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Am J Physiol Lung Cell Mol Physiol 274: L621-L635, 1998;
1040-0605/98 $5.00
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Vol. 274, Issue 4, L621-L635, April 1998

Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells

Xiao-Jian Yuan1,2, Jian Wang1, Magdalena Juhaszova2, Vera A. Golovina2, and Lewis J. Rubin1,2

1 Division of Pulmonary and Critical Care Medicine, Department of Medicine, and 2 Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

K+-channel activity-mediated alteration of the membrane potential and cytoplasmic free Ca2+ concentration ([Ca2+]cyt) is a pivotal mechanism in controlling pulmonary vasomotor tone. By using combined approaches of patch clamp, imaging fluorescent microscopy, and molecular biology, we examined the electrophysiological properties of K+ channels and the role of different K+ currents in regulating [Ca2+]cyt and explored the molecular identification of voltage-gated K+ (KV)- and Ca2+-activated K+ (KCa)-channel genes expressed in pulmonary arterial smooth muscle cells (PASMC). Two kinetically distinct KV currents [IK(V)], a rapidly inactivating (A-type) and a noninactivating delayed rectifier, as well as a slowly activated KCa current [IK(Ca)] were identified. IK(V) was reversibly inhibited by 4-aminopyridine (5 mM), whereas IK(Ca) was significantly inhibited by charybdotoxin (10-20 nM). K+ channels are composed of pore-forming alpha -subunits and auxiliary beta -subunits. Five KV-channel alpha -subunit genes from the Shaker subfamily (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), a KV-channel alpha -subunit gene from the Shab subfamily (KV2.1), a KV-channel modulatory alpha -subunit (KV9.3), and a KCa-channel alpha -subunit gene (rSlo), as well as three KV-channel beta -subunit genes (KVbeta 1.1, KVbeta 2, and KVbeta 3) are expressed in PASMC. The data suggest that 1) native K+ channels in PASMC are encoded by multiple genes; 2) the delayed rectifier IK(V) may be generated by the KV1.1, KV1.2, KV1.5, KV1.6, KV2.1, and/or KV2.1/KV9.3 channels; 3) the A-type IK(V) may be generated by the KV1.4 channel and/or the delayed rectifier KV channels (KV1 subfamily) associated with beta -subunits; and 4) the IK(Ca) may be generated by the rSlo gene product. The function of the KV channels plays an important role in the regulation of membrane potential and [Ca2+]cyt in PASMC.

potassium channel; polymerase chain reaction; fluorescence microscopy; patch clamp; cytoplasmic calcium

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

K+ channels play a critical role in the regulation of pulmonary vasomotor tone by governing membrane potential (Em) (39). Inhibition of K+ channels depolarizes pulmonary arterial (PA) smooth muscle cells (PASMC) to a threshold that opens voltage-gated Ca2+ channels and thus increases the cytoplasmic free Ca2+ concentration ([Ca2+]cyt) (39, 40, 67). In contrast, activation of K+ channels hyperpolarizes PASMC and inhibits the evoked rise in [Ca2+]cyt (4, 72). The elevation of [Ca2+]cyt in PASMC is a major trigger for pulmonary vasoconstriction and an important stimulus for vascular smooth muscle cell proliferation, which leads to vascular remodeling. K+ channels in vascular smooth muscle cells are also potential targets of vasoactive neurotransmitters or therapeutic agents (e.g., nitric oxide) (4, 72). Altered K+-channel function has been implicated in the pathogenesis of several cardiovascular diseases including primary pulmonary hypertension (69) and systemic arterial hypertension (32).

K+ channels are ubiquitously expressed in almost all excitable or nonexcitable cells (11, 52). With the use of electrophysiological approaches, three types of K+ currents have been identified in PASMC: voltage-gated K+ (KV) current [IK(V)] (16, 18, 47, 67, 70), Ca2+-activated K+ (KCa) current [IK(Ca)] (2, 4, 18, 46), and ATP-sensitive K+ (KATP) current [IK(ATP)] (13). IK(V) is an important regulator of resting Em, whereas IK(Ca) functions as a critical negative feedback pathway in the regulation of Em and vascular contractility (7, 17-19, 29, 67).

KV channels are composed of pore-forming alpha -subunits and associated cytoplasmic beta -subunits, which act mainly as a regulatory moiety (26). The KV-channel alpha -subunit gene encoded by the Shaker locus (KV1) in Drosophila was first isolated in 1987 (44); three related fly KV-channel genes, Shab (KV2), Shaw (KV3), and Shal (KV4), were subsequently isolated using molecular biological approaches (9). At least 18 vertebrate genes encoding KV-channel alpha -subunits have been isolated from mammals and frogs with Shaker probes (11). Each of these KV-channel genes produced functionally distinct KV channels (11, 24). The slowpoke gene (Slo) encoding the KCa channel [high-conductance (BK) channel] was first identified in the Drosophila Slo (dSlo) locus (5). The subsequent cloning and expression of the genes encoding KCa channels from Drosophila (5), mouse (mSlo) (8), and human (hSlo) (33) demonstrate that the protein shares extensive homology with the KV channels of the Shaker subfamily.

Recently, four new subfamilies of the electrically silent KV-channel alpha -subunits have been cloned: KV5 (74), KV6 (49), KV8 (25, 54), and KV9 (45, 58). Expression of these modulatory alpha -subunits (e.g., KV9.3 or KV8.1) per se does not produce K+-channel activity; however, coexpression of the modulatory alpha -subunits with other functional KV-channel alpha -subunits (e.g., KV2.1) significantly modulates kinetics, expression level, and voltage dependence of the functional KV channels (45, 54, 55).

The beta -subunits of KV channels were identified as 38- to 41-kDa polypeptides associated with the alpha -subunits (64). Studies on cloning of the cDNAs encoding three KV-channel beta -subunits (KVbeta 1.1, KVbeta 2, and KVbeta 3) and coexpression with KV-channel alpha -subunits indicate that the beta -subunits are highly conserved and play an important role in modulating gating properties of certain alpha -subunits (23, 24, 26, 50). It has recently been reported that the Shaker KV-channel beta -subunits belong to an NAD(P)H-dependent oxidoreductase superfamily (34), suggesting that beta -subunits may play a critical role in sensing changes in redox status (3, 72), oxidoreductive metabolism (72), and oxygen tension (30, 42, 45, 47, 48, 59, 66, 71).

Although the electrophysiological properties of K+ channels have been extensively studied (10, 40), the molecular identity of K+-channel genes in PASMC has not yet been elucidated. In this study, we used patch-clamp techniques, digital-imaging fluorescence microscopy, PCR, and immunoblotting to define the distinct K+ currents and their associated function in regulating [Ca2+]cyt and to identify the corresponding K+-channel genes expressed in PASMC. Because KV-channel beta -subunits specifically bind with Shaker-related KV-channel alpha -subunits (KV1 family) (24, 56), the molecular study focused on the KV1-channel alpha -subunits and KV-channel beta -subunits.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell preparation. Primary cultures of rat PASMC were prepared as previously described (70). Briefly, the intrapulmonary arterial branches as well as the right and left branches of the main PA were incubated for 20 min in Hanks' balanced salt solution containing 1.5 mg/ml of collagenase (Worthington Biochemical, Freehold, NJ). After the incubation, a thin layer of adventitia was carefully stripped off with fine forceps, and the endothelium was removed by gently scratching the intimal surface with a surgical blade. The remaining PA smooth muscle was then digested with 1.5 mg/ml of collagenase, 0.5 mg/ml of elastase (Sigma, St. Louis, MO), and 1 mg/ml of bovine albumin (Sigma) for 45 min at 37°C to create a single-cell suspension of PASMC. The cells were then resuspended and plated onto 25-mm coverslips (for electrophysiological and fluorescent microscopy experiments) or 10-cm petri dishes (for molecular biological experiments) and incubated in a humidified atmosphere of 5% CO2 in air at 37°C in 10% fetal bovine serum culture medium. Before each experiment and extraction of total RNA and protein, the primary cultured PASMC were incubated in 0.3% fetal bovine serum culture medium for 12-24 h to stop cell growth. This treatment would also minimize the effects of growth factors, DNA synthesis, and tyrosine kinase activation on channel expression.

