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Am J Physiol Cell Physiol 292: C1837-C1853, 2007. First published January 31, 2007; doi:10.1152/ajpcell.00405.2006
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VASCULAR BIOLOGY

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

Carmelle V. Remillard,* Donna D. Tigno,* Oleksandr Platoshyn,* Elyssa D. Burg, Elena E. Brevnova, Diane Conger, Ann Nicholson, Brinda K. Rana, Richard N. Channick, Lewis J. Rubin, Daniel T. O'Connor, and Jason X.-J. Yuan

Departments of Medicine and Psychiatry, Center for Molecular Genetics, School of Medicine, University of California–San Diego, La Jolla, California

Submitted 26 July 2006 ; accepted in final form 24 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

voltage-gated K+ channel; single-nucleotide polymorphism; drug response; etiology

MEMBRANE POTENTIAL plays an important role in regulating cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) and thus pulmonary vascular tone by controlling Ca2+ influx through voltage-dependent Ca2+ channels (VDCC). The resting membrane potential is primarily determined by activities of Na+-K+-ATPase (Na+ pumps) and K+ channels in the plasma membrane. In human PASMC, activity of voltage-gated K+ (Kv) channels contributes to the regulation of membrane potential, [Ca2+]cyt, and, as a consequence, of excitation-contraction coupling in pulmonary vascular smooth muscle (66, 78). Inhibition of Kv channels in PASMC causes membrane depolarization, which then opens VDCC, increases [Ca2+]cyt by promoting Ca2+ influx, and induces pulmonary vasoconstriction. Inhibition of Kv channel activity is also implicated in stimulating PASMC proliferation by increasing [Ca2+]cyt (50) and in attenuating PASMC apoptosis by decelerating apoptotic volume decrease and decreasing cytoplasmic caspase activity (28). Conversely, activation of Kv channels in PASMC, such as induced by nitric oxide (NO) (80), causes membrane hyperpolarization, inhibits VDCC activity, and cause pulmonary vasodilation. Furthermore, activation of K+ channels is also involved in mediating apoptotic volume decrease, an early hallmark of apoptosis (7), and facilitating apoptosis (9).

Kv1.5 is an important pore-forming {alpha}-subunit that forms functional Kv channels in human PASMC and other smooth muscle cell types (5, 26, 44, 47, 81). Overexpression of the human Kv1.5 channel gene, KCNA5, in human PASMC and other cell lines (e.g., HEK-293 and COS cells) causes membrane hyperpolarization, accelerated apoptotic cell shrinkage, and enhanced cell apoptosis (8). Downregulation of KCNA5 expression using antisense oligonucleotides or short interfering RNA causes membrane depolarization and increases [Ca2+]cyt (5, 17), while pharmacological agents that block Kv1.5 channels [e.g., 4-aminopyridine (4-AP), correolide, and pergolide] stimulate PASMC contraction (5, 21), migration (56), and proliferation (50), but inhibit apoptosis (8).

Idiopathic pulmonary arterial hypertension (IPAH), previously referred to as primary pulmonary hypertension, is a fatal and progressive disease that predominantly affects young women. Increased pulmonary arterial pressure (PAP) in IPAH patients, due to heightened pulmonary vascular resistance (PVR), is mainly caused by sustained pulmonary vasoconstriction, pulmonary vascular wall remodeling (e.g., medial hypertrophy), and obliteration of small arteries in association with in situ thrombosis (24). Pulmonary vascular medial hypertrophy results mainly from increased proliferation and/or decreased apoptosis of PASMC (24).

Decreased K+ channel expression and activity may contribute to the excessive PASMC proliferation in IPAH patients (77, 81). Indeed, PASMC from IPAH patients exhibit downregulated expression and inhibited function of Kv channel {alpha} pore-forming subunits (e.g., Kv1.5) compared with PASMC from normal subjects and secondary pulmonary hypertensive patients (77, 81). Decreased Kv currents result in membrane depolarization, promote Ca2+ influx through VDCC, and increase [Ca2+]cyt. Similar phenomena have also been documented in PASMC from patients treated with appetite suppressants (e.g., aminorex and fenfluramine) (45, 69), which have been linked to the development of IPAH, as well as in PASMC from chronically hypoxic animals (52, 70, 71). Expression and function of the Kv1.5 channel are inhibited in PASMC from IPAH patients (81), in normal PASMC treated with fenfluramine (45), and in PASMC isolated from rats with chronic hypoxia-mediated pulmonary hypertension (6, 52, 55, 70, 71). Therefore, Kv1.5 channel dysfunction or downregulation may represent a predisposing factor in the development of pulmonary vasoconstriction and vascular medial hypertrophy in patients with IPAH, in conjunction with other factors and genetic defects.

The aims of this study were 1) to characterize biophysical and pharmacological properties of Kv1.5 (KCNA5) channels, 2) to define the sequence and transcription factor binding sites in the putative promoter region of the human Kv1.5 channel gene (KCNA5), and 3) to identify novel single-nucleotide polymorphisms (SNPs) in KCNA5 and its immediate upstream and downstream flanking sequences from IPAH patients.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell preparation and culture. Primary cultured PASMC from transplant patients and normal human PASMC purchased from Cambrex were used for electrophysiological experiments in this study. Lung tissues were obtained from patients during lung/heart transplantation. Peripheral muscular pulmonary arteries (150–300 µm diameter) isolated from the explanted lung tissues were first incubated in Hanks' balanced salt solution that contained 2 mg/ml collagenase (Worthington Biochemical) for 20 min to remove adventitia with fine forceps and to remove endothelium by a surgical scalpel. The remaining smooth muscle was then digested with (in mg/ml) 2.25 collagenase, 0.5 elastase, and 1 albumin (Sigma) at 37°C to make a cell suspension of PASMC. The cells were resuspended in the smooth muscle growth medium (SmGM; Cambrex) and cultured in an incubator under a humidified atmosphere of 5% CO2-95% air at 37°C. Human PASMC from normal subjects were also cultured in SmGM and used at passages 4–6 for experimentation. The medium was changed after 24 h and every 48 h thereafter. The SmGM was composed of smooth muscle basal medium supplemented with 5% FBS, 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor, and 5 µg/ml insulin. Cells were subcultured or plated onto 25-mm coverslips using trypsin-EDTA buffer (Cambrex) when 70–90% confluence was achieved.

Human embryonic kidney epithelial cells (HEK-293) and COS-7 (monkey kidney fibroblast-like cells) cells (American Type Culture Collection), and human coronary arterial smooth muscle cells (CASMC; Cambrex) were cultured in high-glucose (4.5 g/l) DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (BioFluids) and incubated in 5% CO2 at 37°C in a humidified atmosphere. Rat PASMC and mesenteric artery smooth muscle cells (MASMC) were isolated from intrapulmonary arteries removed from Sprague-Dawley rats (125–250 g) according to a previously published protocol (78, 79), plated onto 25-mm coverslips in 10% FBS-DMEM, and cultured (at 37°C) in an incubator equilibrated with 5% CO2. Cells were subcultured or split using 1 mg/ml trypsin (Sigma) when 70–90% confluence was achieved.

Constructs. In the KCNA5/pBK construct (kindly provided by Dr. M. Tamkun from Colorado State University), the coding sequence of the human KCNA5 gene was subcloned into XbaI and KpnI sites of multiple cloning site of the phagemid expression vector pBK-CMV (Stratagene). For electrophysiological experiments, a KCNA5/green fluorescent protein (GFP) construct was designed to visualize the transfected cells. In the KCNA5/GFP construct, the coding sequence of the human KCNA5 gene was subcloned into EcoRI and XbaI sites of the pCMS-EGFP mammalian expression vector (Clontech). In the pCMS-EGFP vector, the EGFP gene (which encodes the enhanced green fluorescent protein, a red-shifted variant of wild-type GFP from Aquorea victoria) is expressed separately from the gene of interest and is used as a transfection marker.

