Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns.

The activity of voltage-gated K(+) (K(V)) channels plays an important role in regulating pulmonary artery smooth muscle cell (PASMC) contraction, proliferation, and apoptosis. The highly conserved NH(2)-terminal tetramerization domain (T1) of K(V) channels is important for proper channel assembly, association with regulatory K(V) beta-subunits, and localization of the channel to the plasma membrane. We recently reported two nonsynonymous mutations (G182R and E211D) in the KCNA5 gene of patients with idiopathic pulmonary arterial hypertension, which localize to the T1 domain of KCNA5. To study the electrophysiological properties and expression patterns of the mutants compared with the wild-type (WT) channel in vitro, we transfected HEK-293 cells with WT KCNA5, G182R, E211D, or the double mutant G182R/E211D channel. The mutants form functional channels; however, whole cell current kinetic differences between WT and mutant channels exist. Steady-state inactivation curves of the G182R and G182R/E211D channels reveal accelerated inactivation; the mutant channels inactivated at more hyperpolarized potentials compared with the WT channel. Channel protein expression was also decreased by the mutations. Compared with the WT channel, which was present in its mature glycosylated form, the mutant channels are present in greater proportion in their immature form in HEK-293 cells. Furthermore, G182R protein level is greatly reduced in COS-1 cells compared with WT. Immunostaining data support the hypothesis that, while WT protein localizes to the plasma membrane, mutant protein is mainly retained in intracellular packets. Overall, these data support a role for the T1 domain in channel kinetics as well as in KCNA5 channel subcellular localization.

Combination of sildenafil and simvastatin ameliorates monocrotaline-induced pulmonary hypertension in rats.

Sildenafil, a phosphodiesterase-5 inhibitor, and simvastatin, a cholesterol lowering drug, both have therapeutic effects on PAH; however, the combination of these drugs has not been tested in the treatment of PAH. The purpose of this study was to determine whether the combination of sildenafil and simvastatin is superior to each drug alone in the prevention of MCT-induced PAH. Phosphorylated Smad levels were decreased in lung tissue in MCT-injected rats, whereas ERK protein levels were increased. This indicates a possible role for an increase in mitogenic ERK activity in addition to decreased proapoptotic Smad signaling in the MCT model of PAH. Combination sildenafil and simvastatin treatment prevented the MCT-induced increases in right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH), exerted an anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC). Our results indicate that combination therapy with sildenafil and simvastatin attenuated the development of pulmonary hypertension more than either treatment alone.

Perturbing dynamin reveals potent effects on the Drosophila circadian clock.

BACKGROUND: Transcriptional feedback loops are central to circadian clock function. However, the role of neural activity and membrane events in molecular rhythms in the fruit fly Drosophila is unclear. To address this question, we expressed a temperature-sensitive, dominant negative allele of the fly homolog of dynamin called shibire(ts1) (shi(ts1)), an active component in membrane vesicle scission. PRINCIPAL FINDINGS: Broad expression in clock cells resulted in unexpectedly long, robust periods (>28 hours) comparable to perturbation of core clock components, suggesting an unappreciated role of membrane dynamics in setting period. Expression in the pacemaker lateral ventral neurons (LNv) was necessary and sufficient for this effect. Manipulation of other endocytic components exacerbated shi(ts1)'s behavioral effects, suggesting its mechanism is specific to endocytic regulation. PKA overexpression rescued period effects suggesting shi(ts1) may downregulate PKA pathways. Levels of the clock component PERIOD were reduced in the shi(ts1)-expressing pacemaker small LNv of flies held at a fully restrictive temperature (29 degrees C). Less restrictive conditions (25 degrees C) delayed cycling proportional to observed behavioral changes. Levels of the neuropeptide PIGMENT-DISPERSING FACTOR (PDF), the only known LNv neurotransmitter, were also reduced, but PERIOD cycling was still delayed in flies lacking PDF, implicating a PDF-independent process. Further, shi(ts1) expression in the eye also results in reduced PER protein and per and vri transcript levels, suggesting that shibire-dependent signaling extends to peripheral clocks. The level of nuclear CLK, transcriptional activator of many core clock genes, is also reduced in shi(ts1) flies, and Clk overexpression suppresses the period-altering effects of shi(ts1). CONCLUSIONS: We propose that membrane protein turnover through endocytic regulation of PKA pathways modulates the core clock by altering CLK levels and/or activity. These results suggest an important role for membrane scission in setting circadian period.

Hypoxia selectively inhibits KCNA5 channels in pulmonary artery smooth muscle cells.