Immunofluorescence labeling. The primary cultured PASMC were fixed in 95% ethanol and stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (DAPI; 5 µM; Molecular Probes); the blue fluorescence emitted at 461 nm was used to visualize the cell nuclei and estimate total cell numbers in the cultures. The specific monoclonal antibody raised against alpha -smooth muscle actin (Boehringer Mannheim, Indianapolis, IN) was used to evaluate cellular purity of the cultures, and a secondary antibody conjugated with indocarbocyanine (Cy3; Jackson ImmunoResearch, West Grove, PA) was used to display the fluorescent image (emitted at 570 nm). The cells were mounted in 10% 1 M Tris · HCl-90% glycerol (pH 8.5) containing 1 mg/ml of p-phenylenediamine. The cell images were processed by a MetaMorph Imaging System (Universal Imaging, West Chester, PA); indocarbocyanine fluorescence was colored red and DAPI fluorescence was colored green to display images with a red-green overlay. All the DAPI-stained cells in the primary cultures also cross-reacted with the smooth muscle cell alpha -actin antibody, indicating that the cultures were all smooth muscle cells.

Recording of K+ current. Whole cell and single-channel K+ currents (IK) were recorded with an Axopatch-1D amplifier and a TL-1 DMA digital interface (Axon Instruments, Foster City, CA) with the patch-clamp technique as described (67, 70). Patch pipettes (2-4 MOmega ) were fabricated from microhematocrit tubes (VWR Scientific, Bridgeport, NJ) and were fire polished on a Narishige microforge. Step-pulse protocols and data acquisition were performed with pCLAMP software. Currents were filtered at 1-2 kHz (-3 dB) and digitized at 2-4 kHz with the Axopatch-1D amplifier. For whole cell current recording, series resistance and capacitance were routinely compensated for (40-70%) by adjusting the internal circuitry of the patch-clamp amplifier. Leakage currents were subtracted with the P/4 protocol in pCLAMP software. In experiments with cell-attached patches, the actual transpatch potential (Epatch) was unknown; however, it was assumed that Epatch equals the difference between the resting Em and the applied pipette command potential (Eapp); i.e., Epatch = Eapp - Em. The resting Em in the cells used in this study was approximately -40 mV (67, 70) when the cells were bathed in a solution containing 4.7 mM K+. Thus the single-channel IK measured in the cell-attached patches did not reverse at 0 mV, although the K+ equilibrium potential was ~0 mV (the pipette solution contained 135 mM K+). The currents actually reversed at approximately +35 to +45 mV. To make them clear, the voltages shown in the figures are expressed as Eapp values. All experiments were performed at room temperature (24°C).

Measurement of Em. The Em in single PASMC was measured in a current-clamp configuration with the conventional patch-clamp technique. The extracellular and intracellular solutions were the same as those for the whole cell current recording. Voltage measurement was performed when the cell was held at zero current. Data were acquired by the TL-1 DMA digital interface coupled to a computer and the Axopatch-1D amplifier and analyzed with pCLAMP software (67, 70).

Measurement of [Ca2+]cyt. Details of the digital-imaging methods employed for measuring [Ca2+]cyt have been previously published (20). Briefly, PASMC grown on 25-mm coverslips were incubated in culture medium containing 3.3 µM fura 2-AM for 30-40 min at room temperature (22-24°C) under an atmosphere of 5% CO2 in air. The fura 2-loaded cells were then superfused with a standard bath solution for 20-30 min at 32-34°C to wash away extracellular dye and permit intracellular esterases to cleave cytosolic fura 2-AM into active fura 2. Fura 2 fluorescence (510-nm emission; 380- and 360-nm excitation) from the cells and background fluorescence were imaged with a Nikon Diaphot microscope equipped for epifluorescence. Fluorescent images were obtained with a microchannel plate-image intensifier (Amperex XX1381, Opelco, Washington, DC) coupled by fiber optics to a Pulnix charge-coupled device video camera (Stanford Photonics, Stanford, CA).

Image acquisition and analysis were performed with a MetaMorph Imaging System (Universal Imaging). Video frames containing images of fura 2 fluorescence from cells as well as the corresponding background images (fluorescence from fields devoid of cells) were digitized at a resolution of 512 horizontal × 480 vertical pixels and 8 bits with a Matrix LC imaging board operating in an IBM-compatible computer (66 MHz, 486). To improve the signal-to-noise ratio, 8-32 consecutive video frames were usually averaged at a video frame rate of 30 frames/s. Images were acquired at a rate of one averaged image every 3 s when the [Ca2+]cyt was changing and every 60 s when the [Ca2+]cyt was relatively constant. The [Ca2+]cyt was calculated from fura 2 fluorescent emission excited at 380 and 360 nm using the ratio method (20). In most experiments, multiple cells (usually 6-10) were imaged in a single field, and one arbitrarily chosen peripheral cytosolic area (4-6 × 4-6 pixels) from each cell was spatially averaged.

Solution and reagents. A coverslip containing the cells was positioned in the recording chamber (approx 0.75 ml) and superfused (2-3 ml/min) with the standard extracellular (bath) physiological salt solution (PSS) for either recording IK or measuring [Ca2+]cyt. The PSS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, buffered to pH 7.4 with 5 M NaOH. In Ca2+-free PSS, CaCl2 was replaced by equimolar MgCl2 and 1 mM EGTA was added to chelate residual Ca2+. The internal (pipette) solution for recording whole cell IK(V) contained (in mM) 125 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 Na2ATP, buffered to pH 7.2 with 1 M KOH. In the experiments for recording whole cell IK(Ca) [together with IK(V)], 8.8 mM CaCl2 was added to yield ~2.65 M free Ca2+ in the pipette solution at room temperature (24°C). In experiments with cell-attached patches, the pipette was filled with a 135 mM K+ solution (with 4 mM MgCl2, 10 mM HEPES, 2 mM EGTA, and 10 mM glucose, buffered to pH 7.4 with 1 M KOH). The bath solution was the same as that used for whole cell current recording.

4-Aminopyridine (4-AP; Sigma) was directly dissolved in PSS on the day of use. The pH of the solution containing 4-AP was adjusted to 7.4 with saturated HCl before each experiment. Charybdotoxin (ChTX; Accurate Chemical and Scientific) was dissolved in water to make a stock solution of 100 µM; an aliquot of the stock solution was diluted 1:1,000-10,000 in PSS to make a final concentration of 10-100 nM.

Total RNA isolation. Total RNA was prepared from freshly isolated PA rings, primary cultured PASMC (on days 6-8), and brain tissues by the acid guanidinium thiocyanate-phenol-chloroform extraction method (12). Briefly, the cultured cells were washed with phosphate-buffered solution (Sigma) and scraped into 1 ml/dish of denaturing solution (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, and 0.1 M 2-mercaptethanol, pH 7.0). The DNA was sheared by propelling the solution through a 21-gauge needle 5-10 times. The total RNA was subsequently phenol extracted from the homogenate by adding 0.1 ml of 2 M sodium acetate (pH 4.0), 1 ml of water-saturated phenol, and 0.2 ml of a chloroform-isoamyl alcohol mixture (49:1). The samples were then mixed vigorously and centrifuged at 10,000 g for 20 min. The RNA was precipitated from the aqueous phase by isopropanol (1:1). The total RNA yield was calculated from the absorption of the RNA preparation at 260 nm. The quality of the RNA was determined from the absorbance ratio of the optical density (OD) at 260 nm to the OD at 280 nm (OD260/OD280 > 1.7) and by electrophoresis of the denaturated RNA samples through a 1% agarose-formaldehyde gel (integrity of the 28S and 18S rRNA bands stained with ethidium bromide). Isolated total RNA was dissolved in diethyl pyrocarbonate water at 1 µg/µl and stored at -70°C.

RT-PCR. RT was performed with the First-Strand cDNA Synthesis Kit (Pharmacia Biotech). Four micrograms of total RNA were reverse transcribed with random hexamers [pd(N)6 primer]. The reaction mixture (33-µl final volume) contained 11 µl of the Bulk First-Strand Reaction Mix [consisting of 45 mM Tris, pH 8.3, 68 mM KCl, 15 mM dithiothreitol, 9 mM MgCl2, 0.08 mg/ml of BSA, 1.8 mM each deoxynucleotide triphosphate (dNTP), and 55 units of FPLCpure murine reverse transcriptase], 1 µl of pd(N)6, and 20 µl of the total RNA solution (including 1-5 µg of total RNA). The reaction mixture was incubated for 1 h at 37°C and heated at 90°C for 5 min to inactivate the reverse transcriptase.

