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Am J Physiol Lung Cell Mol Physiol 287: L226-L238, 2004. First published March 26, 2004; doi:10.1152/ajplung.00438.2003
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Diversity of voltage-dependent K+ channels in human pulmonary artery smooth muscle cells

Oleksandr Platoshyn,* Carmelle V. Remillard,* Ivana Fantozzi, Mehran Mandegar, Tiffany T. Sison, Shen Zhang, Elyssa Burg, and Jason X.-J. Yuan

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Diego, California 92103

Submitted 9 December 2003 ; accepted in final form 19 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Electrical excitability, which plays an important role in excitation-contraction coupling in the pulmonary vasculature, is regulated by transmembrane ion flux in pulmonary artery smooth muscle cells (PASMC). This study examined the heterogeneous nature of native voltage-dependent K+ channels in human PASMC. Both voltage-gated K+ (KV) currents and Ca2+-activated K+ (KCa) currents were observed and characterized. In cell-attached patches of PASMC bathed in Ca2+-containing solutions, depolarization elicited a wide range of K+ unitary conductances (6–290 pS). When cells were dialyzed with Ca2+-free and K+-containing solutions, depolarization elicited four components of KV currents in PASMC based on the kinetics of current activation and inactivation. Using RT-PCR, we detected transcripts of 1) 22 KV channel {alpha}-subunits (KV1.1–1.7, KV1.10, KV2.1, KV3.1, KV3.3–3.4, KV4.1–4.2, KV5.1, KV 6.1–6.3, KV9.1, KV9.3, KV10.1, and KV11.1), 2) three KV channel {beta}-subunits (KV{beta}1–3), 3) four KCa channel {alpha}-subunits (Slo-{alpha}1 and SK2–SK4), and 4) four KCa channel {beta}-subunits (KCa{beta}1–4). Our results show that human PASMC exhibit a variety of voltage-dependent K+ currents with variable kinetics and conductances, which may result from various unique combinations of {alpha}- and {beta}-subunits forming the native channels. Functional expression of these channels plays a critical role in the regulation of membrane potential, cytoplasmic Ca2+, and pulmonary vasomotor tone.

membrane potential; calcium; proliferation; heterogeneity; calcium-activated potassium channel

ELECTRIC EXCITABILITY, which plays an important role in excitation-contraction (EC) coupling in the pulmonary vasculature (12), is mainly controlled by transmembrane ion flux in vascular smooth muscle cells. The expression and functionality of plasma membrane ion channels not only regulate smooth muscle excitability and contractility but also modulate cell secretion, differentiation, motility, migration, apoptosis, and proliferation by governing cytoplasmic free Ca2+ concentration ([Ca2+]cyt) (8, 12, 39, 75). Indeed, many vasoactive substances alter membrane potential (Em) by affecting ion channel activity in vascular smooth muscle cells (5).

In pulmonary artery smooth muscle cells (PASMC), a rise in [Ca2+]cyt triggers pulmonary vasoconstriction and stimulates cell proliferation (51) and migration (46), leading to pulmonary vascular remodeling. The mechanisms involved in the regulation of [Ca2+]cyt in PASMC directly control pulmonary vasomotor tone and vascular wall thickness, two major determinants of pulmonary vascular resistance (PVR), itself an indicator of pulmonary arterial pressure (PAP) based on Poiseuille's law. In patients with pulmonary hypertension, PVR and, thus, PAP will be enhanced due to medial hypertrophy. Ion channel dysfunction has been implicated in a variety of cardiopulmonary diseases, including primary pulmonary hypertension (PPH) (79, 83) and spontaneous genetic hypertension (19, 32).

EC coupling in the pulmonary vasculature requires a change in Em in PASMC. In excitable cells, resting Em is predominantly regulated by the permeability and the concentration gradient of K+ across the plasma membrane, although other cations (Na+ and Ca2+) and anions (Cl–) may also be involved. The transmembrane flux of K+ occurs primarily via sarcolemmal K+ channels. Although at least five K+ channel subtypes have been identified in various vascular smooth muscle cells (31, 40), voltage-gated (KV) and Ca2+-activated potassium (KCa) channels appear to be mainly responsible for controlling Em in human PASMC. Deregulation of K+ flux across the membrane, due either to altered channel activity or expression, may therefore have a serious impact on function and structure of the human pulmonary vasculature. The matter is further complicated when one considers that multiple subunits with unique biophysical characteristics (15) make up the native K+ channels, four {alpha}- and four {beta}-subunits in the case of KV channels (17, 84). Characterizing native K+ currents thus becomes an arduous task indeed.

