptibility or dopamine reactivity in dopaminergic neuronal death is enhanced by the neuroprotection demonstrated in transgenic mice overexpressing ferritin or the use of iron chelators in MPTP-induced PD mouse. It will be important to dissect and understand the contributions of genes, environment and intrinsic cellular states in the generation and progression of the pathophysiology of PD. IUBMB Life, 55: 323-327, 2003 [ABSTRACT FROM AUTHOR]

The mouse and human CD3G and D genes are organized in opposite transcriptional orientation, their 5′ ends being separated by about 1.6 kilobases (kb) of DNA. The molecular basis of the tissue-specific regulation of expression of the human CD3G and D genes was examined using DNase I hypersensitivity and CpG methylation analysis. Two T cell-specific DNase I hypersensitivity sites were defined within the intergenic region. A third hypersensitive site (DHS3) was detected 0.4 kb 3′ to the CD3D gene. This latter site was present in all T cells, but was absent in all other committed cell types examined. DHS3 was also detected in the lympho-myeloid progenitor cell KG1, but was absent when this line was induced to differentiate to the macrophage lineage. The intergenic region was undermethylated in T cells expressing CD3, but was in general more extensively methylated in other cell types. Importantly, however, in KG1 sublines which express the CD3 genes the intergenic region remains extensively methylated. These results define areas 3′ to the D gene and within the intergenic region which contain regulatory elements that influence both CD3D and G expression. They further show that transcription form the CD3D and G genes may occur initially from a methylated promoter. Significantly, the 3′ regulatory region was shown to adopt an open chromatin structure prior to lineage commitment and before CD3 transcription. [ABSTRACT FROM AUTHOR]

The prolongation of QT intervals in both mothers and fetuses during the later period of pregnancy implies that higher levels of progesterone may regulate the function of the human ether-α-go-go-related gene (HERG) potassium channel, a key ion channel responsible for controlling the length of QT intervals. Here, we studied the effect of progesterone on the expression, trafficking, and function of HERG channels and the underlying mechanism. Treatment with progesterone for 24 h decreased the abundance of the fully glycosylated form of the HERG channel in rat neonatal cardiac myocytes and HERG-HEK293 cells, a cell line stably expressing HERG channels. Progesterone also concentration-dependently decreased HERG current density, but had no effect on voltage-gated L-type Ca2+ and K+ channels. Immunofluorescence microscopy and Western blot analysis show that progesterone preferentially decreased HERG channel protein abundance in the plasma membrane, induced protein accumulation in the dilated endoplasmic reticulum (ER), and increased the protein expression of C/EBP homologous protein, a hallmark of ER stress. Application of 2-hydroxypropyl-β-cyclodextrin (a sterol-binding agent) or overexpression of Rab9 rescued the progesterone-induced HERG trafficking defect and ER stress. Disruption of intracellular cholesterol homeostasis with simvastatin, imipramine, or exogenous application of cholesterol mimicked the effect of progesterone on HERG channel trafficking. Progesterone may impair HERG channel folding in the ER and/or block its trafficking to the Golgi complex by disrupting intracellular cholesterol homeostasis. Our findings may reveal a novel molecular mechanism to explain the QT prolongation and high risk of developing arrhythmias during late pregnancy. [ABSTRACT FROM AUTHOR]

L-type Cav1.2 Ca2+ channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Cav1.2 Ca2+ channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Cav1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Cav1.233L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Cav1.2 channels, Cav1.233L channels reduced the current density and altered the electrophysiological properties of the wild type Cav1.2 channels. Interestingly, the truncated 3.5-domain Cav1.233L channels also yielded a dominant negative effect on Cav1.3 channels, but not on Cav3.2 channels, suggesting that Cavβ subunits is required for Cav1.233L regulation. A biochemical study provided evidence that Cav1.233L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Cav1.233L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Cav1.2 channels. Moreover, the human Cav1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Cav1.2 channel. An electrophysiological study showed that human Cav1.233L channel is a functional channel but conducts Ca2+ ions at a much lower level. [ABSTRACT FROM AUTHOR]

The small hydrophobic (SH) protein is encoded by the human respiratory syncytial virus. Its absence leads to viral attenuation in the context of whole organisms, and it prevents apoptosis in infected cells. Herein, we have examined the structure of SH protein in detergent micelles and in lipid bilayers, by solution NMR and attenuated total reflection-Fourier transform infrared spectroscopy, respectively. We found that SH protein has a single α-helical transmembrane domain and forms homopentamers in several detergents. In detergent micelles, the transmembrane domain is flanked N-terminally by an α-helix that forms a ring around the lumen of the pore and C-terminally by an extended β-turn. SH protein was found in the plasma membrane of transiently expressing HEK 293 cells, which showed pH-dependent (acid-activated) channel activity. Channel activity was abolished in mutants lacking both native His residues, His22 and His51, but not when either His was present. Herein, we propose that the pentameric model of SH protein presented is a physiologically relevant conformation, albeit probably not the only one, in which SH contributes to RSV infection and replication. Viroporins are short (∼100 amino acids) viral membrane proteins that form oligomers of a defined size, act as proton or ion channels, and in general enhance membrane permeability in the host. However, with some exceptions, their precise biological role of their channel activity is not understood. In general, viroporins resemble poorly specialized proteins but are nevertheless critical for viral fitness. In vivo, viruses lacking viroporins usually exhibit an attenuated or weakened phenotype, altered tropism, and diminished pathological effects. We have chosen to study the SH protein, 64 amino acids long, found in the human respiratory syncytial virus because of the effect of RSV on human health and the lack of adequate antivirals. We show that SH protein forms oligomers that behave as ion channels when activated at low pH. This study adds SH protein to a growing group of viroporins that have been structurally characterized. Although the precise biological role of this pentameric channel is still unknown, this report is nevertheless essential to fill some of the many gaps that exist in the understanding of SH protein function. [ABSTRACT FROM AUTHOR]

