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VASCULAR BIOLOGY

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

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 -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

Kv1.5 is an important pore-forming -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 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 [I_K(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) 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 pipettes, the series resistance was at a range of 4–9 M 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.

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 -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.

	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 -subunit that associates with other Kv channel - and -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 : and : association, and 4) a cytoplasmic COOH-terminus that differs between Kv channel -subunits (Fig. 1B). Functional Kv channels are homo- or heterotetramers composed of the same number of - and -subunits (₄₄) (Fig. 1C).

View larger version (57K): [in this window] [in a new window]   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 -subunit protein folding motif and regulatory -subunit binding; the pore region (P) is shown between TM-5 and TM-6. C: cross-sectional view of the arrangement of - and -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).

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 I_K(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 -smooth muscle-actin, calponin, and SM-22 (Fig. 2C).

View larger version (54K): [in this window] [in a new window]   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--actin, calponin, and SM-22) confirms that the cultured cells are PASMC.

View larger version (51K): [in this window] [in a new window]   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).

View larger version (33K): [in this window] [in a new window]   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.

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.

View larger version (27K): [in this window] [in a new window]   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 (), during () and after () 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 () application of CRL. Summarized time constants (b) of current activation (act) and deactivation (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.

View larger version (26K): [in this window] [in a new window]   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 () application of ET-1. Summarized time constants (b) of act and 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.

View larger version (28K): [in this window] [in a new window]   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 (), during (), and after () 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 () application of bepridil. Summarized time constants (b) of act and 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.

View larger version (27K): [in this window] [in a new window]   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 (), during () and after () 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 () application of nicotine. Summarized time constants (b) of act and 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.

View larger version (25K): [in this window] [in a new window]   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.

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.

View larger version (34K): [in this window] [in a new window]   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.

View larger version (31K): [in this window] [in a new window]   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.

View larger version (59K): [in this window] [in a new window]   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).

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-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- response element (-IRE) binding site (nt. +62). SNP no. 12 (C62t) coincides with the loss of the -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 -repressor element (that may alter NF-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).

View larger version (19K): [in this window] [in a new window]   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).

View larger version (31K): [in this window] [in a new window]   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.

View larger version (25K): [in this window] [in a new window]   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

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 I_K(V) and causes membrane hyperpolarization but also enhances PASMC apoptosis (8). Furthermore, in vivo transfer of KCNA5, by increasing I_K(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 -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 -subunits, the native Kv channels also contain four regulatory -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 -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 -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 -subunit, it can directly interact with the Kv1 channel -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 -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, (ET_A and/or ET_B) (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 I_K(V) is likely due to activation of nicotinic acetylcholine receptors on the surface membrane. However, it is unclear why nicotine, applied internally, augments I_K(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 -subunits to decelerate inactivation of the pore-forming -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 - 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 - and -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-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 -IRE binding site. Since the -IRE is involved in urokinase plasminogen activator-mediated cell migration and activation of STAT-1 (15), it is possible that loss of the -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 I_K(V).

In our previous studies, we have shown that 1) the amplitude of endogenous I_K(V) is decreased, the inactivation kinetics of whole cell I_K(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 I_K(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 p