This study focuses on the properties of the KV and KCa channels responsible for controlling Em in human PASMC. More specifically, we examine 1) the diverse electrophysiological properties of native voltage-dependent K+ channels and 2) the mRNA expression of various cloned {alpha}- and {beta}-subunits. This study provides an important basis for beginning to identify the molecular components of native K+ channels in human PASMC, which is an initial but necessary step not only in understanding normal EC coupling mechanisms, but also in defining the pathogenic roles of K+ channels in pulmonary vascular disease and in developing new therapeutic approaches for patients with pulmonary arterial hypertension.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell preparation and culture. Primary cultured PASMC from transplant patients who do not have pulmonary hypertension were used in this study (79). Muscular pulmonary arteries isolated from excised tissues were incubated for 20 min in Hanks' balanced salt solution containing 2 mg/ml collagenase. The adventitia was stripped, and endothelium was removed. The remaining smooth muscle was digested with 2.25 mg/ml collagenase, 0.5 mg/ml elastase, and 1 mg/ml albumin at 37°C. Single PASMC in suspension were plated onto coverslips and incubated in a humidified atmosphere of 5% CO2 in air at 37°C in smooth muscle growth medium (SMGM, Cambrex) 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. In some experiments, human PASMC purchased from Cambrex also were seeded and cultured in SMGM. The medium was changed initially after 24 h and then every 48 h thereafter until confluence. Cells were subcultured or plated onto coverslips with trypsin-EDTA buffer when 70–90% confluence was achieved; cells at passages 5–7 were used for experiments.

The purity of PASMC in primary cultures was confirmed by the specific monoclonal antibody raised against smooth muscle {alpha}-actin (Boehringer Mannheim). Briefly, the cells were fixed in 95% ethanol and stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (DAPI, 5 µM); the blue fluorescence emitted at 461 nm was used first to visualize the cell nuclei and to estimate total cell numbers in the cultures. An {alpha}-actin antibody was used to evaluate cellular purity of the DAPI-stained cells in cultures, and a secondary antibody conjugated with indocarbocyanine (Jackson ImmunoResearch) was used to display fluorescent images. The cells were mounted in a solution containing 10% 1 M Tris·HCl, 90% glycerol (pH 8.5), and 1 mg/ml p-phenylenediamine. The cell images were processed by the MetaMorph Imaging System (Universal Imaging). All the DAPI-stained cells also cross-reacted with the smooth muscle cell {alpha}-actin antibody, indicating that the primary cultures comprise only smooth muscle cells.

Electrophysiological measurements. All experiments were performed at room temperature (22–24°C). Currents were recorded from human PASMC with an Axopatch 1D amplifier and a DigiData 1200 interface (Axon Instruments) by conventional whole cell and cell-attached voltage clamp techniques. Cells were plated on glass coverslips, mounted on a Plexiglas bath on a Nikon inverted microscope, and bathed in physiological saline solution (Table 1). Borosilicate patch pipettes (2–4 M{Omega}) were fabricated on a model P-97 electrode puller (Sutter Instruments) and polished with a MF-83 microforge (Narashige Scientific Instruments Laboratories). Step-pulse protocols and data acquisition were performed with pCLAMP software (Axon Instruments). Currents were filtered at 1–2 kHz (–3 dB) and digitized at 2–4 kHz.


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  		Table 1. Ionic composition of extracellular and intracellular solutions used for measurement of various ion channel currents

 
We distinguished whole cell currents mainly by changing ionic compositions of the bath and pipette solutions (Table 1). To record whole cell KV currents [IK(V)], Ca2+ was omitted from the K+-containing pipette and bath solutions, and residual intracellular Ca2+ was buffered by adding 10 mM EGTA in the pipette to eliminate KCa currents [IK(Ca)]. Extracellular Ca2+ was chelated by including 1 mM EGTA in the bath solution. ATP (5 mM) was added to all pipette solutions to minimize ATP-sensitive K+ currents. Series resistance compensation was performed in all whole cell experiments. Leak and capacitative currents were subtracted by the P/4 protocol in pCLAMP software. Voltage protocols (as described in the text) used to distinguish current components were designed to measure current amplitude, activation, inactivation, and deactivation. Em was measured in single PASMC in current-clamp (I = 0) mode.