The sense and antisense PCR oligonucleotide primers chosen to amplify cDNA are listed in Table 1. The sense and antisense primers for the KV-channel alpha -subunits of KV1.1, KV1.2, KV1.3, KV1.4, KV1.5, KV1.6, KV2.1, and KV9.3 were designed from coding regions of RCK1 (GenBank accession no. X12589), BK2 (GenBank accession no. J04731), KV3 (GenBank accession no. M31744), RCK4 (GenBank accession no. X16002), KV1 (GenBank accession no. M27158), KV2 (GenBank accession no. M27159), RSDRK1PC (GenBank accession no. X16476), and Kv9.3 (GenBank accession no. AF029056), respectively (Table 1). The sense and antisense primers for the KCa-channel alpha -subunit of Slo were designed from the coding region of rat Slo (rSlo; GenBank accession no. U55995). Sequences of the sense and antisense primers for KVbeta 1.1, KVbeta 2, and KVbeta 3 were kindly provided by Dr. S. Reinhardt (University of Mainz, Germany) (Table 1). The fidelity and specificity of the sense and antisense oligonucleotides were examined with the BLAST program.

                              
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  		Table 1.   Oligonucleotide sequences of primers used for RT-PCR

PCR was performed by a GeneAmp PCR system (Perkin-Elmer, Norwalk, CT) with Taq polymerase and accompanying buffers. Two to three microliters of the first-strand cDNA reaction mixture were used in a 50-µl PCR consisting of 0.2 nM each primer, 10 mM Tris · HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 200 M each dNTP, and 2 units of Taq DNA polymerase (Perkin-Elmer). The cDNA samples were amplified in a DNA thermal cycler (model 2400, Perkin-Elmer) under the following conditions: the mixture was annealed at 50-61°C (1 min), extended at 72°C (2.0 min), and denatured at 94°C (1 min) for 30-35 cycles. This was followed by a final extension at 72°C (10 min) to ensure complete product extension. The PCR products were electrophoresed through a 1% agarose gel, and amplified cDNA bands were visualized by ethidium bromide staining.

To quantify the PCR products (the amounts of mRNA) of the KV channels (alpha - and beta -subunits), an invariant mRNA of beta -actin was used as an internal control. Immediately after each experiment, the OD values for each band on the gel were measured by a gel documentation system (UVP, Upland, CA). The OD values of K+-channel signals were normalized to the OD values of the beta -actin signals, which are expressed in arbitrary units (the ratio of the KV-channel mRNA levels to the alpha -actin mRNA levels) for quantitative comparison (66). Because PCR amplification is an exponential process, the extent of amplification is not only dependent on the initial amount of target mRNAs (or cDNAs) but is also related to the efficiency and cycle number. Although invariant beta -actin mRNA was used as an internal control, the possible difference in efficiencies between the primer pairs for beta -actin and the target mRNAs can still lead to different yields of PCR products. Therefore, the PCR study provides only a relative comparison of the amounts of mRNA.

Immunoblotting. The primary cultured PASMC were washed with PBS, scraped into PBS (2 ml/dish), and centrifuged at 3,500 rpm. The cell pellet as well as freshly isolated rat heart (ventricular muscle only) and brain tissues was homogenized in 10 mM HEPES-KOH (pH 7.0) containing the protease inhibitor cocktail (Complete tablets, Boehringer Mannheim, Indianapolis, IN) with a Polytron (Brinkmann) for 10 s at 7,000 rpm. Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL) with BSA as a standard. The samples of homogenates were used for immunoblotting.

Proteins solubilized in SDS buffer were separated by SDS-PAGE. The 10% gels were calibrated with prestained protein molecular-weight markers (Bio-Rad, Richmond, CA). The proteins were then transferred to Hybond-C extra nitrocellulose membrane (Amersham) as previously described (63). The efficiency of the transfer was verified by Ponceau-S staining. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and 0.1% Tween 20. The blots were then incubated with the affinity-purified polyclonal antibodies specific for KV1.2-, KV1.3-, KV1.4 (Alomone Labs, Jerusalem, Israel)-, KV1.5-, and KV2.1 (Upstate Biotechnology, Lake Placid, NY)-channel alpha -subunits for >= 1 h. The membranes were washed (3 × 5 min) and incubated with anti-rabbit horseradish peroxidase-conjugated IgG for 1 h, and an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL) was used for detection of the bound antibody.

Statistical analysis. The composite data are expressed as means ± SE. Statistical analyses were performed with paired and unpaired Student's t-test and one-way ANOVA, as indicated. Differences were considered to be significant when P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Electrophysiological differentiation of rapidly inactivating and delayed rectifier IK(V). When cells were superfused with the Ca2+-free, 1 mM EGTA-containing bath solution and dialyzed with the Ca2+-free, 10 mM EGTA-containing pipette solution (with 5 mM ATP), IK(V) was elicited by depolarization to test potentials ranging from -40 to +80 mV (Fig. 1, A and B). IK(Ca) and IK(ATP) were minimized under these conditions (29, 46, 67). On depolarization to +80 mV, the current was rapidly activated and consisted of a transient component [IK(tr)] and a steady-state component [IK(ss)] (Fig. 1C). Changing the holding potential from -70 to -40 mV abolished IK(tr) and decreased IK(ss) (Fig. 1C). Subtraction of the two current records obtained from different holding potentials revealed an IK(tr) that resembles the rapidly inactivating (A-type) IK(V) and isolated an IK(ss) that is similar to the non- or slowly inactivating delayed rectifier IK(V) (16, 18, 46, 47, 67, 70). Both components of IK(V), IK(tr) and IK(ss), were significantly and reversibly inhibited by 5 mM 4-AP (Fig. 1D), whereas ChTX (10-20 nM) had no effect on these currents (67).


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  		Fig. 1.   Electrophysiological differentiation of rapidly inactivating (A-type) and delayed rectifier voltage-gated K+ (KV) currents [IK(V)] and inhibitory effect of 4-aminopyridine (4-AP) on currents in pulmonary arterial smooth muscle cells (PASMC) superfused and dialyzed with Ca2+-free solution. A: family of currents obtained by depolarizing the cell from a holding potential of -70 mV to a series of command potentials ranging from -40 to +80 mV. Pipette (intracellular) solution contained 10 mM EGTA and 5 mM ATP. B: current-voltage (I-V) relationship curve assembled from data shown in A. Transient IK(V) [IK(tr)] was measured at 10-50 ms. Steady-state IK(V) [IK(ss)] was measured at 250-290 ms (test pulse duration was 300 ms). C: predepolarization to -40 mV inactivated IK(tr). Inset: current component that was inactivated by predepolarizing the cells from -70 to -40 mV before +80-mV test pulse was applied. D: representative current trace elicited by depolarizing the cell from a holding potential of -70 to +60 mV recorded before (Control), during (4-AP), and after (Recovery) extracellular application of 5 mM 4-AP. Inset: 4-AP-sensitive component of IK(V).

Activation of IK(Ca) by increasing [Ca2+]cyt. When cells were superfused with the 1.8 mM Ca2+-containing bath solution and dialyzed with the 8.8 mM Ca2+-containing pipette solution (with 5 mM ATP and 10 mM EGTA present), both IK(V) and IK(Ca) were elicited by depolarization to test potentials ranging from -40 to +80 mV (Fig. 2A, left). The activation kinetics of the currents was slower than the IK(V) shown in Fig. 1, but the amplitude of the currents was much greater (compare Fig. 2 with Fig. 1). Extracellular application of 10 nM ChTX significantly inhibited the currents from 9.1 ± 1.7 to 4.1 ± 1.1 nA (P < 0.001; n = 5 cells) at the test potential of +80 mV (Fig. 2, A and B), mainly due to the inhibitory effect of ChTX on IK(Ca). Subtraction of the current records obtained before and during introduction of ChTX revealed the ChTX-sensitive IK(Ca) (Fig. 2A, bottom). The relatively slow activation kinetics of the ChTX-sensitive IK(Ca) appears to be similar to the IK(Ca) recorded from the hSlo cRNA-injected Xenopus oocytes (33).