Transfection of KCNA5. Cells were transiently transfected with the expression constructs using Lipofectamine reagent according to the manufacturer's instruction. Briefly, cells were first split and then cultured for 24 h. Transfection was performed on 40–80% confluent cells at 37°C in serum-free Opti-MEM I medium (Invitrogen) with 1.6 µg/ml DNA and 4 µl/ml of Lipofectamine reagent. After 5–7 h of exposure to the transfection medium, cells were refed with construct-free serum-containing medium and incubated 12–24 h before experiments were performed. The transfection efficiency was consistently >30% for HEK-293 and COS-7 cells and 5–15% for human PASMC, human CASMC, rat PASMC, and rat MASMC using Lipofectamine reagent.

Electrophysiological measurement of Kv currents in KCNA5-transfected cells. Phagemid constructs containing the GFP-tagged human KCNA5 coding sequence were transfected into cells at first, as described previously (8). Whole cell and single channel Kv currents [IK(V)] were recorded at room temperature (22–24°C) from KCNA5-transfected cells using with an Axopatch-1D amplifier and a DigiData 1200 interface (Axon Instruments) using patch-clamp techniques. Patch pipettes (2–3 M{Omega}) were fabricated on an electrode puller (Sutter Instrument) using borosilicate glass tubes and fire polished on a microforge (Narishige Scientific Instruments). Command voltage protocols and data acquisition were performed using pCLAMP-8 software (Axon Instruments). Using the 2 to 3-M{Omega} pipettes, the series resistance was at a range of 4–9 M{Omega} when the whole cell configuration was formed. Series resistance compensation was performed in most of the whole cell experiments. Leak and capacitative currents were subtracted using the P/4 protocol in pCLAMP software. Cells were superfused with a solution containing (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with Tris). Pipettes contained (in mM) 135 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 2 Na2ATP (pH 7.2). 4-AP (Sigma) was added directly to the bathing solution; the pH was readjusted to 7.4 using 2 M HCl. Bepridil (Sigma) and correolide (kindly provided by Dr. Maria L. Garcia from Merck) were dissolved in DMSO to make stock solutions of 10–20 mM; aliquots of the stock solutions were then diluted to the bath solutions to final concentrations as indicated in RESULTS. Endothelin-1 (ET-1; Sigma) was directly dissolved in the bath solution on the day of use. The pH values of all the solutions were reevaluated after addition of the drugs and readjusted to 7.4.

Human subjects. One hundred and eighty-eight patients diagnosed with IPAH participated in our study. IPAH was diagnosed based on the criteria set forth by the National Institutes of Health Registry for Primary Pulmonary Hypertension. Informed consent, approved by the University of California Institutional Review Board, was obtained from all patients. All patients underwent right-heart catheterization as part of the standard diagnostic procedure for pulmonary hypertension.

Measurement of pulmonary hemodynamics in patients with IPAH. A flow-directed balloon-tipped Swan-Ganz catheter was positioned into the right ventricle or pulmonary artery via the internal jugular vein. PAP was measured by a pressure transducer (Namic) connected to a Mac-Laboratory 7000 hemodynamic and electrocardiographic monitoring system (GE Medical System). Cardiac output (CO) was measured by a thermodilution technique and PVR was calculated according to PAP and CO by the monitoring system. PAP, PVR, and CO were compared before and after inhalation of NO during catheterization to determine pulmonary vascular reactivity.

Isolation of genomic DNA from patients. Blood samples were collected from patients via the pulmonary catheter and stored in EDTA-containing tubes. Genomic DNA was extracted from the blood samples using a DNA isolation kit (PUREGENE, Gentra Systems). DNA purity was measured as the ratio of the absorbance at 260 and 280 nm with a spectrophotometer (model DU 520, Beckman Coulter).

Sequencing of KCNA5 and identification of SNPs. SNP discovery of the KCNA5 gene was performed by Agencourt Bioscience using its proprietary SeeSNP Modeling Suite for assay design, utilizing the GenBank accession number for human chromosome 12 (NT_009759) as input. The software mapped the mRNA and promoter sequence of KCNA5 against GenBank sequence contigs where complete sequence alignment was identified. The software then designed amplification primers and developed an amplicon tiling model spanning both coding and regulatory regions, including all intron/exon borders and the full exon sequence. Only primer pairs yielding high PCR success rates and specificity (Table 1) were passed onto high throughput PCR setup and sequencing. Genomic DNA samples were amplified against the 10 validated amplicons spanning KCNA5, excluding a portion of exon 6, which did not amplify during optimization due to high GC content. The PCR products were sequenced using BigDye version 3.1 reactions on ABI3700 instruments. SNPs identified in the sequence traces were verified using Phred/Phrap/Consed software and compared with known SNPs deposited in the National Center for Biotechnology Information (NCBI) SNP databank.


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  		Table 1. Primers used for sequencing human KCNA5

 
Promoter prediction and identification of transcription factor binding sites. Gene2Promoter and PromoterInspector software (Genomatix) were used to predict and verify the sequence and placement of the KCNA5 promoter. Gene2Promoter mapped our base sequence (NT_009759 [GenBank] ) to human chromosome 12p13 and identified the putative promoter region. This transcript region was confirmed by PromoterInspector, which predicts the genomic context of eukaryotic polymerase II promoter regions with high specificity in mammalian genomic sequences, based on equivalence classes of IUPAC words. The base reference sequence containing KCNA5 and its 5'-upstream flanking sequence corresponded to the input sequence for this analysis. The identified region was marked as a true positive if a transcription start site was located within or up to 200-bp downstream of the predicted promoter region. Vertebrate transcription factor binding sites were identified using MatInspector software (Genomatix).

RT-PCR. Total RNA was isolated from human PASMC and RT-PCR was performed according to protocols described previously (51). The sequences of sense and antisense primers (Table 2) were specifically designed from the coding regions of smooth muscle {alpha}-actin, calponin, and SM-22 (59). As a control for integrity of total RNA, primers specific for GAPDH were used. An electrophoresis documentation system (Eastman Kodak) was used to visualize the PCR product and to quantitate the intensity of the PCR product bands.


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  		Table 2. Oligonucleotide sequences of the primers used for RT-PCR

 
Statistical analysis. Data are expressed as means ± SE. Statistical differences of hemodynamic changes were assessed using Student's t-test. We used {chi}2-analyses to assess differences between genotype frequencies in drug-response groups. One-way ANOVA with post hoc analysis was performed to compare hemodynamics between different patient groups. For electrophysiological and pharmacological experiments, n refers to the number of cells used. For patient hemodynamics and SNP analysis, numbers of patients are given.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Biophysical properties of human Kv1.5 channels. The KCNA5 gene, located in chromosome 12p13, contains a single exon, which encodes for the Kv1.5 channel subunit, a pore-forming {alpha}-subunit that associates with other Kv channel {alpha}- and beta-subunits to form native channels (Fig. 1, A–C). Topology of the translated regions of Kv1.5 channel indicates that the channel contains 1) six transmembrane (TM) domains, 2) a pore (P) region between TM5 and TM6, 3) a cytoplasmic NH2 terminus containing a polymerization domain that is responsible for {alpha}:{alpha} and {alpha}:beta association, and 4) a cytoplasmic COOH-terminus that differs between Kv channel {alpha}-subunits (Fig. 1B). Functional Kv channels are homo- or heterotetramers composed of the same number of {alpha}- and beta-subunits ({alpha}4beta4) (Fig. 1C).