For single channel KCa [iK(Ca)] and KV current [iK(V)] recordings in the cell-attached configuration, we held the membrane patches at a potential of –70 mV before applying pulse protocols. Individual recordings lasted a minimum of 30 s at each potential. Channel open probability (Popen), amplitude, and open and closed durations were measured with Fetchan and PStat analysis programs (Axon Instruments). The pipette solution used to measure iK(V) contained 10 mM EGTA to chelate Ca2+ and to prevent Ca2+ influx-mediated activation of KCa channels. High-K+ pipette solutions were used in recording both iK(V) and iK(Ca) so that the K+ equilibrium potential (EK) would be near 0 mV (pipette [K+] {approx} intracellular [K+]). Results are presented as a function of the command potential (Ecomm), which is the inverse of the applied potential. Care should be taken in interpreting Ecomm. Because of a negative resting Em, the actual transmembrane potential across the patched area is equal to the difference between the Ecomm and resting Em (which is approximately –40 mV in cultured human PASMC). This rightward shift explains why the single channel current-voltage curves do not reverse at 0 mV (EK) in our experiments.

Measurement of [Ca2+]cyt. The cells were loaded with the membrane-permeable acetoxymethyl ester form of fura 2 (fura 2-AM, 3 µM) for 30 min at room temperature (24°C) under an atmosphere of 5% CO2 in air. The fura 2-AM-loaded cells were then superfused with standard bath solution for 20 min at 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, 340- and 380-nm excitation) from the cells and background fluorescence were collected at 32°C. The fluorescence signals emitted from the cells were monitored continuously with an Intracellular Imaging fluorescence microscopy system and recorded on an IBM-compatible computer for later analysis. [Ca2+]cyt was calculated from fura 2 fluorescence emission excited at 340 and 380 nm by the ratio method based on the following equation: [Ca2+]cyt = Kd x (Sf2/Sb2) x (R – Rmin)/(Rmax – R), where Kd (225 nM) is the dissociation constant for Ca2+, Sf2 and Sb2 are emission fluorescence values at 380-nm excitation in the presence of EGTA and Triton X-100, respectively, R is the measured fluorescence ratio, and Rmin and Rmax are minimal and maximal ratios, respectively (23). In most experiments, multiple cells were imaged in single field, and one arbitrarily chosen peripheral cytosolic area from each cell was spatially averaged.

RNA extraction and RT-PCR. Total RNA was isolated from human PASMC with the RNeasy Mini Kit (Qiagen). Human brain total RNA was purchased from GIBCO-BRL. Genomic DNA was removed with RNase-free DNase as per the manufacturer's instructions. cDNA was synthesized using SuperScript RT (Invitrogen). Briefly, RNA (2 µg) was incubated with 1 µl of oligo(dT) (0.5 µg/µl) at 70°C for 10 min. Eight microliters of a solution containing 10x buffer, 10 mM dNTP, 20 mM MgCl2, 0.1 M dithiothreitol, 40 U/µl RNaseOUT, and 50 U/µl SuperScript II RT were added to the samples and incubated for 10 min at 30°C, 60 min at 42°C, and 5 min at 95°C. RNase-H (1 µl at 2 U/µl, GIBCO) was added to each reaction, and the samples were incubated for 20 min at 37°C.

Sense and antisense primers were specifically designed from the coding regions of various channel genes as described in Table 2. The fidelity and specificity of the sense and antisense oligonucleotides were examined with a basic local alignment search tool program (BLAST). PCR was performed by a GeneAmp PCR System (Perkin Elmer) using a Platinum PCR Supermix (GIBCO). The first-strand cDNA reaction mixture (1 µl) was used in a 50-µl PCR reaction consisting of 1 µl of each primer (10 µM), 50 mM KCl, 2 mM MgCl2, 10 mM Tris·HCl (pH 8.3), 200 µM of each dNTP, and 2 units of Taq DNA polymerase. cDNA samples were amplified in a DNA thermal cycler under the following conditions: annealing at 55°C (30 s), extension at 72°C (10 min), and denaturation at 94°C (30 s) for 32 cycles. This was followed by a final extension at 72°C (10 min) to ensure complete product extension. Amplified products were separated on 1.5% agarose gels and visualized by ethidium bromide staining. Sense (5'-GAGCCAAAAGGGTCATCATCTC-3') and antisense (5'-AGGGTCTCTCTCTTCCTCTT-3', 719 bp) primers specific for GAPDH were used as an internal control to verify the integrity of total RNA and to quantify the PCR products.


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

 
Solutions and reagents. 4-Aminopyridine (4-AP) was directly dissolved in the bath solution on the day of use; the pH value was readjusted to 7.4 with KOH. Carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP) and cyclopiazonic acid (CPA) were dissolved in DMSO to make 10 and 20 mM stock solutions, respectively. Aliquots of the FCCP and CPA stocks were then diluted to make the final concentrations of 10 and 20 µM, respectively. All chemicals were obtained from Sigma Chemical unless otherwise noted.