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  		Fig. 2.   Whole cell Ca2+-activated K+ currents in PASMC dialyzed with pipette solution containing high concentration of free Ca2+ ([Ca2+]). A: family of currents elicited by depolarizing the cell from a holding potential of -70 mV to a series of command potentials ranging from -40 to +80 mV when [Ca2+] in pipette solution was increased to ~2.65 µM (8.8 mM CaCl2, 10 mM EGTA, and 5 mM ATP, pH 7.2, at 24°C). Currents were recorded before (Control) and during extracellular application of 10 nM charybdotoxin (ChTX). ChTX-sensitive component of the currents was obtained by subtracting the currents recorded during application of ChTX from the currents recorded under control condition (Subtraction). IK, K+ current. B: I-V relationship curves for current records shown in A.

The dose giving half-maximal inhibition of ChTX for IK(Ca) is 2-15 nM in smooth muscle cells (10, 40). Thus 10 nM ChTX did not completely block IK(Ca); the remaining currents shown in Fig. 2A, right, included both IK(V) and IK(Ca). Because of the large fraction of IK(Ca), the remaining currents during application of 10 nM ChTX displayed similar kinetics as IK(Ca). The large, long-lasting inward tail currents elicited under these conditions might be due to the contamination of Ca2+-regulated nonselective cation (e.g., Na+) currents (10) and/or Ca2+-activated Cl- currents, which have been described in these cells (68), although we do not know why the currents were somehow inhibited by ChTX. It has been demonstrated that many K+-channel blockers also block the Ca2+-activated nonselective cation channels (10). It is not clear, however, whether ChTX can affect the Ca2+-activated nonselective cation currents in PASMC.

Single-channel IK(Ca) and IK(V) in PASMC. In cell-attached membrane patches of PASMC superfused with a Ca2+-containing solution, a large-amplitude IK was elicited by depolarizing the patch to +70 mV (Fig. 3A). The slope conductance of the large-amplitude IK, calculated from the current-voltage relationships, was 214 pS (n = 8 patches; Fig. 3C). Therefore, the large-amplitude IK corresponds to the large-conductance IK(Ca) that is activated by an increased [Ca2+]cyt (2, 7, 18, 19, 48, 73). In some cell-attached membrane patches of PASMC superfused with a Ca2+-free solution, several small-amplitude IK were elicited by depolarizing the patches to +70 mV (Fig. 3B). These small-amplitude IK represent the small-conductance IK(V) that were previously described in both animal and human PASMC (18, 19, 40, 46, 73). As shown in Fig. 3, B and C, there appeared to be at least three classes of KV channels in PASMC based on the calculated slope conductances: a 24-pS channel (n = 14 patches; a), a 40-pS channel (n = 9 patches; b), and a 67-pS channel (n = 8 patches; c). It has been demonstrated that there are multiple IK(V) in colonic smooth muscle cells, with conductances of 82, 42, 8, and <4 pS (27). These results indicate that smooth muscle cells functionally express multiple KV channels.


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  		Fig. 3.   Single-channel IK recorded from cell-attached membrane patches of PASMC superfused with Ca2+-containing (A) and Ca2+-free (B) solutions. Patch command potential [holding potential (HP)] was held at +70 mV. A large-amplitude current (A) and 3 small-amplitude currents (Ba-Bc) were observed on depolarization. Dotted lines, current levels when channels are closed. Currents were recorded from different PASMC. C: I-V relationship curves for large-amplitude current {Ca2+-activated current [IK(Ca)]} and the 3 small-amplitude currents [IK(V); a-c]. Calculated slope conductances are 214 (n = 8 patches; square ), 24 (n = 14 patches; black-triangle), 40 (n = 9 patches; open circle ), and 67 pS (n = 8 patches; black-square). Resting membrane potential (Em) of these cells is about -40 mV. Data are means ± SE.

Functional roles of KV and KCa channels in regulating Em and [Ca2+]cyt. Application of 4-AP (5 mM) depolarized PASMC and elicited Ca2+-dependent action potentials (Fig. 4A). Removal of extracellular Ca2+ abolished the transient action potentials but did not affect the steady-state depolarization (data not shown). The KCa-channel blocker ChTX (20 nM), however, had a negligible effect on the resting Em (Fig. 4B). Although the inability of ChTX to elicit depolarization may be partially due to a high-buffering capacity of intracellular Ca2+, it is more likely that KCa channels are relatively closed under resting conditions.


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  		Fig. 4.   Effect of 4-AP and ChTX on Em (A and B) and cytosolic free [Ca2+] ([Ca2+]cyt; C-E) in PASMC. Em was measured using current-clamp mode in cells before, during, and after application of 5 mM 4-AP (A) or 20 nM ChTX (B) in presence of extracellular Ca2+. C: [Ca2+]cyt measured in peripheral areas (small boxes labeled a-c in black-and-white 360-nm image). Right: corresponding [Ca2+]cyt record obtained from each of the cells shown at left. D: summarized data showing change in [Ca2+]cyt induced by 4-AP [5 mM; peak and plateau (Plat)] or ChTX (20 and 100 nM). Data are means ± SE; n = 41 cells. *** P < 0.001 vs. basal [Ca2+]cyt levels. E: pseudocolor images showing [Ca2+]cyt before (a), during [peak (b) and Plat (c)] and after (d) application of 4-AP as well as [Ca2+]cyt before (e), during [20 ( f ) and 100 (g) nM], and after (h) application of ChTX. The 360-nm excitation image of the cells used for measuring [Ca2+]cyt (a-h) is shown in C (left) to clarify size of cells.

In intact PASMC devoid of dialysis with the pipette solution, 4-AP (5 mM) reversibly increased [Ca2+]cyt (Fig. 4, C, D, and Ea-Ed) due, apparently, to the evoked membrane depolarization and Ca2+ action potentials. After application of 4-AP, a slight shift of the resting [Ca2+]cyt was noted (compare Fig. 4Ea with Fig. 4Ee), especially in the perinuclear areas where the sarcoplasmic reticulum is mainly located (20, 65). This suggests that more Ca2+ may be sequestered into the sarcoplasmic reticulum after application of 4-AP, and slow leakage of the stored Ca2+ to the cytosol resulted in the small drift of the resting [Ca2+]cyt in perinuclear areas. Application of the KCa-channel blocker ChTX (20 or 100 nM) had no effect on [Ca2+]cyt (Fig. 4C, right, D, and Ee-h). The cells used for measuring [Ca2+]cyt were then stained with anti-alpha -smooth muscle actin antibody, and the results indicated that all of the cells were smooth muscle cells.

The results for [Ca2+]cyt in the nondialyzed PASMC (Fig. 4, C-E) are consistent with the findings for Em in the dialyzed PASMC (Fig. 4A), indicating that the IK(V) is the major determinant of the Em and thus the [Ca2+]cyt under resting conditions. KCa channels, although relatively closed under resting conditions, certainly contribute to the regulation of Em, Ca2+ homeostasis, and vascular tone when [Ca2+]cyt is increased (7, 16, 17, 29, 38, 46, 67).

Identification of KV- and KCa-channel alpha -subunit gene transcripts in PASMC. Based on the known sequences, K+ channels fall into three superfamilies: the voltage-gated channels, Ca2+-activated channels, and inward rectifier channels. The subsequent experiments were designed to identify the K+-channel genes expressed in PASMC.

RT-PCR was used to test the functional expression of KV and KCa channels in PASMC. Equal amounts of total mRNA isolated from primary cultured PASMC were reversely transcribed and amplified with oligonucleotide primers (Table 1) specifically designed for the alpha -subunits [KV1.1 (RCK1), KV1.2 (BK2), KV1.3 (KV3), KV1.4 (RCK4), KV1.5 (KV1), KV1.6 (KV2), and rSlo] as well as for the beta -subunits (KVbeta 1.1, KVbeta 2, and KVbeta 3). The K+-channel PCR products were separated on a 1% agarose gel and visualized by staining the gel with ethidium bromide. On the basis of the predicted size of the PCR products, PASMC contain KV1.1 (594 bp), KV1.2 (295 bp), KV1.4 (322 bp), KV1.5 (1,111 bp), KV1.6 (394 bp), and rSlo (864 bp) gene transcripts (Fig. 5A). Because the RT-PCR procedures used in these experiments were not quantitative, these results did not provide any information regarding the quantity of the particular KV-channel mRNAs (see Quantitative comparison of KV- and KCa-channel gene transcripts in PASMC).