Figure 1
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  		Fig. 1. Structure of KCNA5 gene and characteristics of Kv1.5 channel currents. A: KCNA5 gene organization showing the start codon (dotted line with arrow), the heteromerization domain (Het. dom.), and 6 transmembrane (TM) segments (TM1-6). Noncoding flanking 5'- and 3'-untranslated regions (UTR) are also indicated by hatched boxes. Right, pictogram of human chromosome 12 highlighting the p13 region containing KCNA5 (see arrow at top). B: Kv channel {alpha}-subunit protein folding motif and regulatory beta-subunit binding; the pore region (P) is shown between TM-5 and TM-6. C: cross-sectional view of the arrangement of {alpha}- and beta-subunits into a tetrameric Kv channel with an ion-selective pore. D: representative single channel Kv1.5 currents (left) at different test potentials (TP) and the current-voltage (I-V) relationship curve (middle) recorded from a cell-attached membrane patch of a human pulmonary artery smooth muscle cell (PASMC) transiently transfected with KCNA5. The fluorescence microscopy image (right) depicts human PASMC transfected with KCNA5, identifiable by the green fluorescence. E: representative single channel Kv1.5 currents at a test potential (TP) of +100 mV from a COS-7 cell, a rat PASMC, a human embryonic kidney (HEK)-293 cell, and a human coronary artery smooth muscle cell (CASMC) transfected with KCNA5 (left). The summarized (n = 6, middle) I-V curve is constructed from single channel Kv1.5 currents recorded from multiple cell types (i.e., COS-7 cells, rat PASMC, HEK-293 cells, and human CASMC). Images identify KCNA5-transfected COS-7 cells, rat PASMC, HEK-293 cells, and rat mesenteric artery smooth muscle cells (rMASMC) by their green fluorescence (right and bottom).

 
In human PASMC transiently transfected with KCNA5, single channel KCNA5 currents had a slope conductance of ~14.7 pS (Fig. 1D). The slope conductance of Kv1.5 channels in human PASMC was not significantly different in KCNA5-transfected COS-7 cells, rat PASMC, HEK-293 cells, and human CASMC (14.4 pS in average) (Fig. 1E). The transfection rate of human KCNA5 was >30% in COS-7 and HEK-293 cells and 5–15% in human PASMC, rat PASMC, and rat MASMC (Fig. 1, D and E, right and bottom images).

Overexpression of KCNA5 in human PASMC increased whole cell outward K+ currents (recorded 24–48 h after initial transfection) by ~29 times; the amplitude of whole cell IK(V) at +60 mV in wild-type and KCNA5-transfected human PASMC was 0.28 ± 0.01 and 8.46 ± 0.79 nA, respectively (Fig. 2 A, a and b). The KCNA5 currents activated and deactivated rapidly; time constants for current activation and deactivation were 1.79 ± 0.02 and 2.12 ± 0.05 ms, respectively (Fig. 2A, d and e). The Kv1.5 currents did not show significant inactivation (Fig. 2A, a). Extracellular application of 5 mM 4-AP significantly and reversibly blocked the Kv1.5 channels; the amplitude of currents at +60 mV was reduced by ~76% (14.33 ± 0.40 vs. 3.45 ± 0.66 nA before and during treatment with 4-AP). The cultured human PASMC in which we transfected KCNA5 for electrophysiological experiments maintained the characteristics of smooth muscle cells. Extracellular application of 100 µM serotonin (5-HT) caused PASMC contraction (Fig. 2B). The cells highly expressed the smooth muscle markers {alpha}-smooth muscle-actin, calponin, and SM-22 (Fig. 2C).


Figure 2
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  		Fig. 2. Overexpression of KCNA5 in human PASMC. A: whole cell K+ currents in wild-type (WT) and KCNA5-transfected human PASMC. Currents were elicited by step depolarizations (–60 to +60 mV in 20-mV increments) from a holding potential of –80 mV. Representative current recordings (a), and summarized I-V (b) and conductance-voltage (c) relationships are shown for WT (n = 6) and KCNA5-transfected (n = 26) PASMC. Kinetics of current activation (d) and deactivation (e) of averaged Kv1.5 currents in KCNA5-transfected human PASMC are shown. B: human PASMC (two shown here at top and bottom) gradually contract when exposed to 5-HT (100 µM). C: mRNA expression of smooth muscle (SM) markers (SM-{alpha}-actin, calponin, and SM-22) confirms that the cultured cells are PASMC.

 
As shown in Fig. 3, whole cell IK(V) recorded in KCNA5-transfected HEK-293 cells exhibit similar properties to the currents recorded in KCNA5-transfected human PASMC (see Fig. 2A). Compared with wild-type cells, transfection of an empty vector negligibly affected the endogenous outward K+ currents in HEK-293 cells, which were at the range of 300–400 pA at +60 mV, while overexpression of KCNA5 in the cells led to a 38-fold increase in IK(V) (Fig. 3, B and C). In KCNA5-transfected HEK-293 cells, the voltage-tail current relationship curve suggested that the currents were voltage dependent with an activation threshold at approximately –35 mV. The voltage that induced half activation (V1/2) was approximately –5.1 mV (Fig. 4).


Figure 3
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  		Fig. 3. Transfection of HEK-293 cells with KCNA5. A: fluorescent images on WT untransfected HEK-293 cells (left) and HEK-293 cells transfected with the enhanced green fluorescent protein (EGFP) vector alone (middle) or the KCNA5-EGFP vector (right). B: basal outward currents measured in WT cells, EGFP vector-transfected cells, and KCNA5-EGFP-transfected cells following step depolarizations ranging between –60 and +60 mV from a holding potential of –80 mV. Currents in WT and vector-transfected HEK-293 cells were very small compared with KCNA5-EGFP-transfected cells and are shown in the inset with enlarged scale (vertical bar denotes 100 pA). C: representative I-V relationships of outward currents measured in WT and vector-transfected cells (left) as well as in WT, vector-transfected cells, and KCNA5-transfected cells (right).

 

Figure 4
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  		Fig. 4. Kinetics of tail currents (Itail generated by K+ efflux through Kv1.5 channels. A: whole cell currents in a KCNA5-transfected HEK-293 cell; the currents were elicited by depolarizing the cell from a holding potential of –70 mV to a series of test potentials ranging from –60 to +60 mV in 10-mV increments. B: Itail recorded from the HEK-293 cell in A following repolarization to –40 mV. C and D: absolute (C) and normalized (D) amplitudes of Itail are plotted as a function of the prepulse potential (–60 to +60 mV in 10-mV increments). Half-activation voltage (V1/2; –5.1 mV) is shown in D.

 
Pharmacological properties of Kv1.5 channels. To examine pharmacological properties of Kv1.5 channels, we transiently transfected the human KCNA5 gene to HEK-293 cells. Extracellular application of 4-AP, a potent Kv channel blocker, significantly and reversible inhibited the amplitude of whole cell Kv1.5 currents (data not shown) (8, 49). 4-AP (5 mM) usually caused >70% inhibition of the amplitude of Kv1.5 currents in different transfection system (8, 49).

Effect of correolide. Extracellular application of correolide (1 µM), a newly synthesized compound that has been demonstrated to be a selective blocker of Kv1.x channels (16, 20), markedly reduced the amplitude of Kv1.5 currents (Fig. 5A) in HEK-293 cells. At +60 mV, correolide decreased the current amplitude by ~67.5% (from 9,449.2 ± 677.9 to 3,066.3 ± 333.4 pA; n = 6; P < 0.001). In addition to reducing the amplitude of whole cell Kv1.5 currents, correolide also decelerated the channel activation; the time constant of channel activation at +60 mV was increased from 0.696 ± 0.114 to 1.803 ± 0.422 ms (n = 6; P < 0.05) after treatment with correolide (Fig. 5, Ba and b). However, correolide did not affect the channel deactivation and inactivation (Fig. 5, B, b and C). These data suggest that correolide blocks the channel conduction by potentially binding with the amino acids around the pore region (16, 19, 20) and interferes with the channel opening gating.