Statistics. Summarized data are plotted as means ± SE unless otherwise stated. Statistical significance was determined using Student’s t-test and ANOVA analysis. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Passive cell membrane properties of human PASMC. Membrane capacitance (Cm) in human PASMC ranges from 15 to 45 pF, with a mean value of 34 ± 5 pF (n = 220) (Fig. 1A, left). Specific Cm, calculated from the mean values of Cm and cell surface (capacitative) area, was 1.25 µF/cm2 in these cells, similar to the 1.3 µF/cm2 reported in rat caudal artery myocytes (66). The mean calculated input resistance from the membrane (Rm) in human PASMC was 5 ± 1 G{Omega} (n = 171) (Fig. 1B, left). Neither Cm nor Rm was significantly altered by time in culture (Fig. 1, A and B, right). Resting Em in cultured human PASMC was –45 ± 5 mV (Fig. 1C), slightly less negative than freshly dissociated PASMC from animals (53). In some PASMC, spontaneous electrical activity was observed under resting conditions (Fig. 1D), suggesting that these cells are electrically excitable (63). Removal of extracellular Ca2+ abolished the electrical transients (Fig. 1D), indicating that PASMC can generate spontaneous action potentials that are dependent on extracellular Ca2+ (81).



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  		Fig. 1. Properties of human pulmonary artery smooth muscle cells (PASMC). A: membrane capacitance (Cm) is plotted as a function of cell number (n = 220) or of number of days (1–7) in culture (means ± SE). B: membrane resistance (Rm) is plotted as a function of cell number (n = 171) or of number of days (1–7) in culture (means ± SE). C: histogram showing the wide distribution of resting membrane potential (Em) in human PASMC. Em was measured in the current-clamp (I = 0) mode. D: representative record showing spontaneous action potentials in human PASMC before, during, and after superfusion with a Ca2+-free solution.

 
Classification of KV currents based on unitary conductance. Functionally, both KV and KCa channels are sensitive to voltage changes. A fundamental difference between KV and KCa channels is their response to Ca2+. Although they can be dissociated by their different sensitivity to cytoplasmic Ca2+ (53), unitary KV [iK(V)] and KCa [iK(Ca)] currents in vascular smooth muscle cells are most easily distinguished by their unitary conductance.

Figure 2 shows some sample cell-attached recordings from human PASMC where multiple channel subtype openings can be recorded from the same patch using identical Ca2+-containing perfusion solutions. As shown in Fig. 2A, large-amplitude K+ currents (Fig. 2Aa) and several small-amplitude currents (Fig. 2A, b–f) were recorded in cell-attached membrane patches. In addition to the various amplitudes of the recorded K+ currents, the duration of the channel openings varies in human PASMC. Examples of long-lasting channel and "flickery" openings iK(V) and iK(Ca) recorded from different patches are shown in Fig. 2, B and C.



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  		Fig. 2. Single channel K+ currents recorded in cell-attached membrane patches of human PASMC. A: recordings from different membrane patches showing the variability of current amplitudes (a–f) within the same patches at +70 mV. The dotted lines denote the current level when channels are closed. B: both sustained (a) and flickery (b) unitary voltage-gated potassium (KV) currents [iK(V)] were recorded at +70 mV in 1 human PASMC patch. C: another membrane patch showing flickery (a) and sustained (b) unitary calcium-activated potassium (KCa) currents [iK(Ca)] recorded at +70 mV. Channel openings indicated by boxes are shown on an expanded time scale to indicate the kinetic difference between flickery and sustained openings.

 
We determined the slope conductance of these multiple amplitudes of unitary outward K+ currents that were elicited by steadily holding the membrane patch at different potentials. The histogram of the single channel conductance indicates a wide range of conductances (from 5 to 300 pS) recorded from the cell-attached membrane patches of human PASMC (Fig. 3). In 54% of the cells, we recorded K+ currents with conductances ranging from 5 to 85 pS, and 26% of the cells exhibited large-conductance (>200 pS) K+ current activity. These results indicate that human PASMC express multiple functional K+ channels; the channels with conductances from 5 to 85 may represent the family of small-conductance KV channels that are composed by multiple heterotetrameric {alpha}- and {beta}-subunits (66) as well as the small-conductance KCa (SKCa) channels, whereas the channels with conductances >200 pS are obviously the large-conductance KCa (Maxi-K or BK) channels (66). Twenty percent of the cells show single channel K+ currents with conductances ranging from 120 to 180 pS (symmetric [K+] inside and outside of the membrane patch), which may include the large-conductance KV channels (66) and intermediate-conductance KCa (IKCa) channels (66). Although any subclassification would be subjective and arbitrary, there is ample evidence in the literature showing that different channels' conductance falls within these subfamilies in both vascular (38, 40, 41, 48, 53) and nonvascular (28, 62, 70) smooth muscles.



