Tuck Wah Soong

Mutations in Potassium Channel Kir2.6 Cause Susceptibility to Thyrotoxic Hypokalemic Periodic Paralysis

Thyrotoxic hypokalemic periodic paralysis (TPP) is characterized by acute attacks of weakness, hypokalemia, and thyrotoxicosis of various etiologies. These transient attacks resemble those of patients with familial hypokalemic periodic paralysis (hypoKPP) and resolve with treatment of the underlying hyperthyroidism. Because of the phenotypic similarity of these conditions, we hypothesized that TPP might also be a channelopathy. While sequencing candidate genes, we identified a previously unreported gene (not present in human sequence databases) that encodes an inwardly rectifying potassium (Kir) channel, Kir2.6. This channel, nearly identical to Kir2.2, is expressed in skeletal muscle and is transcriptionally regulated by thyroid hormone. Expression of Kir2.6 in mammalian cells revealed normal Kir currents in whole-cell and single-channel recordings. Kir2.6 mutations were present in up to 33% of the unrelated TPP patients in our collection. Some of these mutations clearly alter a variety of Kir2.6 properties, all altering muscle membrane excitability leading to paralysis.

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.

RNA Editing of the IQ Domain in Cav1.3 Channels Modulates Their Ca2+-Dependent Inactivation

Adenosine-to-inosine RNA editing is crucial for generating molecular diversity, and serves to regulate protein function through recoding of genomic information. Here, we discover editing within Ca(v)1.3 Ca²⁺ channels, renown for low-voltage Ca²⁺-influx and neuronal pacemaking. Significantly, editing occurs within the channels IQ domain, a calmodulin-binding site mediating inhibitory Ca²⁺-feedback (CDI) on channels. The editing turns out to require RNA adenosine deaminase ADAR2, whose variable activity could underlie a spatially diverse pattern of Ca(v)1.3 editing seen across the brain. Edited Ca(v)1.3 protein is detected both in brain tissue and within the surface membrane of primary neurons. Functionally, edited Ca(v)1.3 channels exhibit strong reduction of CDI; in particular, neurons within the suprachiasmatic nucleus show diminished CDI, with higher frequencies of repetitive action-potential and calcium-spike activity, in wild-type versus ADAR2 knockout mice. Our study reveals a mechanism for fine-tuning Ca(v)1.3 channel properties in CNS, which likely impacts a broad spectrum of neurobiological functions.

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.

CaV1.2 channelopathies: from arrhythmias to autism, bipolar disorder, and immunodeficiency

Mutations of human CaV1.2 channel gene were identified only recently. The gain-of-function mutations were found at two mutually exclusive exons in patients with Timothy syndrome (TS). These patients exhibit prolonged QT interval and lethal cardiac arrhythmias. In contrast, the loss-of-function mutations of CaV1.2 channel in patients with Brugada syndrome produce short QT interval that could result in sudden cardiac death. TS patients also suffer from multi-organ dysfunction that includes neurological disorder such as autism and mental retardation reflecting the wide tissue distribution of CaV1.2 channel. Mutations found on different mutually exclusive exons determine the severity of the disease. Unexpectedly, TS patients may develop recurrent infections and bronchitis that suggests a role of CaV1.2 channel in the immune system. Furthermore, recent reports revealed a linkage of CaV1.2 channel polymorphism with multiple central nervous system disorders including bipolar disorder, depression, and schizophrenia. Here, we will discuss how alternative splicing modulates CaV1.2 channelopathy and the role of CaV1.2 channel in both excitable and non-excitable tissues.

Developmental Activation of Calmodulin-Dependent Facilitation of Cerebellar P-Type Ca2+ Current

P-type (CaV2.1) 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 CaV2.1 and rapidly enhanced during early development.

Enhanced Autophagy from Chronic Toxicity of Iron and Mutant A53T α-Synuclein IMPLICATIONS FOR NEURONAL CELL DEATH IN PARKINSON DISEASE

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 α-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 α-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.