Figure 5
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  		Fig. 5. Inhibition of Kv1.5 currents by correolide (CRL). A: representative Kv1.5 currents (a) elicited by step depolarizations (–60 to +60 mV, holding potential of –80 mV) before (Cont), during (CRL), and after (Wash) application of 1 µM CRL. Summarized (means ± SE) I-V (b, left) and conductance-voltage (b, right) relationship curves from KCNA5-transfected HEK-293 cells (n = 6) before ({circ}), during (bullet) and after ({triangleup}) treatment with CRL are shown. B: normalized currents (I/Imax) at +60 mV are plotted as a function of time showing the kinetics of current activation (a, left) and deactivation (a, right) before (control, gray circles) and during (bullet) application of CRL. Summarized time constants (b) of current activation ({tau}act) and deactivation ({tau}deact) in control (gray bars) and CRL-treated (solid bars) cells are shown. *P < 0.05 vs. control (n = 6). C: I/Imax at +60 mV plotted as a function of time showing inactivation kinetics before (control) and during (CRL) treatment with CRL.

 
Effect of ET-1. In the pulmonary (and systemic) circulation system, ET-1 is a potent endothelium-derived contracting factor and a mitogenic factor for vascular smooth muscle cells. It has been well documented that ET-1 inhibits native Kv currents in rat and human PASMC (61). In KCNA5-transfected HEK-293 cells, acute application of ET-1 (100 nM) caused a 32% reduction of the Kv1.5 channel current (from 10,997.0 ± 1391.4 to 7,473.7 ± 1,566.1 pA, n = 6; P < 0.001) (Fig. 6A). ET-1, however, did not affect the kinetics of channel activation, deactivation, and inactivation (Fig. 6, B and C). These results suggest that ET-1 may directly interact with Kv1.5 channels in native PASMC to reduce whole cell Kv currents. The inhibitory effect of ET-1 on Kv1.5 channels may indirectly result from activation of the endothelin receptors (e.g., ETA and ETB) in the plasma membrane and the downstream signaling cascade.


Figure 6
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  		Fig. 6. Inhibition of Kv1.5 currents by endothelin-1 (ET-1). A: representative Kv1.5 currents (a) elicited by step depolarizations (–60 to +60 mV, holding potential of –80 mV) before (Cont), during (ET-1), and after (Wash) application of 100 nM ET-1. Summarized current amplitude (b, left) and conductance (b, right) at +60 mV from KCNA5-transfected HEK-293 cells (n = 6) before (open bars), during (closed bars), and after (gray bars) treatment with ET-1 are shown. ***P < 0.001 vs. control. B: I/Imax at +60 mV are plotted as a function of time showing the kinetics of current activation (a, left) and deactivation (a, right) before (control, gray circles) and during (bullet) application of ET-1. Summarized time constants (b) of {tau}act and {tau}deact in control (gray bars) and ET-1-treated (solid bars) cells (n = 6) are shown. C: I/Imax at +60 mV plotted as a function of time showing inactivation kinetics before (control) and during (ET-1) treatment with ET-1.

 
Effect of bepridil. As shown in Figs. 5 and 6, correolide and ET-1 predominantly reduced the amplitude of whole cell Kv1.5 currents but negligibly affected the kinetics of current deactivation and inactivation. However, in HEK-293 cells transiently transfected with KCNA5, extracellular application of bepridil (25 µM) not only reduced the current amplitude (by ~82%) but also significantly accelerated the current inactivation (Fig. 7), suggesting that different drugs may affect Kv1.5 channels by different mechanisms. The amplitude of steady-state currents (measured at 250–290 ms) in KCNA5-transfected cells was 8,582.6 ± 794.4 pA at +60 mV before treatment and 1,551.9 ± 312.0 pA after treatment with 25 µM bepridil (n = 6, P < 0.001) (Fig. 7, A, a and b). The 82% decrease in the steady-state current amplitude was associated with a significant acceleration of current inactivation, whereas bepridil did not affect the kinetics of channel activation and deactivation (Fig. 7, B and C). These data, which are in good agreement with those by Kobayashi et al. (27) in HEK-293 cells, suggest that bepridil is a classic open channel pore blocker.


Figure 7
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  		Fig. 7. Inhibition of Kv1.5 currents by bepridil. A: representative Kv1.5 currents (a) elicited by step depolarizations (–60 to +60 mV, holding potential of –80 mV) before (Cont), during, and after (Wash) application of 25 µM bepridil. Summarized I-V (b, left) and conductance-voltage (b, right) relationship curves from KCNA5-transfected HEK-293 cells (n = 6) before ({circ}), during (bullet), and after ({triangleup}) treatment with bepridil are shown. B: I/Imax at +60 mV are plotted as a function of time showing the kinetics of current activation (a, left) and deactivation (a, right) before (control, gray circles) and during (bullet) application of bepridil. Summarized time constants (b) of {tau}act and {tau}deact in control (gray bars) and bepridil-treated (solid bars) cells (n = 6) are shown. C: I/Imax at +60 mV plotted as a function of time showing inactivation kinetics before (control) and during treatment with bepridil.

 
Effect of nicotine. Acute treatment of KCNA5-transfected HEK-293 cells with nicotine (100 nM), a ligand of nicotinic acetylcholine receptors, also reduced the current amplitude by 37% (from 8,391.8 ± 442.7 to 5,191.1 ± 348.7 pA at +60 mV, n = 6; P < 0.001) (Fig. 8, A, a and b). Similar to correolide and ET-1, nicotine had no effect on the kinetics of current activation and deactivation (Fig. 8, B, a and b). Acute exposure of cells to nicotine, however, slightly enhanced (or accelerated) the channel inactivation when cells were depolarized by positive potentials (e.g., +40 and +60 mV) (Fig. 8, A, a and C). Again, it is unclear whether nicotine-mediated inhibition of Kv1.5 channels is due to its direct interaction with the channel protein or to its activation of nicotinic acetylcholine receptors and downstream signaling cascade.


Figure 8
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  		Fig. 8. Inhibition of Kv1.5 currents by nicotine. A: representative Kv1.5 currents (a) elicited by step depolarizations (–60 to +60 mV, holding potential of –80 mV) before (Cont), during, and after (Wash) extracellular application of 100 nM nicotine. Summarized I-V (b, left) and conductance-voltage (b, right) relationship curves from KCNA5-transfected HEK-293 cells (n = 6) before ({circ}), during (bullet) and after ({triangleup}) treatment with nicotine are shown. B: I/Imax at +60 mV are plotted as a function of time showing the kinetics of current activation (a, left) and deactivation (a, right) before (control, gray circles) and during (bullet) application of nicotine. Summarized time constants (b) of {tau}act and {tau}deact in control (gray bars) and nicotine-treated (solid bars) cells (n = 6) are shown. C: I/Imax at +60 mV plotted as a function of time showing inactivation kinetics before (control) and during treatment with nicotine.

 
In human PASMC, we observed that extracellular application of nicotine caused a different effect on native Kv currents compared with intracellular dialysis of nicotine. As shown in Fig. 9, extracellular application of nicotine reduced native IK(V) in human PASMC, whereas introduction of nicotine into the cytosolic space (by diffusion via the pipette) enhanced native IK(V) (Fig. 9, A and B). Our results suggest that nicotine may affect the channel activity via different mechanisms depending on its binding site, and the inhibitory effect of nicotine on Kv1.5 channels may be related to the development of pulmonary hypertension in chronic obstructive pulmonary disease (COPD) patients who have a long history of cigarette smoking.