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  		Fig. 3. Histogram depicting the range and distribution of single channel conductances in different human PASMC. The slope conductance was calculated from the current-voltage (I-V) relationship curves of single channel currents recorded in cell-attached membrane patches with symmetric [K+]. The data are obtained from 89 PASMC examined.

 
In addition to its regulation of current amplitude, Em also affected the channels' Popen. For a 189-pS channel, Popen increased with membrane depolarization from 0.0005 at 0 mV to 0.015 at +50 mV and 0.27 at +90 mV (Fig. 4A). Similarly, Popen for a 33-pS channel increased from 0.04 at +60 mV to 0.43 at +90 mV, from 0.0003 at +50 mV to 0.010 at +90 mV for a 141-pS channel, from 0.0009 at +50 mV to 0.013 at +90 mV for a 81-pS channel, and from 0.007 at +40 mV to 0.02 at +90 mV for a 6-pS channel (Fig. 4B). These examples are representative of the recordings. Therefore, both the single channel amplitude and Popen of KCa and KV channels are voltage dependent and influenced by Em changes in human PASMC.



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  		Fig. 4. Open probability (Popen) is enhanced by membrane depolarization. Two human PASMC membrane patches (A and B) showing multiple amplitude and conductance channel openings at +50, +70, and +90 mV. A: sample traces at each potential. Boxed areas are magnified in B. C: Popen is plotted as a function of the transmembrane potential across the patched area (Epatch) for the respective conductances.

 
Modulation of iK(Ca) by increased [Ca2+]cyt. KCa channel activity is also dependent on a rise in [Ca2+]cyt. Therefore, we further identified iK(Ca) in cell-attached PASMC patches based on their sensitivity to pharmacological agents known to activate KCa channels and to increase [Ca2+]cyt.

FCCP is a protonophore that dissipates the H+ gradient across the inner membrane of mitochondria. As we previously showed in rat and human pulmonary artery myocytes (30), extracellular FCCP (5 µM) caused a significant 11-fold increase of the steady-state open probability (NPopen) of a large-conductance iK(Ca) (220 pS) (Fig. 5A), presumably due to the release of Ca2+ from the mitochondria to the cytoplasm (Fig. 5Ac). Extracellular application of dihydroepiandrosterone, an agent that opens KCa channels via cAMP/cGMP-independent pathways (21), significantly increased NPopen of the large-conductance iK(Ca) (~218 pS) from 0.06 to 0.62 (Fig. 5B). In some PASMC cell-attached patches, increased [Ca2+]cyt induced by CPA (which causes Ca2+ mobilization from intracellular stores) also increased NPopen of a smaller-conductance iK(Ca) (47 pS) at +70 mV (Fig. 5C). These results suggest that at least two types of KCa channels are functionally expressed in human PASMC, and they are synergistically regulated by Em and [Ca2+]cyt.



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  		Fig. 5. Single channel [iK(Ca)] currents in cell-attached membrane patches of human PASMC. A: representative currents (a), elicited by a sustained depolarization at +70 mV, were recorded before (Control), during [p-trifluoromethoxyphenylhydrazone (FCCP)], and after (Washout) extracellular application of 5 µM FCCP. Bar graphs showing the steady-state open probability (NPopen) as a function of time for the sample recordings in a are shown in b. A representative record (c, left) and summarized data (c, right, gray bars; n = 15) showing that FCCP significantly increased cytoplasmic free Ca2+ concentration ([Ca2+]cyt, gray bars) and Popen (for full-length recording, black bars) in PASMC. ***P < 0.001 vs. control (Cont). B: representative currents at +70 mV in a cell were recorded before (Control), during [dihydroepiandrosterone (DHEA)], and after (Washout) extracellular application of 100 µM DHEA. Bar graphs showing the NPopen as a function of time for the sample recordings in a are shown in b. C: representative currents (a) at +70 mV were recorded before (Control), during [cyclopiazonic acid (CPA)], and after (Washout) extracellular application of 5 µM CPA. Bar graphs showing the NPopen as a function of time for the sample recordings in a are shown in b. A representative record (c, left) and summarized data (c, right; n = 21) showing the changes of [Ca2+]cyt (gray bars) and Popen (for full-length recording, black bars) in PASMC before, during, and after treatment with CPA. ***P < 0.001 vs. control.