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  		Fig. 5.   RT-PCR analysis of KV- and Ca2+-activated K+ (KCa)-channel alpha -subunit mRNAs isolated from PASMC. A: PCR-amplified products displayed in agarose gel stained with ethidium bromide for KV1.1 (594 bp; a), KV1.2 (295 bp; b), KV1.4 (322 bp; c), KV1.5 (1,111 bp; d), KV1.6 (394 bp; e), and rat slowpoke gene (rSlo; 864 bp; f ) in PASMC. B and C: PCR-amplified products for KV1.3 (515 bp) and conserved region of all KV channels (KV-all; 227 bp), respectively, in PASMC (PA) and brain tissues (Br). First-strand cDNAs synthesized from 5 µg of total RNA (extracted from PASMC or Br) were amplified with specific sense and antisense primers for the respective alpha -subunits (Table 1). M, molecular-weight marker; +, cDNA added; -, cDNA not added. Same results were repeated in 3-5 independent experiments.

With the use of the primer specifically designed for the KV1.3 alpha -subunit, the PCR product (predicted size is 515 bp) was not detected in PASMC but was perceived in brain tissue (Fig. 5B). To ensure that the cDNAs used for the amplification of KV1.3 channels were not defective, the same cDNAs of PASMC were used in a PCR with another primer that was designed for the conserved region of KV channels (KV-all in Table 1). As shown in Fig. 5C, a 227-bp PCR product was detected in both PASMC and brain tissue, indicating that the mRNAs and cDNAs were both intact. These results suggest that PASMC may not express KV1.3 channels. Because KV1.3 is a KV channel that is sensitive to ChTX (11), these data are consistent with the previous observation by Yuan (67) that ChTX negligibly affected the native IK(V) in PASMC.

Identification of KV-channel beta -subunit gene transcripts in PASMC. KV-channel beta -subunits were recently cloned from rat brain (KVbeta 1.1, KVbeta 2, and KVbeta 3) and human heart (KVbeta 1.2 and KVbeta 1.3) (15, 24, 26, 50). Association of the beta -subunits with KV-channel pore-forming alpha -subunits significantly contributes to the KV-channel diversity and its various functions in vivo (24, 26, 36, 50, 56). Figure 6 illustrates that PASMC also express KVbeta 1.1, KVbeta 2, and KVbeta 3; the predicted sizes of the PCR products are 150, 141, and 178 bp, respectively.


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  		Fig. 6.   RT-PCR analysis of KV-channel beta -subunit mRNA in PASMC and Br. PCR-amplified products are displayed in agarose gel stained with ethidium bromide for KVbeta 1.1 (150 bp; A), KVbeta 2 (141 bp; B), and KVbeta 3 (178 bp; C). First-strand cDNAs, synthesized from 5 µg of total RNAs extracted from PASMC and Br, were amplified with specific sense and antisense primers for the respective beta -subunits (Table 1). Same results were repeated in 3-4 independent experiments.

In another set of experiments, a sham cDNA control (i.e., the RNA that was treated exactly like the RT reactions except that no reverse transcriptase was used) was included in the PCR experiments to ensure that all the RNA samples prepared from PASMC were not contaminated by DNA. As shown in Fig. 7, even though RNA was used for PCR (i.e., RT reaction was blocked by eliminating reverse transcriptase from the reaction mixture) as a no-RT control, there were no PCR products detected when the specific primers for K+-channel alpha - and beta -subunits were used.


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  		Fig. 7.   RT-PCR analyses of KV1.2, KV1.4, rSlo, and KVbeta 1.1 mRNA in PASMC. PCR-amplified products are displayed in agarose gel stained with ethidium bromide for KV1.2 (295 bp; A), KV1.4 (322 bp; B), rSlo (864 bp; C), KVbeta 1.1 (150 bp; D), and beta -actin (244 bp; A-D). +, RT performed in presence of reverse transcriptase (cDNA); -, RT performed in absence of reverse transcriptase (RNA). Same results were repeated in 3-4 independent experiments.

Quantitative comparison of KV- and KCa-channel gene transcripts in PASMC. In previous experiments by Wang et al. (66), the quantity of PCR products for beta -actin and KV1.2 correlated linearly with the change in cycle numbers between 23 and 28 cycles when 3.0 µg of total RNA and 3.0 µl of cDNA were used in RT-PCR. Under conditions of 25 cycles and 3.0 µg of total RNA being used for amplifying the messages in PCR, the change in the cDNA level of beta -actin and KVbeta 3 between 1.5 and 5.0 µl correlated linearly with the amount of the PCR products. When 3.0 µl of cDNA and 25 cycles were used in the PCR, the change in total RNA levels between 1.5 and 5.0 µg also correlated linearly with the quantity of the PCR products of beta -actin, KV1.2, and KVbeta 3. These results indicate that the experimental protocol for RT-PCR, 3 µg of total RNA for RT and 3 µl of cDNA and 25 cycles for PCR, was appropriate to quantify the mRNA levels of the KV channels (66).

Total RNA was extracted from the primary cultured PASMC. After RT, the same amount of first-strand cDNA was used in PCR, consisting of the specific primers for K+ channels and beta -actin. Gene transcription (mRNA levels) of the KV-channel alpha -subunits (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), KCa-channel alpha -subunit (rSlo), and KV-channel beta -subunits (KVbeta 1.1, KVbeta 2, and KVbeta 3) was examined and compared, whereas the beta -actin mRNA level was used as the control level (Fig. 8). The mRNA levels of KV1.2, KV1.5, and rSlo were significantly greater than those of KV1.1, KV1.4, and KV1.6 (Fig. 8A), whereas the mRNA level of KVbeta 1.1 was significantly greater than those of KVbeta 2 and KVbeta 3 (Fig. 8B). These results suggest that the transcriptional levels of various K+-channel alpha - and beta -subunits are different in PASMC. KV1.2, KV1.5, and rSlo appear to be the major K+ channels expressed in PASMC.


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  		Fig. 8.   Quantitative comparison of KV-channel alpha - and beta -subunit and KCa-channel alpha -subunit mRNA levels in PASMC. A: PCR-amplified products displayed in agarose gels for KV1.1 (594 bp), KV1.2 (295 bp), KV1.4 (322 bp), KV1.5 (283 bp), KV1.6 (394 bp), rSlo (864 bp), and beta -actin (244 bp) when first-strand cDNAs, synthesized from total RNA extracted from PASMC, were amplified with specific sense and antisense primers (Table 1). Bottom: data that were normalized to amount of beta -actin expressed as means ± SE (experiments were repeated 4 times independently). B: PCR analyses for KVbeta 1.1 (150 bp), KVbeta 2 (141 bp), KVbeta 3 (178 bp), and beta -actin when first-strand cDNAs were amplified with specific sense and antisense primers (Table 1). Bottom: data that were normalized to amount of beta -actin expressed as means ± SE (experiments were repeated 3 times independently).

The quantitative RT-PCR experiments, however, were performed with different primers for the respective channel genes, and different primers may have different binding efficiencies with the corresponding channel cDNAs. Therefore, the data may not reflect the true differences in the transcriptional levels of the various channels in PASMC.

Identification of KV- and KCa-channel gene transcripts in freshly isolated PA rings. Total RNA was extracted from freshly isolated, endothelium-denuded PA rings. After RT, the same amount of first-strand cDNA was used in PCR, consisting of the specific primers for K+ channels (Table 1) and beta -actin. Consistent with the results obtained from primary cultured PASMC, the gene transcripts of KV1.1, KV1.2, KV1.4, KV1.5, KV1.6, and rSlo as well as those of KVbeta 1.1, KVbeta 2 and KVbeta 3 were also detected in the isolated PA rings (Fig. 9).