The CaV1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type CaV1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native CaV1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the CaV1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the Cterminus. The CaV1.443*channel exhibited robust CDI, alarger current density, a hyperpolarized shift in activation potential by ~10mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible formodulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length CaV1.4. Naturally occurring alternativ esplice variants may in part contribute to the properties of the native CaV1.4 channels. [ABSTRACT FROM AUTHOR]

Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain, Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant a-synuclein. We found that DMT1 overexpression dramatically enhances Fe2+ uptake, which concomitantly promotes cell death. This Fe2+ mediated toxicity is aggravated by the presence of mutant z-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe2+-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe2+ overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe2+ -mediated cell death. [ABSTRACT FROM AUTHOR]

Native smooth muscle L-type Cav1.2 calcium channels have been shown to support a fraction of Ca2+ currents with a window current that is close to resting potential. The smooth muscle L-type Ca2+ channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Cav1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Cav1.2 channels. Here, we report a smooth muscle L-type Cav1.2 calcium channel splice variant, Cav1.2SM (1/8/9*/32//Δ33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Cav1.2b clone (1/8/9*/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Cav1.2CM clone (1a/8a/Δ9*/32/33) that is different from the established Cav1.2a (1a/8a/Δ9*/31/33). Importantly, Cav1.2SM channels were shown to be more sensitive to nifedipine blockade than Cav1.2b and cardiac Cav1.2CM channels when currents were recorded in either 5 mM Ba2+ or 1.8 mM Ca2+ external solutions. This is the first time that a smooth muscle Cav1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP. [ABSTRACT FROM AUTHOR]

Voltage-gated calcium channels play a major role in many important processes including muscle contraction, neurotransmission, excitation-transcription coupling, and hormone secretion. To date, 10 calcium channel α1-subunits have been reported, of which four code for L-type calcium channels. In our previous work, we uncovered by transcript-scanning the presence of 19 alternatively spliced exons in the L-type Cav1.2 α1-subunit. Here, we report the smooth muscle-selective expression of alternatively spliced exon 9* in Cav1.2 channels found on arterial smooth muscle. Specific polyclonal antibody against exon 9* localized the intense expression of 9*-containing Cav1.2 channels on the smooth muscle wall of arteries, but the expression on cardiac muscle was low. Whole-cell patch clamp recordings of the 9*-containing Cav1.2 channels in HEK293 cells demonstrated −9 and −11-mV hyperpolarized shift in voltage-dependent activation and current-voltage relationships, respectively. The steady-state inactivation property and sensitivity to blockade by nifedipine of the ±exon 9* splice variants were, however, not significantly different. Such cell-selective expression of an alternatively spliced exon strongly indicates the customization and fine tuning of calcium channel functions through alternative splicing of the pore-forming α1-subunit. The generation of proteomic variations by alternative splicing of the calcium channel Cav1.2 α1-Subunit can potentially provide a flexible mechanism for muscle or neuronal cells to respond to various physiological signals or to diseases. [ABSTRACT FROM AUTHOR]

The L-type (Cav1.2) voltage-gated calcium channels play critical roles in membrane excitability, gene expression, and muscle contraction. The generation of splice variants by the alternative splicing of the poreforming Cavl.2 α1-subunit (α11.2) may thereby provide potent means to enrich functional diversity. To date, however, no comprehensive scan of α11.2 splice variation has been performed, particularly in the human context. Here we have undertaken such a screen, exploiting recently developed "transcript scanning" methods to probe the human gene. The degree of variation turns out to be surprisingly large; 19 of the 55 exons comprising the human α11.2 gene were subjected to alternative splicing. Two of these are previously unrecognized exons and two others were not known to be spliced. Comparisons of fetal and adult heart and brain uncovered a large IVS3-S4 variability resulting from combinatorial utilization of exons 31-33. Electrophysiological characterization of such IVS3-S4 variation revealed unmistakable shifts in the voltage dependence of activation, according to an interesting correlation between increased IVS3-S4 linker length and activation at more depolarized potentials. Steady-state inactivation profiles remained unaltered. This systematic portrait of splice variation furnishes a reference library for comprehending combinatorial arrangements of Cav1.2 splice exons, especially as they impact development, physiology, and disease. [ABSTRACT FROM AUTHOR]