 
Classification of IK(V) based on their activation and inactivation kinetics. To record optimal IK(V), the cells were superfused with Ca2+-free bath solution (plus 1 mM EGTA) and dialyzed with Ca2+-free pipette solution (plus 10 mM EGTA). Depolarizing the cells from a holding potential of –70 mV to a series of test potentials ranging from –60 to +80 mV elicited outward K+ currents with a threshold potential of activation at approximately –45 mV. Four families of IK(V) could be distinguished based on their activation and inactivation kinetics: rapidly activating and slowly inactivating IK(V) (Fig. 6A), rapidly activating and noninactivating IK(V) (Fig. 6B), slowly activating and noninactivating IK(V) (Fig. 6C), and rapidly activating and rapidly inactivating IK(V) (Fig. 6D). Activation time constants ({tau}act) could be separated into two categories corresponding to the rapidly and slowly activating currents (<=5 and >5 ms, respectively, mean = 2.67 ± 0.09 ms) (Fig. 6Ea). Inactivation constants ({tau}inact) were much more variable (Fig. 6Eb) with the midpoint between rapid and slow inactivation being ~100 ms (mean = 122.7 ± 5.25 ms). The {tau}act did not appear to be correlated with the {tau}inact (Fig. 6Ec; correlation coefficient, r2 = 0.00371).



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  		Fig. 6. Whole cell (or macroscopic) KV currents [IK(V)] in human PASMC. A–D: 4 different types of KV currents were elicited by step depolarizations from a holding potential of –70 mV to test potentials between –80 and +80 mV in 20-mV increments using the solutions described in Table 1. Representative families of currents (left), enlarged trace segments showing steady-state activation (middle, top) and inactivation (middle, bottom), and I-V curves are presented for each type of current. For steady-state activation and inactivation panels, current amplitude (y-axis) is plotted as a function of time (x-axis). E: histograms of activation (a, {tau}act) and inactivation time constants (b, {tau}inact) of IK(V) in human PASMC. Correlation of {tau}inact and {tau}act is plotted in c. The correlation coefficient (r2) was calculated by a linear regression fit to the data.

 
Regardless of their activation and inactivation kinetics, extracellular application of 5 mM 4-AP reversibly decreased all the four families of whole cell currents (Fig. 7), suggesting that 4-AP-sensitive KV channels are responsible for generating the currents. Although the slow inactivation kinetics of three of the kinetically different currents are typical of most native delayed-rectifier K+ currents recorded in vascular smooth muscle cells (40, 81), 4-AP-sensitive rapidly activating and rapidly inactivating currents (Fig. 7D) may represent a different class of K+ current less commonly observed in systemic vascular smooth muscle cells. On the basis of its rapid inactivation (93.23 ms) kinetics, this component closely resembles the transient A-type current that has been observed in phasic smooth muscle cells (7, 71) and PASMC (47, 48).



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  		Fig. 7. Inhibitory effect of 4-aminopyridine (4-AP) on IK(V) in human PASMC. Rapidly activating and slowly inactivating (A), rapidly activating and noninactivating (B), slowly activating and noninactivating (C), and rapidly activating and rapidly inactivating (D) Kv currents were elicited by step depolarizations from a holding potential to test potentials between –80 and +80 mV. Representative currents are shown before (Cont), during (4-AP), and after (Wash) extracellular application of 5 mM 4-AP. I-V curves of the 4-AP-sensitive currents are presented for each current type. The 4-AP-sensitive current components depicted to the right were obtained by subtracting the currents recorded during 4-AP application from the currents recorded under control conditions.

 
KV channel genes that are expressed in human PASMC. Native KV channels are believed to be heteromeric tetramers composed of the pore-forming {alpha}-subunits and regulatory cytoplasmic {beta}-subunits ({alpha}4/{beta}4) (Fig. 8A). We detected transcripts of KV channel genes by RT-PCR on human PASMC mRNA using primers targeted against each KV channel subunit (Table 2). All of the KV channel {alpha}- and {beta}-subunits tested appear to be strongly expressed in brain cells, verifying that brain tissue results serve as proper controls for comparison.



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  		Fig. 8. Molecular biology of KV channels in human PASMC. A: structural arrangement of KV channel {alpha}- and {beta}-subunits (a), the tetrameric association of {alpha}-subunits (b, top), and the proposed ball-and-chain inactivation mechanism for IK(V) (b, bottom). B: mRNA expression of KV1 (a), KV{beta} (b), KV2 (c), KV3 (d), KV4 (e), KV5 (f), KV6 (g), KV9 (h), and KV11 (i) channel subunits in human PASMC (P) and brain tissues (B) was verified using the primers listed in Table 2. RT-PCR amplified products for GAPDH (j) in human PASMC are shown as a control. Data are representative for 3–6 PASMC samples tested. M, 100-bp DNA ladder.