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  		Fig. 9.   RT-PCR analyses of KV-channel alpha - and beta -subunit and KCa-channel alpha -subunit mRNAs in freshly isolated pulmonary arteries. A: PCR-amplified products displayed in agarose gels for KV1.1 (594 bp), KV1.2 (295 bp), KV1.4 (322 bp), KV1.5 (283 bp), KV1.6 (394 bp), and rSlo (864 bp) when first-strand cDNAs, synthesized from total RNA extracted from rat endothelium-denuded pulmonary arteries, were amplified with specific sense and antisense primers (Table 1). B: PCR analysis for KVbeta 1.1 (150 bp), KVbeta 2 (141 bp), and KVbeta 3 (178 bp) when first-strand cDNAs were amplified with the specific sense and antisense primers for KV-channel beta -subunits (Table 1).

Identification of KV-channel proteins by immunoblotting in PASMC. The expression of three KV-channel proteins (KV1.2, KV1.4, and KV1.5) from the Shaker family was verified by immunoblotting (Fig. 10). In control experiments, the immunoblot was incubated in rabbit normal serum; the serum did not cross-react with KV-channel proteins (data not shown).


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  		Fig. 10.   Western blotting analyses of KV1.2 (A)-, KV1.3 (B)-, KV1.4 (C)-, and KV1.5 (D)-channel proteins in PASMC. Immunoblots of PASMC (PA), Br, and heart (H) tissue proteins (10 µg/lane) were incubated with affinity-purified anti-KV1.2, anti-KV1.3, anti-KV1.4, and anti-KV1.5 polyclonal antibodies. Nos. on left, molecular-mass markers. Control blot, incubated with rabbit normal serum, was blank and is not shown.

The anti-KV1.2 antibody recognized a single band at ~75 kDa in PASMC (Fig. 10A). A band of almost the same size was also detected in brain and heart tissues (Fig. 10A). The predicted molecular mass of the KV1.2-channel protein is 56.7 kDa (35). The slower mobility of the KV1.2-channel protein on immunoblots was probably caused by extensive glycosylation of this protein; similar results were also observed by other investigators (57). The anti-KV1.3 antibody did not recognize a band in PASMC and heart ventricular muscle (Fig. 10B). However, a double band at ~68 kDa and a single band at ~82 kDa were detected in brain tissue (Fig. 10B). The molecular sizes of the two bands are in good agreement with the results (68 and 88 kDa) by researchers from the Alomone Labs (from which we purchased the KV1.3 antibody) but are larger than the predicted molecular mass (58.4 kDa) (61). This is likely due to the glycosylation of the KV1.3-channel protein, which is a typical feature for most of the KV channels (57). These results are consistent with RT-PCR experiments that showed that KV1.3 may not be expressed in PASMC and heart ventricles (61). The anti-KV1.4 antibody recognized a single band at ~72 kDa in PASMC and brain and heart samples (Fig. 10C). The size of this band is in good agreement with the predicted molecular mass of the KV1.4-channel protein (73.4 kDa; Ref. 60) but is smaller than the KV1.4-channel proteins identified by Maletic-Savatic et al. (95 kDa; Ref. 31) and Takimoto et al. (96 kDa; Ref. 62). The discrepancies in the sizes of the KV1.4-channel protein detected by immunoblots can be due to different experimental conditions used for sample preparation and immunoblotting (e.g., gel percentage, sample boiling). The anti-KV1.5 antibody recognized a sharp band at ~63 kDa in all three tested samples (PASMC and brain and heart tissues) (Fig. 10E). This result is consistent with the predicted molecular mass (66.3 kDa) of the KV1.5-channel protein in rat brain (61) and canine colonic smooth muscle (43).

Identification of KV2.1 and KV9.3 channels in PASMC and freshly isolated PA. Patel et al. (45) recently cloned KV2.1 and a novel KV channel, KV9.3, from rat PASMC. Expression of KV2.1 and coexpression of KV2.1 and KV9.3 (KV2.1/KV9.3) into COSm6 cells significantly altered the resting Em (from +4 to -31 and -51 mV, respectively). KV2.1 and KV2.1/KV9.3 were both reversibly inhibited by hypoxia and activated by intracellular ATP, suggesting that the KV2.1/KV9.3 heteromultimer in PASMC may play an important role in hypoxia-mediated membrane depolarization (3, 42, 45, 47, 48, 59, 71) and pulmonary vasoconstriction (28, 37).

With the use of the primers specifically designed for KV2.1 and KV9.3 (45), 496- and 569-bp PCR products were identified from both PASMC (Fig. 11A) and the endothelium-denuded PA rings (Fig. 11B). The expression of KV2.1 was confirmed in PASMC by Western blot analysis with the polyclonal antibody for KV2.1. The anti-KV2.1 antibody recognized a single band at ~108 kDa (58) in PASMC (Fig. 11C). A band at the same size was also detected in rat brain and heart (ventricle) tissues (Fig. 11C). The predicted molecular mass of the KV2.1 channel from the deduced primary sequence is 95 kDa (58). The increased size of KV2.1 in PASMC and brain and heart tissues is probably due to posttranslational modification (58). KV9.3 is an electrically silent KV-channel alpha -subunit that modulates the electrophysiological properties of the functional KV-channel KV2.1 (45). Whether KV9.3 is heteromultimerized with other subfamilies of KV-channel alpha -subunits (e.g., KV1) is unknown.


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  		Fig. 11.   Identification of KV2.1 and KV9.3 in PASMC and pulmonary arterial (PA) rings. A and B: RT-PCR-amplified products displayed in agarose gels for KV2.1 (496 bp; left) and KV9.3 (569 bp; right). First-strand cDNAs, synthesized from total RNA extracted from rat PASMC (A) and endothelium-denuded PA rings (B), were amplified with specific sense and antisense primers (Table 1). +, RT performed in presence of reverse transcriptase (cDNA); -, RT performed in absence of reverse transcriptase (RNA). Same results were repeated in 3 independent experiments. C: Western blot analysis of KV2.1 in PASMC. Immunoblots of PASMC (PA; 11 µg/lane), Br (5 µg/lane), and H (18 µg/lane) tissue proteins were incubated with affinity-purified anti-KV2.1 polyclonal antibody. Nos. on left, molecular-mass markers.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Activity of K+ channels, by governing Em, plays an important role in the regulation of [Ca2+]cyt and pulmonary vascular tone. Inhibition of KV channels by 4-AP or hypoxia depolarizes PASMC, thereby increasing [Ca2+]cyt and causing pulmonary vasoconstriction (3, 16, 23, 42, 45-48, 59, 67). Activation of KV channels by nitric oxide hyperpolarizes PASMC, thus inhibiting the evoked increase in [Ca2+]cyt and inducing pulmonary vasodilation (4, 73). In this study, two kinetically distinct IK(V), IK(tr) [the A-type IK(V)] and IK(ss) [the non- or slowly inactivating delayed rectifier IK(V)], as well as a large-amplitude slowly activating IK(Ca) were described in PASMC using electrophysiological approaches.

Because IK(tr) is almost completely inactivated at a potential close to the resting Em (approximately -40 mV) in PASMC, IK(tr) is mainly involved in regulating duration of the action potential and limiting agonist-induced depolarization. IK(ss), however, is active at the resting Em (19 ± 3 pA at -40 mV; n = 28 patches) and thus plays an important role in controlling the resting Em (16-19, 29, 47, 59, 67). In vascular smooth muscle cells, including PASMC, the resting [Ca2+]cyt ranges from 50 to 150 nM, and most KCa channels are closed when [Ca2+]cyt is <= 300 nM at 0 mV (2, 46). Thus, under resting conditions in which [Ca2+]cyt is ~100 nM and Em is approximately -40 mV, KCa channels are largely inactive. However, when [Ca2+]cyt in PASMC is increased and the cells are depolarized, activation of KCa channels provides a critical negative feedback pathway to control vascular tone and stimulation-induced active tension in the pulmonary vasculature (7, 46).