Acute hypoxia induces pulmonary vasoconstriction and chronic hypoxia causes pulmonary vascular remodeling characterized by significant vascular medial hypertrophy. Electromechanical and pharmacomechanical mechanisms are involved in regulating pulmonary vasomotor tone, while changes in cytosolic Ca2+ concentration ([Ca2+](cyt)) are an important signal in regulating contraction and proliferation of pulmonary artery smooth muscle cells (PASMC). Hypoxia-induced increases in [Ca2+](cyt) are, in part, mediated by selective inhibition of voltage-gated K+ (Kv) channels in PASMC. Kv1.5, encoded by the KCNA5 gene, is a Kv channel alpha subunit that forms functional homotetrameric and heterotetrameric Kv channels in PASMC. Activity of Kv channels contributes to the regulation of resting membrane potential. Overexpression of the human KCNA5 gene in rat PASMC and other cell types increases whole-cell Kv currents and causes membrane hyperpolarization. However, acute hypoxia only reduced Kv currents in KCNA5-transfected PASMC. These results provide compelling evidence that Kv1.5 is an important hypoxia-sensitive Kv channel in PASMC, contributing to regulation of membrane potential and intracellular Ca2+ homeostasis during hypoxia. This hypoxia-sensitive mechanism essential for inhibiting Kv1.5 channel activity is exclusively present in PASMC.

Functional characterization of voltage-gated K+ channels in mouse pulmonary artery smooth muscle cells.

Mice are useful animal models to study pathogenic mechanisms involved in pulmonary vascular disease. Altered expression and function of voltage-gated K(+) (K(V)) channels in pulmonary artery smooth muscle cells (PASMCs) have been implicated in the development of pulmonary arterial hypertension. K(V) currents (I(K(V))) in mouse PASMCs have not been comprehensively characterized. The main focus of this study was to determine the biophysical and pharmacological properties of I(K(V)) in freshly dissociated mouse PASMCs with the patch-clamp technique. Three distinct whole cell I(K(V)) were identified based on the kinetics of activation and inactivation: rapidly activating and noninactivating currents (in 58% of the cells tested), rapidly activating and slowly inactivating currents (23%), and slowly activating and noninactivating currents (17%). Of the cells that demonstrated the rapidly activating noninactivating current, 69% showed I(K(V)) inhibition with 4-aminopyridine (4-AP), while 31% were unaffected. Whole cell I(K(V)) were very sensitive to tetraethylammonium (TEA), as 1 mM TEA decreased the current amplitude by 32% while it took 10 mM 4-AP to decrease I(K(V)) by a similar amount (37%). Contribution of Ca(2+)-activated K(+) (K(Ca)) channels to whole cell I(K(V)) was minimal, as neither pharmacological inhibition with charybdotoxin or iberiotoxin nor perfusion with Ca(2+)-free solution had an effect on the whole cell I(K(V)). Steady-state activation and inactivation curves revealed a window K(+) current between -40 and -10 mV with a peak at -31.5 mV. Single-channel recordings revealed large-, intermediate-, and small-amplitude currents, with an averaged slope conductance of 119.4 +/- 2.7, 79.8 +/- 2.8, 46.0 +/- 2.2, and 23.6 +/- 0.6 pS, respectively. These studies provide detailed electrophysiological and pharmacological profiles of the native K(V) currents in mouse PASMCs.

Function of Kv1.5 channels and genetic variations of KCNA5 in patients with idiopathic pulmonary arterial hypertension.

The pore-forming alpha-subunit, Kv1.5, forms functional voltage-gated K(+) (Kv) channels in human pulmonary artery smooth muscle cells (PASMC) and plays an important role in regulating membrane potential, vascular tone, and PASMC proliferation and apoptosis. Inhibited Kv channel expression and function have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). Here, we report that overexpression of the Kv1.5 channel gene (KCNA5) in human PASMC and other cell lines produced a 15-pS single channel current and a large whole cell current that was sensitive to 4-aminopyridine. Extracellular application of nicotine, bepridil, correolide, and endothelin-1 (ET-1) all significantly and reversibly reduced the Kv1.5 currents, while nicotine and bepridil also accelerated the inactivation kinetics of the currents. Furthermore, we sequenced KCNA5 from IPAH patients and identified 17 single-nucleotide polymorphisms (SNPs); 7 are novel SNPs. There are 12 SNPs in the upstream 5' region, 2 of which may alter transcription factor binding sites in the promoter, 2 nonsynonymous SNPs in the coding region, 2 SNPs in the 3'-untranslated region, and 1 SNP in the 3'-flanking region. Two SNPs may correlate with the nitric oxide-mediated decrease in pulmonary arterial pressure. Allele frequency of two other SNPs in patients with a history of fenfluramine and phentermine use was significantly different from patients who have never taken the anorexigens. These results suggest that 1) Kv1.5 channels are modulated by various agonists (e.g., nicotine and ET-1); 2) novel SNPs in KCNA5 are present in IPAH patients; and 3) SNPs in the promoter and translated regions of KCNA5 may underlie the altered expression and/or function of Kv1.5 channels in PASMC from IPAH patients.

Acute hypoxia selectively inhibits KCNA5 channels in pulmonary artery smooth muscle cells.