 
In human PASMC, we identified 22 KV {alpha}-subunits and three KV {beta}-subunits (representative gels shown in Fig. 8B). Eight Shaker-type KV channel {alpha}-subunits (KV1.1–1.7, and 1.10) were identified in PASMC. The bands representing KV1.1–1.6 and KV1.10 are less intense in PASMC compared with brain tissues; the band representing KV1.7 in PASMC seems to be of equal intensity to those in brain samples (Fig. 8Ba). Bands associated with KV{beta}1.1, KV{beta}2.1, and KV{beta}3 are slightly more intense in brain than in PASMC (Fig. 8Bb). The bands associated with Shab-type KV2.1 channels are considerably more intense in brain than in PASMC (Fig. 8Bc); KV2.2 appears to be absent in PASMC. Shaw-type KV3.4 shows a band of similar intensity in both PASMC and brain cells, whereas KV3.1 and 3.3 have a more intense band in brain tissues (Fig. 8Bd). Shal-type KV4.1 shows bands of equal intensity in both PASMC and brain, whereas the KV4.3 band is very faint in PASMC compared with brain tissues (Fig. 8Be).

Electrically silent modulatory {alpha}-subunits were also identified in PASMC. The bands associated with KV5.1 (Fig. 8Bf) and KV6.1 and Kv6.3 (KV10.1) (Fig. 8Bg) are also less dense in PASMC compared with brain tissues, whereas KV6.2 shows bands of slightly higher intensity in PASMC (Fig. 8Bg). KV9.1 and 9.3 (Fig. 8Bh) have bands of lower intensity in PASMC than in brain tissue. Bands for KV11.1 were moderately more intense in brain vs. PASMC (Fig. 8Bi). Overall these results illustrate the possibility that most of the known KV channels {alpha}-subunits, with the exception of KV2.2 and KV4.3, are expressed to some extent in human PASMC. Because RT-PCR only gives us the mRNA expression of the subunit genes, the next step should be to confirm the expression of the proteins generated by these genes. However, we did not proceed with Western blot analysis or immunocytochemistry experiments to confirm the protein expression of KV channel subunits, mainly because the commercially available antibodies are not reliable in terms of their specificity for the given subunits.

Molecular identities of KCa channels in human PASMC. In addition to the pore-forming {alpha}-subunit for KCa channels, several {beta}-subunits have been identified in human vascular smooth muscle cells. The primers we used were aimed at identifying the {alpha}-subunits for both Maxi-KCa and SKCa channels as well as the regulatory {beta}-subunits. Maxi-KCa {alpha}1 (hSlo-{alpha}1) was highly expressed in human PASMC, significantly more than in brain tissues (Fig. 9A). Four {beta}-subunits (Maxi-KCa{beta}1–4) were also detected, although {beta}2 was very faint in PASMC (Fig. 9A). In contrast, {beta}4 mRNA was not detected in human brain tissue. Three (SK2–4) pore-forming subunits were identified corresponding to IKCa and SKCa channels (Fig. 9B). SK4 was more highly expressed in PASMC than in brain, whereas the opposite can be said for SK2 and SK3. SK1 mRNA was detected in brain but not in PASMC. For a similar reason as that listed above for KV channel subunits, we did not determine the protein expression of KCa {alpha}- and {beta}-subunits.



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  		Fig. 9. Molecular biology of KCa channels. A, top: structural arrangement of {alpha}- and {beta}-subunits for the large-conductance (Maxi-K) KCa channels (16). The putative binding site for Ca2+ is shown on the COOH-terminal region of the {alpha}-subunit. Bottom: mRNA expression of Maxi-K channel {alpha}1- and {beta}1–4-subunits in human PASMC (hPASMC) and brain tissues (hBrain) were confirmed by RT-PCR analysis. B, top: structural arrangement of the small-/intermediate-conductance KCa channels (SK-Ca, IK-Ca). Bottom: mRNA expression of SK/IK channels in human PASMC and brain tissues (hBrain) were confirmed by RT-PCR analysis. RT-PCR amplified products of GAPDH are shown as controls.

 

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The expression and proper functioning of K+ channels in PASMC are essential to the regulation of vascular tone and cell viability under pathological conditions. Dysfunction and downregulation of K+ channels have been demonstrated to play an important role in the development of pulmonary vasoconstriction and vascular remodeling in patients and animals with pulmonary hypertension (64, 79, 83). At least two types of K+ channels, KV and KCa, have been involved in the regulation of pulmonary vascular tone and cell proliferation/apoptosis in various animal models (16, 21, 25, 30, 47, 51, 61). However, knowledge of the molecular basis and biophysical properties of these K+ channels in human pulmonary artery myocytes is limited. Therefore, our goal was to describe the mRNA expression of various pore-forming and regulatory subunits of voltage-gated KV and KCa channels, as well as describe the basic electrophysiological properties of the native KV and KCa currents in human PASMC. The single channel and whole cell data identified multiple K+ currents belonging to different families based on their unitary conductances and on their activation and inactivation parameters. RT-PCR analysis confirmed the mRNA expression of 22 KV channel {alpha}- and {beta}-subunits, as well as five KCa channel {alpha}- and {beta}-subunits (Table 3). Functional expression of the multiple K+ channel subunits and the formation of heterotetrameric channels in vivo may account for the diversity and complexity of native K+ current amplitudes and kinetics in human PASMC.


