The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

The L-type (Ca v 1.2) 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 Ca v 1.2 ␣ 1 -subunit (␣ 1 1.2) may thereby provide potent means to enrich functional diversity. To date, however, no comprehensive scan of ␣ 1 1.2 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 ␣ 1 1.2 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 Ca v 1.2 splice exons, especially as they impact development, physiology, and disease.Rapid influx of Ca 2ϩ through the Ca v 1.2 channels initiates physiological responses like gene expression, neurotransmitter release, cardiac or smooth muscle contraction, and regulation of Ca 2ϩ -dependent ion channels (1-4). In these capacities, the functional profile of Ca v 1.2 calcium channels can be customized by combinatorial assembly of the Ca v 1.2 ␣ 1 with several different auxiliary ␤-and ␣ 2 ␦-subunits (5). Even greater flexibility in functional tuning could arise from alternative splicing of the ␣ 1 -subunit genes (6); splicing of the human ␣ 1 1.2 subunit gene is known to generate variants with tissue-specific biases and with distinct pharmacological properties (7). At present, 15 of 53 known exons (Fig. 1) of the human ␣ 1 1.2 gene have been reported to be subjected to alternative splicing (8). However, the full set of possible splice loci and variations and their distributions in heart and brain could far exceed this initial view.Recently, genome-wide analyses suggest that as high as 74% of human genes are alternatively spliced (9). Alternative splicing of pre-mRNA has been implicated in development, physiology, and pathophysiology, and the inclusion or exclusion of exons can be regulated in a tissue-specific or temporal manner (10, 11). Splice variations of human ␣ 1 1.2 subunit confer on the channel isoforms altered properties such as sensitivity to blockade by antagonists, regulation by protein kinase, current density, and activation and inactivation characteristics (12)(13)(14). However, these studies have reported generally on the impact of al...

Background: Few effective treatments exist for human respiratory syncytial virus infection. The absence of small hydrophobic (SH) protein in RSV leads to viral attenuation. Results: SH protein forms pentamers and shows pH-dependent ion channel activity. Conclusion: SH protein forms pentameric ion channels. Significance: The SH protein and its channel activity constitute a potential drug target.

Background: Alternative splicing generates calcium channel splice variants with altered electrophysiological properties. Results: Exclusion of exons encoding the IQb domain or proximal/distal domains attenuates Ca 2ϩ -dependent inactivation of the Ca V 1.3 channels. Conclusion: Alternative splicing at the C terminus alters the critical Ca 2ϩ inhibitory feedback property of Ca V 1.3 channels. Significance: Alternative splicing is an exquisite mechanism for customizing channel function within diverse biological niches.

Native smooth muscle L-type Ca v 1.2 calcium channels have been shown to support a fraction of Ca 2؉ currents with a window current that is close to resting potential. The smooth muscle L-type Ca 2؉ channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca v 1.2 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 Ca v 1.2 channels. Here, we report a smooth muscle L-type Ca v 1.2 calcium channel splice variant, Ca v 1.2SM (1/8/9*/32/⌬33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca v 1.2b 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 Ca v 1.2CM clone (1a/8a/⌬9*/32/33) that is different from the established Ca v 1.2a (1a/8a/⌬9*/31/33). Importantly, Ca v 1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca v 1.2b and cardiac Ca v 1.2CM channels when currents were recorded in either 5 mM Ba 2؉ or 1.8 mM Ca 2؉ external solutions. This is the first time that a smooth muscle Ca v 1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.

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 ␣ 1 -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 Ca v 1.2 ␣ 1 -subunit. Here, we report the smooth muscle-selective expression of alternatively spliced exon 9* in Ca v 1.2 channels found on arterial smooth muscle. Specific polyclonal antibody against exon 9* localized the intense expression of 9*-containing Ca v 1.2 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 Ca v 1.2 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 ␣ 1 -subunit. The generation of proteomic variations by alternative splicing of the calcium channel Ca v 1.2 ␣ 1 -subunit can potentially provide a flexible mechanism for muscle or neuronal cells to respond to various physiological signals or to diseases.

Since its initial publication in 2002, the genome of Ciona intestinalis type A (Ciona robusta), the first genome sequence of an invertebrate chordate, has provided a valuable resource for a wide range of biological studies, including developmental biology, evolutionary biology, and neuroscience. The genome assembly was updated in 2008, and it included 68% of the sequence information in 14 pairs of chromosomes. However, a more contiguous genome is required for analyses of higher order genomic structure and of chromosomal evolution. Here, we provide a new genome assembly for an inbred line of this animal, constructed with short and long sequencing reads and Hi-C data. In this latest assembly, over 95% of the 123 Mb of sequence data was included in the chromosomes. Short sequencing reads predicted a genome size of 114–120 Mb; therefore, it is likely that the current assembly contains almost the entire genome, although this estimate of genome size was smaller than previous estimates. Remapping of the Hi-C data onto the new assembly revealed a large inversion in the genome of the inbred line. Moreover, a comparison of this genome assembly with that of Ciona savignyi, a different species in the same genus, revealed many chromosomal inversions between these two Ciona species, suggesting that such inversions have occurred frequently and have contributed to chromosomal evolution of Ciona species. Thus, the present assembly greatly improves an essential resource for genome-wide studies of ascidians.

Native Ca V 1.3 channels within cochlear hair cells exhibit a surprising lack of Ca 2ϩ -dependent inactivation (CDI), given that heterologously expressed Ca V 1.3 channels show marked CDI. To determine whether alternative splicing at the C terminus of the Ca V 1.3 gene may produce a hair cell splice variant with weak CDI, we transcript-scanned mRNA obtained from rat cochlea. We found that the alternate use of exon 41 acceptor sites generated a splice variant that lost the calmodulin-binding IQ motif of the C terminus. These Ca V 1.3 IQ⌬ ("IQ deleted") channels exhibited a lack of CDI, which was independent of the type of coexpressed ␤-subunits. Ca V 1.3 IQ⌬ channel immunoreactivity was preferentially localized to cochlear outer hair cells (OHCs), whereas that of Ca V 1.3 IQfull channels (IQ-possessing) labeled inner hair cells (IHCs). The preferential expression of Ca V 1.3 IQ⌬ within OHCs suggests that these channels may play a role in processes such as electromotility or activity-dependent gene transcription rather than neurotransmitter release, which is performed predominantly by IHCs in the cochlea.