Dejie Yu

A Smooth Muscle Cav1.2 Calcium Channel Splice Variant Underlies Hyperpolarized Window Current and Enhanced State-dependent Inhibition by Nifedipine

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.

Alternative Splicing of the CaV1.3 Channel IQ Domain, a Molecular Switch for Ca2+-Dependent Inactivation within Auditory Hair Cells

Native CaV1.3 channels within cochlear hair cells exhibit a surprising lack of Ca2+-dependent inactivation (CDI), given that heterologously expressed CaV1.3 channels show marked CDI. To determine whether alternative splicing at the C terminus of the CaV1.3 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 CaV1.3IQΔ (“IQ deleted”) channels exhibited a lack of CDI, which was independent of the type of coexpressed β-subunits. CaV1.3IQΔ channel immunoreactivity was preferentially localized to cochlear outer hair cells (OHCs), whereas that of CaV1.3IQfull channels (IQ-possessing) labeled inner hair cells (IHCs). The preferential expression of CaV1.3IQΔ 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.

The small hydrophobic protein of the human respiratory syncytial virus forms pentameric ion channels

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. 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.

Smooth muscle-selective alternatively spliced exon generates functional variation in Cav1.2 calcium channels

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.

Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel

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.

Functional Characterization of Alternative Splicing in the C Terminus of L-type CaV1.3 Channels

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 Ca2+-dependent inactivation of the CaV1.3 channels. Conclusion: Alternative splicing at the C terminus alters the critical Ca2+ inhibitory feedback property of CaV1.3 channels. Significance: Alternative splicing is an exquisite mechanism for customizing channel function within diverse biological niches. CaV1.3 channels are unique among the high voltage-activated Ca2+ channel family because they activate at the most negative potentials and display very rapid calcium-dependent inactivation. Both properties are of crucial importance in neurons of the suprachiasmatic nucleus and substantia nigra, where the influx of Ca2+ ions at subthreshold membrane voltages supports pacemaking function. Previously, alternative splicing in the CaV1.3 C terminus gives rise to a long (CaV1.342) and a short form (CaV1.342A), resulting in a pronounced activation at more negative voltages and faster inactivation in the latter. It was further shown that the C-terminal modulator in the CaV1.342 isoforms modulates calmodulin binding to the IQ domain. Using splice variant-specific antibodies, we determined that protein localization of both splice variants in different brain regions were similar. Using the transcript-scanning method, we further identified alternative splicing at four loci in the C terminus of CaV1.3 channels. Alternative splicing of exon 41 removes the IQ motif, resulting in a truncated CaV1.3 protein with diminished inactivation. Splicing of exon 43 causes a frameshift and exhibits a robust inactivation of similar intensity to CaV1.342A. Alternative splicing of exons 44 and 48 are in-frame, altering interaction of the distal modulator with the IQ domain and tapering inactivation slightly. Thus, alternative splicing in the C terminus of CaV1.3 channels modulates its electrophysiological properties, which could in turn alter neuronal firing properties and functions.

Alternative Splicing at C Terminus of CaV1.4 Calcium Channel Modulates Calcium-dependent Inactivation, Activation Potential, and Current Density

Background: Alternative splicing diversifies calcium channel structure to change channel properties. Results: Extensive C-terminal alternative splicing generates channels differing in activation potential and voltage- and calcium-dependent inactivation properties. Conclusion: Diversification of channel function through altered structure is fine-tuned by alternative splicing. Significance: CaV1.4 C-terminal splice variations recapitulate some aspects of native photoreceptor calcium currents. 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 C terminus. The CaV1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ∼10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating 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 alternative splice variants may in part contribute to the properties of the native CaV1.4 channels.

Progesterone Impairs Human Ether-a-go-go-related Gene (HERG) Trafficking by Disruption of Intracellular Cholesterol Homeostasis

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-a-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.

C-Terminal Alternative Splicing of CaV1.3 Channels Distinctively Modulates Their Dihydropyridine Sensitivity

The transcripts of L-type voltage-gated calcium channels (CaV) 1.3 undergo extensive alternative splicing. Alternative splicing, particularly in the C terminus, drastically modifies gating properties of the channel. However, little is known about whether alternative splicing could modulate the pharmacologic properties of CaV1.3 in a manner similar to the paralogous CaV1.2. Here we undertook the screening of different channel splice isoforms harboring splice variations in either the IS6 segment or the C terminus. Unexpectedly, while inclusion of exon 8a or 8, which code for IS6, did not alter dihydropyridine (DHP) sensitivity, distinct pharmacologic properties were observed for the various C-terminal splice isoforms. In the presence of external Ca2+, fast inactivating splice variants including CaV1.342a and CaV1.343s with intact calmodulin-IQ domain interaction showed consistently low DHP sensitivity. Interestingly, attenuation of calcium-dependent inactivation with overexpression of calmodulin34 did not enhance the sensitivity of CaV1.342a, suggesting that the low DHP sensitivity may not be a result of fast channel inactivation. Alternatively, disruption of calmodulin-IQ domain binding in the CaV1.3Δ41 and full-length CaV1.342 channels was associated with heightened DHP sensitivity. In distinct contrast to the well-known modulation of DHP blockade of CaV1.2 channels, this study has therefore uncovered a novel mechanism for modulation of the pharmacologic properties of CaV1.3 channels through posttranscriptional modification of the C terminus.

Modest CaV1.342-selective inhibition by compound 8 is β-subunit dependent.

Two voltage-gated calcium channel subtypes—CaV1.2 and CaV1.3—underlie the major L-type Ca2+ currents in the mammalian central nervous system. Owing to their high sequence homology, the two channel subtypes share similar pharmacological properties, and at high doses classic calcium channel blockers, such as dihydropyridines, phenylalkylamines and benzothiazepines, do not discriminate between the two channel subtypes. Recent progress in treating Parkinson’s disease (PD) was marked by the discovery of synthetic compound 8, which was reported to be a highly selective inhibitor of the CaV1.3 L-type calcium channels (LTCC). However, despite a previously reported IC50 of ~24 μM, in our hands inhibition of the full-length CaV1.342 by compound 8 at 50 μM reaches a maximum of 45%. Moreover, we find that the selectivity of compound 8 towards CaV1.3 relative to CaV1.2B15 channels is greatly influenced by the β-subunit type and its splice isoform variants.