Figure 9
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  		Fig. 9. Differential effect of extracellular and intracellular application of nicotine on native Kv currents in human PASMC. A: representative currents (a) and summarized I-V relationships (n = 5 cells, b) of IK(V) recorded from human PASMC before (control), during (nicotine), and after (washout) extracellular application of 100 nM nicotine. Currents were elicited by step depolarizations ranging between –60 and +80 mV from a holding potential of –70 mV. Time course (c) of the changes in IK(V) at +80 mV before, during, and after extracellular application of nicotine indicates that the effect of nicotine occurs within 2 min of exposure. B: representative currents (a) and summarized I-V relationships (n = 5 cells, b) of IK(V) recorded from human PASMC immediately after breaking in (0 min) and 26 min after intracellular dialysis of 100 nM nicotine (via pipette). Currents were elicited by a step protocol identical to that in A.

 
As mentioned earlier, activity of Kv channels in human PASMC plays a critical role in excitation-contraction coupling of pulmonary arterial smooth muscle and in regulating PASMC proliferation and apoptosis (9, 78). The Kv1.5 (or KCNA5) channel has been demonstrated to be an important Kv channel {alpha}-subunit in PASMC that 1) contributes to regulating the resting membrane potential (5, 49); 2) functions as an important Kv channel subunit (or effector) to sense hypoxia and induce hypoxic pulmonary vasoconstriction (5, 49); and 3) serves as a modulator for apoptotic volume decrease and apoptosis (8). Downregulated KCNA5 expression and inhibited Kv1.5 channel function have been demonstrated to be important causes for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in patients with IPAH and hypoxia-mediated pulmonary hypertension (55, 70, 71, 81). Indeed, overexpression of the KCNA5 gene has been successfully used as an effective gene therapy approach in animal models (55), and opening of Kv1.5 channels is a target for many drugs (e.g., NO, prostacyclin, and sildenafil) clinically used for patients with familial and idiopathic pulmonary arterial hypertension (38, 68).

Previously, we showed that S-nitroso-N-acetyl penicillamine, an NO donor, enhanced the activity of large-conductance Ca2+-activated K+ channels and voltage-gated K+ channels (29, 80). As shown in Fig. 10, extracellular application of 0.1 mM S-nitroso-N-acetyl penicillamine also enhanced Kv1.5 current amplitude in KCNA5-transfected HEK-293 cells. Therefore, KCNA5-encoded Kv channels may represent a putative target for NO and its vasodilatory action. The next set of experiments was designed to identify novel SNPs in the KCNA5 gene from patients with IPAH and to explore the potential association of the SNPs in KCNA5 with different responses to NO in these patients.


Figure 10
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  		Fig. 10. Nitric oxide (NO) activates Kv1.5 channels. A: phase-contrast and fluorescent images of a patched HEK-293 cell transfected with KCNA5-EGFP. B: representative (a) and summarized (n = 8 cells, b) currents recorded from KCNA5-transfected HEK 293 cells before and after treatment with 0.1 mM S-nitroso-N-acetyl penicillamine (SNAP). Currents were elicited by a step depolarization to potentials ranging between –60 and +80 mV from a holding potential of –70 mV. Current amplitudes were significantly greater at all membrane potentials, including –60 mV (c). However, relative current enhancement (ISNAP/IControl) did no vary significantly with test potential (d). *P < 0.05 vs. control.

 
Identification of SNPs in the KCNA5 gene and its flanking sequences in IPAH patients. The average age of the participating IPAH patients was 44.6 ± 1.1 yr when the first right-heart or pulmonary catheterization was performed. Average mean PAP was 53.9 ± 1.0 mmHg, whereas PVR was 999.5 ± 38.9 dyn·s·cm5. In all patients, CO was within the normal range of 2–9 l/min (4.14 ± 0.12 l/ml) (Fig. 11). IPAH predominantly affects women; 76% of the IPAH patients in our study were women. Caucasians accounted for 83% and Hispanics for 12% of the IPAH patients; there were 2 African-Americans and 1 Native American, and 5 patients who identified themselves as of "other" ethnic origin. Although the majority of IPAH patients were women, there was no significant difference in terms of age, mean PAP, PVR, or CO between men and women (Fig. 11).


Figure 11
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  		Fig. 11. Distribution and comparison of hemodynamics between female and male patients with idiopathic pulmonary arterial hypertension (IPAH). Histograms showing distribution of age (A), mean pulmonary arterial pressure (PAP) (B), pulmonary vascular pressure (PVR) (C), and cardiac output (CO) (D) in patients with IPAH (left). Averaged values (means ± SE) of age, mean PAP, PVR, and CO for female (W, closed bars) and male (M, open bars) patients are shown at right.

 
Using the primers shown in Table 1, a ~7,000-bp sequence corresponding to the human KCNA5 gene and its ±2,000-bp flanking sequence was sequenced in genomic DNA samples from IPAH patients. We identified 17 SNPs in these samples (Fig. 12A), 7 of which have not been reported in the NCBI SNP database or in the literature (Table 3). Twelve of the SNPs (5 novel) were in the 5'-upstream region, two (1 novel) in the translated region, two (one novel) in the 3'-untranslated (UTR) region, and one downstream of KCNA5 (Fig. 12A).


Figure 12
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  		Fig. 12. Location of SNPs in the human KCNA5 gene. A: location of SNPs in the 7,000-bp sequence encompassing the human KCNA5 gene. Point 0 (below the gene) denotes the start of the KCNA5 gene. The start and stop codons are marked as * and **, respectively. The numbers above the gene denote the 17 SNPs identified in IPAH patients (see Table 3 for descriptions of each SNP). B: transcription factor binding sites within the putative KCNA5 promoter region. SNPs identified within the ~600-bp sequence are indicated by boxes and SNP number. Transcription factor binding sites are underlined and identified by name. C: location (a) of two nonsynonymous SNPs (G773a and G861c) that correlate with changes of amino acids (E211D and G182R) in the NH2-terminal region (see b for sequence).

 

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  		Table 3. SNPs in the KCNA5 gene identified in IPAH patients

 
Two nonsynonymous SNPs (nos. 13 and 14) were located in the heteromerization or polymerization domain of KCNA5 channel; SNP no. 13 (G773a) changed the hydrophobic glycine to a basic arginine (G182R), while SNP no. 14 (G861c, NCBI ID: rs35853292) incurred a glutamate-to-aspartate change (E211D), both of which are negatively charged and hydrophilic (Fig. 12, A and C). These two mutations, particularly the 773G/A SNP, may have important physiological implications. They are located in a singularly important region of KCNA5, which coordinates {alpha}-subunit tetramerization as well as association of {alpha}-subunits with regulatory beta-subunits (60, 76); altered Kv1.5 channel assembly would clearly change its function. SNPs nos. 15 and 16 (A2578t, A2806t or g) were identified downstream of the UGA stop codon (nt. 2069), but within the 3'-UTR region of KCNA5. The latter SNPs are probably involved in modulating the channel expression. The function of SNP no. 17 (G2870a), located beyond the end (nt. 2864) of the KCNA5 gene, is unclear.

SNPs nos. 1–11 are located in regions upstream of the KCNA5 gene, which includes its putative promoter (Fig. 12, A and B). We determined that part of the putative KCNA5 promoter is located in a 600-bp sequence between residues –500 and +100. Sequence analysis of this region highlighted many transcription factor binding sites and motifs (Fig. 12B), such as two c-Myb binding sequences (nt. –449 and +15), an NF-{kappa}B consensus binding site (nt. –269), a SP1-VGF/SP1-Erk1 site (nt. –216), an E2A/E box binding motif (nt. –158), a CreA site (nt. –149), two c/EBP-apoB-intron enhancer binding sites (nt. –105 and –94), a CAP site (nt. –23), two AP-2 binding sites (nt. –362 and nt. +19), a CACCC box (nt. –139), a CREB/c-Jun consensus site (nt. +64), and an interferon-{gamma} response element ({gamma}-IRE) binding site (nt. +62). SNP no. 12 (C62t) coincides with the loss of the {gamma}-IRE binding site. SNP no. 10 (C–136t) did not alter the CACCC box sequence (nt. –139). Regions upstream from the putative promoter also contained numerous binding sites: a TATA box (nt. –1669), a myoD-MCK binding site (nt. –1087), a CAP site (nt. –828), a c-Myc responsive region (nt. –805), and a number of AP-1 consensus sites (nt. –1767, –652, and –546) (data not shown). SNP no. 2 (G-140Ga) introduced a {delta}-repressor element (that may alter NF-{kappa}B function) binding site, and SNP no. 3 (C-1087t) caused the loss of the myoD-MCK binding site. The presence of other upstream SNPs did not delete or introduce other transcription factor binding sites.