Molecular identification of KV channels in PASMC. There are at least eleven subfamilies of the KV-channel alpha -subunits KV1 (KV1.1-KV1.7, Shaker), KV2 (KV2.1-KV2.2, Shab), KV3 (KV3.1-KV3.4, Shaw), KV4 (KV4.1-KV4.3, Shal), KVLQT, EAG (ether-a-go-go), KV5 (KV5.1), KV6 (KV6.1), KV7, KV8 (KV8.1), and KV9 (KV9.1-KV9.3) that have been cloned in mammals (11). Four of these, KV5, KV6, KV8, and KV9, are known to be electrically silent KV-channel modulatory alpha -subunits, whereas the remainders are functional KV-channel alpha -subunits (with electrical activity) (11, 14, 25, 45, 49, 54, 55, 74). In addition, there are three subfamilies of the KV-channel beta -subunits KVbeta 1 (KVbeta 1.1-KVbeta 1.3), KVbeta 2 (KVbeta 2.1), and KVbeta 3 (KVbeta 3.1) (15). At the molecular level, the native K+ channels are heteromultimers composed of four large pore-forming alpha -subunits and four smaller cytoplasmic beta -subunits (alpha 4beta 4) (11, 26). The biophysical properties of K+ channels encoded by certain alpha -subunits can be dramatically altered by association with beta -subunits (24, 26, 50, 56).

By using RT-PCR, five KV-channel alpha -subunit genes from the Shaker subfamily (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), a KV-channel alpha -subunit gene from the Shab subfamily (KV2.1), and a modulatory alpha -subunit gene from the KV9 subfamily (KV9.3) as well as a KCa-channel alpha -subunit gene (rSlo) were identified in PASMC. In addition, transcripts of three beta -subunits, KVbeta 1.1, KVbeta 2, and KVbeta 3, were also identified in PASMC. By using immunoblotting, expression of KV1.2-, KV1.4-, KV1.5-, and KV2.1-channel alpha -subunits was confirmed. The specifically cross-reacting bands of KV1.2-, KV1.4-, KV1.5-, and KV2.1-channel proteins have molecular masses of ~75, 72, 63, and 108 kDa, respectively, which are comparable in size to the respective KV-channel bands from the brain and heart (43, 57, 58, 60, 61). These Western blotting results also indicate that the KV1.2, KV1.4, KV1.5, and KV2.1 channels expressed in PASMC are immunologically similar to the corresponding KV channels expressed in the brain.

The data from the present study suggest that the native K+ channels in PASMC are encoded by multiple alpha - and beta -subunit genes. The homo- and/or heteromultimeric assembly of alpha -subunits as well as the association of alpha - and beta -subunits both contribute to the remarkable diversity of K+ channels and the numerous electrophysiological and pharmacological properties of K+ channels in vivo (11, 26, 50, 52).

Although extensively studied in the brain (26, 61) and heart (6, 15, 51), the molecular basis for KV channels in smooth muscle has only recently been elucidated (1, 22, 43, 45). In canine colonic smooth muscle (22, 43), KV1.2 and KV1.5 have been cloned, and the sequences revealed significant homology to KV1.2 and KV1.5 in the brain and heart. The colonic KV1.5 (cKV1.5) was also detected in the canine PA (43). By using a Northern blot and PCR, Adda et al. (1) recently identified KV1.1, KV1.2, and KV1.5 transcripts in human airway smooth muscle cells. The electrophysiological and functional studies indicated that the gene products of KV1.1, KV1.2, and KV1.5 play an important role in regulating airway smooth muscle contractility in humans (1).

The Shab KV channel KV2.1 and a novel KV-channel alpha -subunit, KV9.3, have been recently cloned in rat PASMC (45). The KV2.1/KV9.3 heteromultimer, expressed in COSm6 cells or Xenopus oocytes, opens at the voltage range of the resting Em in PASMC and is regulated by oxygen tension and intracellular ATP (45).

Biophysical and pharmacological properties of KV channels. In PASMC, the native IK(V) is composed of at least an A-type IK(V) and a non- or slowly inactivating delayed rectifier IK,V. Both of the currents are activated at relatively negative potentials (-40 to -30 mV), and the single-channel conductance is 5-75 pS (17-19, 27, 47, 73). 4-AP is a potent blocker of KV channels (41, 43); the IC50 is 0.3-0.7 mM at 10 mV (18, 41), and the dissociation constant (Kd) ranges from 0.2 to 9 mM for expressed KV channels (11). KV channels, however, are relatively insensitive to ChTX, iberiotoxin, and a low dose of tetraethylammonium (TEA; <= 1 mM) (10, 40).

Of the seven mammalian Shaker KV channels that have been biophysically characterized, only KV1.4 displays rapid (N-type) inactivation, and the remainder (KV1.1, KV1.2, KV1.3, KV1.5, KV1.6, and KV1.7) are non- or slowly inactivating delayed rectifiers (11). Inactivation of the A-type IK(V) [IK(tr)] can be explained by the "ball-and-chain" model (11, 26, 50, 53). In KV channels, the ball corresponds to the first 20 NH2-terminal amino acids in the channel alpha -subunit protein (53) or the associated KVbeta 1 subunit (50). Oxidation of a cysteine residue located in the ball sequence of the KV1.4 channel or the KVbeta 1 subunit eliminates, whereas application of the reducing agent glutathione restores, rapid inactivation of this channel (50, 53). The rapid inactivating property and the sensitivity to hypoxia and the reducing agents of the native IK(tr) in PASMC (71, 72) as well as the long NH2-terminal amino acid sequence of the KV1.4 gene-encoded protein product (11) support the contention that IK(tr) is generated by KV1.4 channels. Nevertheless, the possibility that beta -subunits are associated with delayed rectifier KV-channel alpha -subunits in PASMC cannot be excluded because the association confers the fast N-type inactivation on the slowly inactivating, delayed rectifier KV channels (50).

The cloned delayed rectifier KV channels share some electrophysiological and pharmacological properties with the native KV channels described in smooth muscle cells. The activation threshold of KV1.1, KV1.2, KV1.5, and KV2.1/KV9.3 is -50 to -30 mV, and the single-channel conductance is 8-30 pS (11, 22, 43, 45). The cloned delayed rectifier channels are sensitive to 4-AP; the Kd ranges from 0.2 to 9 mM (11). Similarly, the activation threshold of the native delayed rectifier IK(V) in PASMC is approximately -50 to -30 mV (47, 67), and the currents are also sensitive to 4-AP; the Kd ranges from 0.2 to 10 mM (40, 52). The single-channel conductance of the delayed rectifier KV channels, however, is somehow greater (20-104 pS) (17-19, 40, 47, 73) than that of the cloned channels (11, 22, 43, 45).

Although we have demonstrated that both alpha - and beta -subunits are expressed in PASMC, their stoichiometry and whether the native A-type KV channels and/or delayed rectifier KV channels in PASMC are homogenous (monomer) or heteromultimeric (multimer) tetramers are unclear and need further study. In this study, the KV1-family alpha -subunits were emphatically examined in PASMC because the KV-channel beta -subunits only bind to the Shaker-related subfamily (KV1 alpha -subunits) (56). Other KV-channel genes that were not examined in the study are also very likely involved in encoding the channels that contribute to the functionally and kinetically distinct IK(V) in PASMC.

Biophysical and pharmacological properties of KCa channels. KCa channels (BK or maxi-K channels) have been described in smooth muscle cells obtained from a variety of systemic and pulmonary arteries and veins (2, 4, 10, 46). These channels are often activated by relatively more positive test potentials when the membrane is exposed to a physiological [Ca2+]cyt (50-150 nM) (2, 10, 18, 29). IK(Ca) can be significantly blocked by ChTX (IC50 = 2 nM), iberiotoxin (IC50 = 10 nM), or a low dose of TEA (IC50 = 0.16 mM) and is extremely sensitive to [Ca2+]cyt (2, 10). Other characteristics of KCa channels include their large conductance (200-250 pS with a symmetrical K+ gradient) (2, 10, 18, 41) and relatively slow activation kinetics (33).