Acute hypoxia causes pulmonary vasoconstriction in part by inhibiting voltage-gated K(+) (Kv) channel activity in pulmonary artery smooth muscle cells (PASMC). The hypoxia-mediated decrease in Kv currents [I(K(V))] is selective to PASMC; hypoxia has little effect on I(K(V)) in mesenteric artery smooth muscle cells (MASMC). Functional Kv channels are homo- and/or heterotetramers of pore-forming alpha-subunits and regulatory beta-subunits. KCNA5 is a Kv channel alpha-subunit that forms functional Kv channels in PASMC and regulates resting membrane potential. We have shown that acute hypoxia selectively inhibits I(K(V)) through KCNA5 channels in PASMC. Overexpression of the human KCNA5 gene increased I(K(V)) and caused membrane hyperpolarization in HEK-293, COS-7, and rat MASMC and PASMC. Acute hypoxia did not affect I(K(V)) in KCNA5-transfected HEK-293 and COS-7 cells. However, overexpression of KCNA5 in PASMC conferred its sensitivity to hypoxia. Reduction of Po(2) from 145 to 35 mmHg reduced I(K(V)) by approximately 40% in rat PASMC transfected with human KCNA5 but had no effect on I(K(V)) in KCNA5-transfected rat MASMC (or HEK and COS cells). These results indicate that KCNA5 is an important Kv channel that regulates resting membrane potential and that acute hypoxia selectively reduces KCNA5 channel activity in PASMC relative to MASMC and other cell types. Because Kv channels (including KCNA5) are ubiquitously expressed in PASMC and MASMC, the observation from this study indicates that a hypoxia-sensitive mechanism essential for inhibiting KCNA5 channel activity is exclusively present in PASMC. The divergent effect of hypoxia on I(K(V)) in PASMC and MASMC also may be due to different expression levels of KCNA5 channels.

Suggestive evidence for association of the circadian genes PERIOD3 and ARNTL with bipolar disorder.

Bipolar affective disorder (BPAD) is suspected to arise in part from malfunctions of the circadian system, a system that enables adaptation to a daily and seasonally cycling environment. Genetic variations altering functions of genes involved with the input to the circadian clock, in the molecular feedback loops constituting the circadian oscillatory mechanism itself, or in the regulatory output systems could influence BPAD as a result. Several human circadian system genes have been identified and localized recently, and a comparison with linkage hotspots for BPAD has revealed some correspondences. We have assessed evidence for linkage and association involving polymorphisms in 10 circadian clock genes (ARNTL, CLOCK, CRY2, CSNK1epsilon, DBP, GSK3beta, NPAS2, PER1, PER2, and PER3) to BPAD. Linkage analysis in 52 affected families showed suggestive evidence for linkage to CSNK1epsilon. This finding was not substantiated in the association study. Fifty-two SNPs in 10 clock genes were genotyped in 185 parent proband triads. Single SNP TDT analyses showed no evidence for association to BPAD. However, more powerful haplotype analyses suggest two candidates deserving further studies. Haplotypes in ARNTL and PER3 were found to be significantly associated with BPAD via single-gene permutation tests (PG = 0.025 and 0.008, respectively). The most suggestive haplotypes in PER3 showed a Bonferroni-corrected P-value of PGC = 0.07. These two genes have previously been implicated in circadian rhythm sleep disorders and affective disorders. With correction for the number of genes considered and tests conducted, these data do not provide statistically significant evidence for association. However, the trends for ARNTL and PER3 are suggestive of their involvement in bipolar disorder and warrant further study in a larger sample.

Diversity of voltage-dependent K+ channels in human pulmonary artery smooth muscle cells.

Electrical excitability, which plays an important role in excitation-contraction coupling in the pulmonary vasculature, is regulated by transmembrane ion flux in pulmonary artery smooth muscle cells (PASMC). This study examined the heterogeneous nature of native voltage-dependent K(+) channels in human PASMC. Both voltage-gated K(+) (K(V)) currents and Ca(2+)-activated K(+) (K(Ca)) currents were observed and characterized. In cell-attached patches of PASMC bathed in Ca(2+)-containing solutions, depolarization elicited a wide range of K(+) unitary conductances (6-290 pS). When cells were dialyzed with Ca(2+)-free and K(+)-containing solutions, depolarization elicited four components of K(V) currents in PASMC based on the kinetics of current activation and inactivation. Using RT-PCR, we detected transcripts of 1) 22 K(V) channel alpha-subunits (K(V)1.1-1.7, K(V)1.10, K(V)2.1, K(V)3.1, K(V)3.3-3.4, K(V)4.1-4.2, K(V)5.1, K(V) 6.1-6.3, K(V)9.1, K(V)9.3, K(V)10.1, and K(V)11.1), 2) three K(V) channel beta-subunits (K(V)beta 1-3), 3) four K(Ca) channel alpha-subunits (Slo-alpha 1 and SK2-SK4), and 4) four K(Ca) channel beta-subunits (K(Ca)beta 1-4). Our results show that human PASMC exhibit a variety of voltage-dependent K(+) currents with variable kinetics and conductances, which may result from various unique combinations of alpha- and beta-subunits forming the native channels. Functional expression of these channels plays a critical role in the regulation of membrane potential, cytoplasmic Ca(2+), and pulmonary vasomotor tone.