Minor allele frequencies of the SNPs varied greatly (Table 3). SNPs within the KCNA5 gene had a relatively low allele frequency except for SNP no. 15 (A2578t), which had an allele frequency of 0.266. For SNP no. 15, 40% of the patients exhibited a heterozygous (AA/AT) genotype. A similar case could be made for SNP no. 12 (C62t), with an allele frequency of 0.425. Upstream SNPs no. 1 (G-1951t), no. 3 (C-1087t), and no. 10 (C-136t) also occurred frequently (allele frequencies: 0.583, 0.347, and 0.900, respectively). SNP no. 1 may not interfere greatly with Kv1.5 expression and function because it is so far upstream of the KCNA5 gene, and it does not appear to alter any transcription factor binding sites. However, as we stated above, SNPs no. 3 and no. 10 may alter a myoD-MCK binding site and a CACCC box, respectively, potentially influencing KCNA5 transcription in IPAH patients.

Correlation of SNPs in KCNA5 to drug response in IPAH patients. Inhalation of NO is used clinically to treat IPAH patients based on its vasodilatory effect on pulmonary arteries and antiproliferative/proapoptotic effects on PASMC (24, 29). One of the mechanisms involved in the relaxant and proapoptotic effects of NO is activation of Kv1.5 channels (as shown in Fig. 10) and other K+ channels in PASMC (7, 9, 29, 37, 80). Furthermore, inhaled NO has also been used to identify IPAH patients with pulmonary vascular reactivity; the clinical outcome of these patients treated with other drugs (e.g., prostacyclin and bosentan) is better than that of patients with little vascular reactivity (24, 64).

Among the IPAH patients participated in this study, we determined pulmonary vascular reactivity by measuring PAP and PVR during pulmonary catheterization in patients before and after inhalation of NO. There were 42 patients who responded to inhaled NO (>+20% increased in PAP), whereas 37 patients did not (–2% to +2% change in PAP). Acute inhalation of NO reduced PAP by 17.4 ± 1.8% in the 42 responders but negligibly changed PAP by 0.2 ± 0.2% in the 37 nonresponders (Fig. 13A). NO-induced PAP changes paralleled PVR in both groups (Fig. 13, B and C). The allele frequency of SNP no. 4 (T-937a) was significantly different (P = 0.01) between responders and nonresponders; ~7% of NO responders were the –937T genotype, while ~24% of nonresponders were the altered –937A genotype. The allele frequency of SNP no. 17 (G2870a) was also significantly different (P = 0.049) between responders and nonresponders (Fig. 13C).


Figure 13
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  		Fig. 13. Correlation of the SNPs in KCNA5 with hemodynamic responses to inhaled NO. A: summarized mean PAP (A) and PVR (B) in NO responders (Resp, n = 42 patients) and nonresponders (Non-Resp, n = 37 patients) before (Cont, open bars) and after (NO, solid bars) inhalation of NO. ***P < 0.001 vs. Cont. Allele frequencies of SNP no. 4 and no. 17 were significantly different between NO responders and nonresponders (C).

 
To examine the possibility that the different response to NO in IPAH patients is due to disease severity, we compared the values of mean PAP, PVR, and CO in responders and nonresponders before treatment. As shown in Fig. 14, the averaged PAP was comparable between NO responders and nonresponders, whereas the averaged PVR was significantly higher in nonresponders than in responders. Furthermore, the averaged CO was lower in nonresponders than in responders. These results seem to be consistent with the data reported by Sitbon et al. (64) showing that PVR in nonresponders is significantly higher than in responders (15.3 ± 6.6 vs. 12.2 ± 5.3 Wood units; P < 0.001), whereas CO is lower in nonresponders than in responders (3.7 ± 1.1 vs. 4.5 ± 1.4 l/min; P < 0.001). In their study, the IPAH patients were tested by acute intravenous application of epoprostenol or inhalation of NO (65).


Figure 14
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  		Fig. 14. Distribution and comparison of hemodynamics between NO responders and nonresponders in IPAH patients. Histograms show distribution of mean PAP (A), PVR (B), and CO (C) in IPAH patients whose PAP/PVR were reduced by inhalation of NO (responders) and patients whose PAP/PVR were not changed by NO (nonresponders). Averaged values (means ± SE) of mean PAP, PVR, and CO for responders (Resp) and nonresponders (Non-Resp) are shown at right. **P < 0.01 vs. responders.

 
Among all IPAH patients that participated in this study, 37 patients had a history of taking fenfluramine and phentermine (Fen-Phen) for >3 mo. Averaged PAP in patients who had never taken Fen-Phen was ~7 mmHg higher than for Fen-Phen users (Fig. 15A), while NO-mediated changes in PAP were comparable between non-Fen-Phen patients and Fen-Phen users (Fig. 15B). Having previously reported that treatment of human PASMC with fenfluramine downregulated KCNA5 expression and decreased IK(V), we compared the allele frequency of the SNPs in KCNA5 between IPAH patients with or without history of Fen-Phen use. SNP no. 5 (C-828t) and no. 10 (C-136t) showed significantly different allele frequencies (P = 0.027 and 0.043, respectively) between these two groups of IPAH patients. SNP no. 5 was completely absent in patients who used Fen-Phen, while it was present in 12% of the patients with no history of Fen-Phen use. SNP no. 10, although it possessed a high allele frequency in both groups, was less prominent in the Fen-Phen users than in the non-Fen-Phen users (Fig. 15C).


Figure 15
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  		Fig. 15. Correlation of the SNPs in KCNA5 with use of fenfluramine and phentermine (Fen-Phen). A and B: mean PAP (A) and NO-mediated changes in PAP (B) in IPAH patients with (+) or without (–) a history of taking Fen-Phen. Allele frequencies of SNP nos. 5 and 10 were significantly different in (+) and (–) Fen-Phen patients (C).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Kv channel function plays an important role in the regulation of excitation-contraction coupling in vascular smooth muscle. Blockade of Kv channels causes membrane depolarization and triggers pulmonary vasoconstriction by opening VDCC and elevating [Ca2+]cyt (54, 79), whereas activation of Kv channels causes membrane hyperpolarization and vasodilation by closing VDCC (80). Kv1.5 channel is a pore-forming {alpha}-subunit that participates in forming native Kv channels in PASMC (6, 55). Downregulated KCNA5 channel expression and decreased IK(V) have been demonstrated in PASMC from patients with IPAH (77, 81) and animals with chronic hypoxia-mediated pulmonary hypertension (52, 70). The resultant membrane depolarization and [Ca2+]cyt elevation in PASMC contribute to causing sustained pulmonary vasoconstriction (54, 78, 79) and stimulating PASMC proliferation (50, 52, 77).