Correspondingly, the cloned KCa channels (hSlo, mSlo, or dSlo) expressed in Xenopus oocytes also have a large conductance (200-282 pS) and relatively positive activation threshold (23-32 mV when [Ca2+]cyt is 10 M; Refs. 8, 33). IK(Ca) in the hSlo cRNA-injected Xenopus oocytes is also very sensitive to ChTX, iberiotoxin [40 nM ChTX or 20 nM iberiotoxin almost abolishes the hSlo IK(Ca); Ref. 33], and TEA (IC50 = 0.14 mM; Refs. 8, 33). The significant similarities of the electrophysiological and pharmacological properties between the native IK(Ca) and the IK(Ca) derived from the Slo gene suggest that rat PASMC express an rSlo gene that gives rise to the large-conductance IK(Ca). Because KCa channels are also composed of alpha - and beta -subunits and the latter contributes to the pharmacological and biophysical properties of IK(Ca) (21, 36), further study is needed to elucidate whether rSlo and KVbeta 1 (or KVbeta 2 and KVbeta 3)-subunit encoding products are associated in PASMC.

Function of KV and KCa channels in regulating Em, [Ca2+]cyt, and pulmonary vasomotor tone. Functionally, the non- or slowly inactivating delayed rectifier KV channels are more likely the major determinants in controlling the resting Em and thus the resting [Ca2+]cyt and tonic tension (16-19, 23, 29, 46, 47, 67). KCa channels, in contrast, comprise a negative-feedback pathway in regulating the Em when the [Ca2+]cyt is increased and thus govern phasic or active tension (7, 19, 29, 46). Indeed, inhibition of KV channels by 4-AP depolarized PASMC, raised [Ca2+]cyt, and significantly increased PA pressure and vascular resistance in an isolated perfused lung (23), whereas inhibition of KCa channels by a low dose of TEA and ChTX negligibly affected the Em, [Ca2+]cyt, or PA pressure (23). The same results were also obtained from an isolated, endothelium-denuded human PA (46).

It has been recently demonstrated that KV channels are regulated by oxygen tension (3, 30, 45-48, 59, 66, 71), cellular metabolism (45, 72), redox status (3, 50, 53, 72), and nitric oxide (4, 73). Hypoxia-induced inhibition of KV channels in PASMC has been demonstrated to be an important trigger of hypoxic pulmonary vasoconstriction (3, 30, 42, 45-48, 59, 66, 71). Furthermore, dysfunctional KV channels have been described in PASMC from patients with primary pulmonary hypertension (69) and in renal arterial smooth muscle cells from genetically hypertensive rats (32). Thus a defect in KV channels may also play an important role in the pathogenesis of primary pulmonary hypertension (69) and genetic systemic hypertension (32).

Due to the large conductance, activation of KCa channels by Ca2+ sparks (originated by Ca2+ release from the ryanodine-sensitive sarcoplasmic reticulum) has been demonstrated to cause relaxation of arterial smooth muscle (38). Because the voltage- and Ca2+-dependent gating for KCa channels are synergistic, a small shift of Em in the direction of depolarization dramatically increases Ca2+ sensitivity of the channel; conversely, a small increase in [Ca2+]cyt markedly increases the sensitivity of the channel to the Em (10). This property of KCa channels explains why KCa channels play a critical role in regulating vasomotion when [Ca2+]cyt is increased and cells are depolarized.

Summary and conclusion. The KV-channel alpha -subunit genes of the Shaker-related subfamily (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), the KV-channel alpha -subunit gene of the Shab subfamily (KV2.1), the KV-channel modulatory alpha -subunit gene (KV9.3), and the KCa-channel alpha -subunit gene (rSlo) are expressed in PASMC. In addition, the KV-channel beta -subunits KVbeta 1.1, KVbeta 2, and KVbeta 3 are also expressed in PASMC. The 4-AP-sensitive non- or slowly inactivating delayed rectifier IK(V), apparently attributed to all of the KV1.1, KV1.2, KV1.5, KV1.6, KV2.1, and KV2.1/KV9.3 gene products, is a critical determinant of resting Em and [Ca2+]cyt in PASMC. The 4-AP-sensitive A-type IK(V), probably conferred by the KV1.4 channel and/or the delayed rectifier KV channels (KV1.1, KV1.2, KV1.5, and KV1.6) associated with the KV-channel beta -subunits, plays an important role in limiting depolarization and controlling duration of the action potentials in PASMC. The ChTX-sensitive, Ca2+-activated IK(Ca), likely endowed by the rSlo gene product, plays a central role in triggering and maintaining repolarization of the Em when the myocytes are stimulated and the [Ca2+]cyt is increased. Association of the cytoplasmic beta -subunits with the pore-forming alpha -subunits of the KV and KCa channels and heteromultimerization between different K+-channel alpha -subunits significantly contribute to the diversity of K+ channels in vivo. Sensitivity of the native KV and KCa channels to hypoxia, redox status change, and metabolic inhibition may be conferred by KV-channel beta -subunits and/or other modulatory alpha -subunits.

    ACKNOWLEDGEMENTS

We gratefully acknowledge A. M. Aldinger and J. E. Seiden for technical assistance; Dr. M. L. Tod for critical review of the manuscript; and Drs. X. Liu, D.-X. Zhou, S. Reinhardt, Q. Zhu, E. Limen, and F. Xia for generous advice on the molecular biological experiments.

    FOOTNOTES

This project was supported by National Heart, Lung, and Blood Institute Grants HL-54043, HL-02659, and HL-32276 and grants from the American Heart Association-Maryland Affiliate, the Primary Pulmonary Hypertension (PPH) Cure Foundation, and the PPH Research Foundation.

X.-J. Yuan is an Established Investigator of the American Heart Association, a Parker B. Francis Fellow in Pulmonary Research, and a recipient of the Giles F. Filley Memorial Award and the Research Career Enhancement Award from the American Physiological Society.

Address for reprint requests: X.-J. Yuan, Division of Pulmonary and Critical Care Medicine, Univ. of Maryland School of Medicine, 10 S. Pine St., Suite 800, Baltimore, MD 21201.

Received 14 May 1997; accepted in final form 7 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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S. Krick, O. Platoshyn, M. Sweeney, S. S. McDaniel, S. Zhang, L. J. Rubin, and J. X.-J. Yuan
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J. X.-J. Yuan
Oxygen-sensitive K+ channel(s): where and what?
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E. A. Coppock and M. M. Tamkun
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L. Perchenet, L. Hilfiger, J. Mizrahi, and O. Clement-Chomienne
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M. Iftinca, G. J. Waldron, C. R. Triggle, and W. C. Cole
State-Dependent Block of Rabbit Vascular Smooth Muscle Delayed Rectifier and Kv1.5 Channels by Inhibitors of Cytochrome P450-Dependent Enzymes
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E. A. Coppock, J. R. Martens, and M. M. Tamkun
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D. Ekhterae, O. Platoshyn, S. Krick, Y. Yu, S. S. McDaniel, and J. X.-J. Yuan
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H. L. Reeve, E. Michelakis, D. P. Nelson, E. K. Weir, and S. L. Archer
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O. Platoshyn, Y. Yu, V. A. Golovina, S. S. McDaniel, S. Krick, L. Li, J.-Y. Wang, Lewis. J. Rubin, and J. X.-J. Yuan
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C. G. Sobey and F. M. Faraci
Knockout Blow for Channel Identity Crisis : Vasodilation to Potassium Is Mediated via Kir2.1
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J.-Y. Wang, J. Wang, V. A. Golovina, L. Li, O. Platoshyn, and J. X.-J. Yuan
Role of K+ channel expression in polyamine-dependent intestinal epithelial cell migration
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C. Xu, Y. Lu, G. Tang, and R. Wang
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L. A. Shimoda, J. T. Sylvester, and J. S. K. Sham
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V. A. Golovina
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J. V. Steidl and A. J. Yool
Differential Sensitivity of Voltage-Gated Potassium Channels Kv1.5 and Kv1.2 to Acidic pH and Molecular Identification of pH Sensor
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P. M. Kerr, O. Clement-Chomienne, K. S. Thorneloe, T. T. Chen, K. Ishii, D. P. Sontag, M. P. Walsh, and W. C. Cole
Heteromultimeric Kv1.2-Kv1.5 Channels Underlie 4-Aminopyridine-Sensitive Delayed Rectifier K+ Current of Rabbit Vascular Myocytes
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K. S. Thorneloe, T. T. Chen, P. M. Kerr, E. F. Grier, B. Horowitz, W. C. Cole, and M. P. Walsh
Molecular Composition of 4-Aminopyridine-Sensitive Voltage-Gated K+ Channels of Vascular Smooth Muscle
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