Another consequence of attenuated Kv channel activity is a decrease in PASMC apoptosis due to decelerated apoptotic volume decrease and inhibited cytoplasmic caspase activity (28). Overexpression of human KCNA5 in vitro not only increases IK(V) and causes membrane hyperpolarization but also enhances PASMC apoptosis (8). Furthermore, in vivo transfer of KCNA5, by increasing IK(V) in PASMC, causes significant regression of pulmonary vascular medial hypertrophy and reduction of PAP and PVR in chronically hypoxic rats (4, 55). These observations provide compelling evidence that 1) normal KCNA5 expression and Kv1.5 function are necessary for maintaining the resting membrane potential in PASMC and regulating pulmonary vascular tone (78), 2) KCNA5 gene downregulation and/or Kv1.5 channel dysfunction in PASMC are involved in the development of PAH (77), and 3) restored KCNA5 expression and/or Kv1.5 function may be a useful therapeutic approach for treatment of pulmonary artery hypertension (55).

Biophysical and pharmacological properties of Kv1.5 channels. Native Kv channels in human PASMC are homotetramers and heterotetramers composed of the same or different Kv channel {alpha}-subunits, respectively. It has been demonstrated that Kv1.5 not only forms homotetrameric channels but also forms heteromeric channels with other Kv1 family members (e.g., Kv1.2 and Kv1.4) (2, 14, 23). In addition to four pore-forming {alpha}-subunits, the native Kv channels also contain four regulatory beta-subunits that play an important role in sensing oxygen tension or redox status (43, 46), protein kinase-mediated phosphorylation (31, 73), and modulation of channel kinetics (e.g., inactivation) (57, 58, 67). Our data, however, indicate that many drugs can directly modulate the function of Kv1.5 channels.

Bepridil, a nonspecific inhibitor of Ca2+, Na+, and K+ channels in cardiomyocytes (11, 42), can significantly accelerate inactivation of homotetrameric Kv1.5 channels that are not associated with beta-subunits (27). In rat atrial cells, inhibition of the bepridil-sensitive ultrarapid delayed rectifier channel (which is believed to be encoded primarily by KCNA5) leads to increased action potential duration, likely due to the change in Kv1.5 inactivation kinetics. Correolide can block all Kv1 channel {alpha}-subunits, although it is most potent for the Kv1.3 isoform (16). By binding in a region near, but not in, the pore of the Kv channel {alpha}-subunit, it can directly interact with the Kv1 channel {alpha}-subunits to decrease current amplitude (6, 16, 47). In many of the cases reported above, Kv1.5 subunits were prominent components of the correolide-sensitive Kv channels.

In comparison to correolide, bepridil-mediated effect on Kv1.5 channels is quite different. Bepridil not only reduced the amplitude of the currents but also significantly accelerated the inactivation of the channels. The data shown in Fig. 7 indicate that bepridil is a classic open channel pore blocker. Accelerating channel inactivation is known to occur by a mechanism similar to the "ball-and-chain" theory in which an inactivating domain (e.g., a cytoplasmic NH2-terminus of the pore-forming subunit, a cytoplasmic regulatory beta-subunit, or a drug like bepridil) physically occludes into the channel pore in the cytoplasmic site. Such a mechanism for inhibition would be consistent with the accelerated inactivation kinetics during bepridil treatment.

Interestingly, our data also demonstrate that extracellular application of ET-1 and nicotine significantly reduced the amplitude of currents generated by K+ efflux through homomeric Kv1.5 channels. ET-1 is an important endothelium-derived constricting factor and mitogenic factor which can cause sustained pulmonary vasoconstriction and pulmonary vascular remodeling (10). The inhibitory effect of ET-1 on Kv1.5 channels shown in this study provides convincing evidence that the contractile and mitogenic effect of ET-1 may partially result from its inhibition of Kv1.5 channels in human PASMC (61). How ET-1 inhibits Kv1.5 channel activity remains unclear, but the potential mechanisms may relate to the following: 1) a direct interaction of the peptide with the extracellular pore region of the channel protein, 2) phosphorylation of the channel protein (via the cytoplasmic PKC-binding domain) mediated by increased protein kinases upon activation of ET receptors, (ETA and/or ETB) (61), and 3) indirect inhibition of the channel activity by Ca2+ mobilization from intracellular stores (53).

As far as nicotine is concerned, studies have shown that direct infusion of nicotine may reduce NO-mediated vasorelaxation, despite the fact that inhaled nicotine may maintain circulating nitric oxide in humans (33, 35, 39). In addition to inhibiting Kv1.5 channels, as shown in this study, nicotine has also been demonstrated to inhibit Ca2+-activated K+ channels in human umbilical vein endothelial cells (30), HERG channels expressed in Xenopus oocytes (74), and ATP-sensitive K+ channels in vascular smooth muscle cells (34). Nicotine may also decrease Kv1.5 current by altering KCNA5 subunit expression. Shin et al. (62) reported such a finding with Kv1.1 subunits in rat gastric mucosal epithelial cells. The inhibitory effect of nicotine on ATP-sensitive K+ channels may result partially from nicotine-mediated production of reactive oxygen species (34). Although Kv channels are modulated by reactive oxygen species in PASMC (40, 72), it remains unclear whether nicotine-mediated inhibition of Kv1.5 channels is related to the production of reactive oxygen species.

Interestingly, our study also demonstrated that extracellular application of nicotine and intracellular dialysis of nicotine had divergent effects on native Kv channels in PASMC. The inhibitory effect of nicotine, applied externally, on IK(V) is likely due to activation of nicotinic acetylcholine receptors on the surface membrane. However, it is unclear why nicotine, applied internally, augments IK(V) and what mechanisms are involved in this effect. One of the possibilities is that nicotine, when applied internally, may bind with the cytoplasmic regulatory beta-subunits to decelerate inactivation of the pore-forming {alpha}-subunits.

In addition to patients with idiopathic and thromboembolic pulmonary hypertension, patients with COPD and emphysema may also develop pulmonary hypertension as a result of sustained pulmonary vasoconstriction, increased stiffness (or decreased compliance) of pulmonary vascular wall, deposition of extracellular matrix proteins around the vessels, and hypoxia-mediated pulmonary vascular remodeling (41). Many COPD patients with secondary pulmonary hypertension have a long history of cigarette smoking. The nicotine-mediated inhibition of Kv1.5 channels, as shown in this study, may serve as an additional mechanism for the development of pulmonary hypertension in COPD patients with history of smoking.

Possible impact of SNPs in KCNA5 on channel expression and function in IPAH patients. Forty-four SNPs are listed in the NCBI SNP database for the human KCNA5 gene and its 2,000-bp 5'- and 3'-flanking sequences. The KCNA5 gene itself contains 23 of these SNPs, with only 5 SNPs within the coding region (i.e., exon 1). Three recent studies have identified another 12 low-frequency SNPs in various populations, with possible linkage to long QT syndrome and drug resistance. Iwasa et al. (25) reported a synonymous S3-S4 loop G383G mutation in Japanese citizens. Simard et al. (63) reported five synonymous and six nonsynonymous (allele frequency <7%) in the cardiac KCNA5 of 190 individuals from three ethnic groups (Caucasian, Asian, and African-American), including the G383G mutation; four SNPs in the NH2 terminus, one in the TM-1 domain, two in the TM-1/TM-2 loop, one in the TM-3/TM-4 loop, one in the TM-6 domain, and two in the COOH-terminus. Expression of the five nonsynonymous SNPs and four nonsynonymous SNPs in Chinese hamster ovary cells did not alter KCNA5 gating properties, whereas the two COOH-terminal variants (P532L and R578K) altered current sensitivity to quinidine and propafenone, both clinically used antiarryhthmic drugs. Plante et al. (48) identified 3 SNPs in a population of 96 French-Canadians; two of the SNPs (A251T and P307S) had been identified previously in the study by Simard et al. (63). When expressed in Chinese hamster ovary cells, two of the SNPs altered channel gating (decreased amplitude, slowed inactivation, accelerated opening); these effects led to slight prolongation of the action potential. Their data also suggested that changes in channel gating associated with these SNPs required the presence of the regulatory Kv beta- subunit. From the latter two studies, it is possible that SNPs in KCNA5 may have serious physiological implications in cardiovascular disease.

As mentioned earlier, decreased Kv channel expression and function are implicated in PASMC from IPAH patients and in PASMC from animals with hypoxia-mediated pulmonary hypertension. IPAH is a rare and fatal disease; the incidence of IPAH is ~1–2 per million of general population (18). Taking advantage of our access to blood and DNA samples from of patients with this rare disease, we also aimed to identify novel SNPs in the KCNA5 gene in the patients with IPAH. We identified 17 SNPs, 7 of which have not been previously reported (Table 2). While the bulk of the SNPs occurred in the upstream region, two nonsynonymous SNPs within the KCNA5 coding region were identified. Because the SNPs (G773a and G861c) are located in the heteromerization or polymerization domain where Kv channel {alpha}- and beta-subunits interacts with each other, they may affect assembly of functional tetrameric Kv channels (60, 76). We are currently evaluating the impact of these two SNPs on Kv1.5 currents. The other two SNPs (A2578t and T2806t/g) in the 3'-UTR may alter the regulatory effects of protein kinases on channel activity or may be involved in posttranslational regulation of Kv1.5 channel expression.

Although we identified a putative KCNA5 promoter in the 5'-upstream sequence, the transcriptional regulation of the gene is not necessarily limited to the transcription factor binding sites in this 600-bp region. It is possible that transcription factors may modulate KCNA5 transcription by binding to sequences farther upstream of our predicted promoter region. In analyzing the putative 600-bp promoter region and the region farther upstream, we identified multiple transcription factor binding sites which suggest that KCNA5 transcription may be regulated by NF-{kappa}B, CREB, c/EBP, c-Myb, Erk1, and AP-2, all of which have been implicated in the regulation of vascular smooth muscle proliferation and apoptosis and, potentially, in the development of PAH (22, 32, 75). SNP no. 12 (C62t) itself may result in loss of a {gamma}-IRE binding site. Since the {gamma}-IRE is involved in urokinase plasminogen activator-mediated cell migration and activation of STAT-1 (15), it is possible that loss of the {gamma}-IRE binding site in KCNA5 promoter influences thrombosis-mediated PASMC proliferation. Although most of the SNPs upstream of KCNA5 do not directly alter the known binding sequences, they may still affect transcriptional regulation of the gene by indirectly altering the tertiary structure of the promoter and the binding of transcription factors to the promoter region. While we identified several SNPs in the putative promoter region of KCNA5 gene, it is, however, still unknown whether these SNPs are functionally involved in the transcriptional regulation of the gene. Another limitation of this study is that we are unable to correlate the SNPs identified from blood samples with Kv channel activity in PASMC and to define how the SNPs lead to a decrease in whole-cell IK(V).

In our previous studies, we have shown that 1) the amplitude of endogenous IK(V) is decreased, the inactivation kinetics of whole cell IK(V) is accelerated, and the mRNA expression of Kv1.5 (KCNA5) is downregulated in PASMC from IPAH patients (77, 81); 2) the basal apoptosis and staurosporine/BMP-2-induced apoptosis are both inhibited in PASMC from IPAH patients compared with PASMC from normal subjects and normotensive patients (82); 3) overexpression of KCNA5 in PASMC increases IK(V) and causes membrane hyperpolarization (49); and 4) overexpression of KCNA5 in PASMC and other cell types (e.g., HEK-293 and COS-7 cells) accelerates apoptotic volume decrease and enhances apoptosis (8). These observations suggest that the expression and function of Kv1.5 (KCNA5) channels are associated with the degree of pulmonary vascular remodeling in IPAH patients. Further study is needed to define whether and how the SNPs identified in KCNA5 gene from IPAH patients affect the transcription, function, and modulation of Kv1.5 channels in PASMC.

Implication of SNP occurrence in KCNA5 with drug response in IPAH patients. Ion channels, such as Kv channels, represent one of the cellular targets for NO-mediated vasodilatory, antiproliferative, and/or proapoptotic effects in the pulmonary vasculature. NO can cause pulmonary vasodilation 1) by activating Ca2+-activated K+ channels and Kv channels in PASMC, causing membrane hyperpolarization and closure of VDCC (3, 29), and 2) by directly blocking VDCC (12, 13). NO also enhances PASMC apoptosis by activating K+ channels, accelerating apoptotic cell shrinkage and increasing cytoplasmic caspase activity, which may potentially cause regression of pulmonary medial hypertrophy (29). The higher allele frequency of the SNP no. 4 (T-937a) in KCNA5 in IPAH patients who do not respond to NO suggests that variations in KCNA5 transcriptional regulation may affect pulmonary vascular reactivity to vasodilators in IPAH patients. However, whether SNP nos. 4 (T-937a) and 17 (G2870a) can be used as a genetic indicator for NO responsiveness to guide therapeutic choices for IPAH patients is uncertain and needs further study in a larger group of patients.

As indicated by the hemodynamic data (Fig. 14), 1) mean PAP (before inhalation of NO) is comparable between responders and nonresponders, 2) basal PVR is higher in nonresponders than in responders, and 3) CO is lower in nonresponders than in responders. These data are similar to the observations in IPAH patients by Sitbon et al. (64). Therefore, the possibility exists that the different response to NO in IPAH patients might also be due to differences in disease severity rather than solely due to the SNPs identified in KCNA5 gene.

Anorexigens, which have been linked to the development of IPAH (1), also inhibit the expression and function of Kv channels (e.g., Kv1.5) in PASMC (45, 69). We identified SNP nos. 5 and 10 whose minor allele frequencies were significantly decreased in IPAH patients with Fen-Phen use. Unfortunately, because of the low number of Fen-Phen users in our study, we were unable to establish a definite correlation between SNP occurrence and Fen-Phen-related IPAH development.

In summary, Kv1.5 channel is one of the important Kv channel subunits that are functionally expressed in human PASMC (14, 21, 36, 81). Kv1.5 channel can form homotetrameric channels that are sensitive to 4-AP, bepridil, and correolide. 4-AP and correolide decrease Kv1.5 currents predominantly by reducing the channel conductance, whereas bepridil decreases the currents largely by conferring (or accelerating) inactivation onto the noninactivating Kv1.5 channels (i.e., bepridil acts as an open channel blocker for Kv1.5 channels). Furthermore, the inhibitory effect of ET-1 and nicotine on Kv1.5 channels suggest that blockade of Kv1.5 in PASMC may play an important role in ET-1 (and nicotine)-mediated pulmonary vasoconstriction and vascular medial hypertrophy. Because of its complex etiology, IPAH is generally treated with a combination of active drugs, such as NO, prostacyclin, ET receptor antagonists, and phosphodiesterase inhibitors (24). Targeting Kv1.5 channels may have potential in developing new therapeutic approaches for IPAH, especially considering the fact that Kv channel dysfunction is closely linked to pulmonary vasoconstriction and PASMC proliferation (4, 52, 66, 69, 77). Indeed, gene transfection of KCNA5 in PASMC causes membrane hyperpolarization and enhanced apoptosis (8) and in vivo gene transfer of KCNA5 in lung tissues reverses both pulmonary vasoconstriction and vascular remodeling in animal models with hypoxia-mediated pulmonary hypertension (55).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-69758, HL-64945, HL-54043, and HL-66012.


   ACKNOWLEDGMENTS
 
We thank Dr. Maria L. Garcia from Merck for providing correolide and Dr. Michael M. Tamkun from Colorado State University for providing the Kv1.5 plasmid.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. X.-J. Yuan, Div. of Pulmonary and Critical Care Medicine, Dept. of Medicine, Univ. of California-San Diego, 9500 Gilman Dr., MC 0725, La Jolla, CA 92093-0725 (e-mail: xiyuan{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* C. V. Remillard, D. D. Tigno, and O. Platoshyn contributed equally to this work. Back


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