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  		Table 3. Biophysical properties and molecular identities of voltage-dependent K+ channels expressed in human PASMC

 
Expression and electrophysiological properties of KV channels in human PASMC. The family of KV channels can be grossly divided into two categories based on the kinetics of current activation and inactivation. Delayed-rectifier KV channels generate slowly activating and non- or slowly inactivating currents. Rapidly activating and rapidly inactivating currents originate from the activation of transient A-type currents similar to those observed in phasic smooth muscle, cardiomyocytes, and neurons (7). In human PASMC, we identified four types of 4-AP-sensitive currents generated by KV channels (Figs. 6, 7) with properties similar to those of both delayed-rectifier and A-type currents. Although the current-voltage relations and pharmacological properties (Fig. 7) of these currents were similar, there were significant differences in the current kinetics, particularly their inactivation time constants (Fig. 6). Heteromeric assembly of K+ channel {alpha}-subunits can account for this diversity of K+ currents within the same cell system. When the electrophysiological properties of PASMC KV currents are compared with those generated by cloned KV channel {alpha}-subunits (15, 16), it is clear that the native channels' electrophysiological and pharmacological properties are intermediaries of those different clones forming the functional channels.

Cytoplasmic KV channel {beta}-subunits associate with the amino-terminal region of KV {alpha}-subunits via their own highly conserved carboxy terminus (67, 77) (Fig. 8A). The association of multiple {beta}-subunits with the functional {alpha}-homo- or heterotetramer may further influence the biophysical properties of native KV currents (17), providing a potential explanation for the diverse nature of the whole cell and unitary KV currents in human PASMC. Whether {beta}-subunit interaction influences KV activity via 1) enhanced interaction of {alpha}-subunits with protein kinases (34), 2) conveyed inactivation onto noninactivating channels (57), or 3) a shifting of the activation curve, slowed deactivation of the current, enhanced slow inactivation, or altered peak current amplitude by acting as an open channel blocker (34, 55) remains unknown in human PASMC and will be investigated further.

The behavior of single channels provides further evidence for the heteromeric assembly of the pore-forming units. Cloned KV channels have a wide range of single channel conductances that do not always match with the conductance of native KV channels (15). For example, the single channel conductances for KV1.1, KV1.2, and KV1.5 channels expressed in heterologous expression systems are reported to be 10, 9–17, and 8 pS, respectively (14, 15, 24). The conductance of native KV channels in vascular smooth muscle cells ranges between 5 and 11 pS (6, 72) at physiological K+ concentrations and between 15 and 70 pS in symmetrical high-K+ conditions (1, 53). Although the differences between native and cloned KV conductances may relate to differences in the expression systems (e.g., pulmonary artery vs. HEK-293 cells), splicing, or postranslational modifications, it is quite likely that native KV channels exist largely as heterotetramers. The wide range of conductances we reported in human PASMC patches may reflect this heterogeneity of subunit association. Given the diversity of roles and properties of KV {alpha}- and {beta}-subunits, it is not surprising that KV channel activity is central to numerous processes, such as hypoxic pulmonary vasoconstriction (43, 53, 61, 82), cell proliferation (51, 78), and myogenic reactivity (29).

Expression and electrophysiological properties of KCa channels in human PASMC. KCa channels are ubiquitously distributed among tissues and play an important role in regulating contractile tone in smooth muscle. In human PASMC, as in other vascular smooth muscle cells, they are easily distinguished from KV channels by their sensitivity to [Ca2+]cyt (Fig. 5) and by their single channel conductance. Unitary conductance also serves to distinguish between the three types of KCa channels (Maxi-K, IKCa, and SKCa). The conductance of a Maxi-KCa (also called BKCa) channel is generally >200 pS in symmetrical [K+]. The single channel openings can be very rapid, exhibiting distinct flickering activity (Fig. 2Ca, open time is 1–3 ms) or be more sustained (Fig. 2Cb, open time is >5 ms). These channels are also quite prominent in vascular smooth muscle cells (40), which explains why more than one channel can often be triggered within a patch at different potentials (Figs. 2A and 5A). The conductances of IKCa and SKCa channels are ~50–70 pS (Fig. 5C) and 10–50 pS, respectively, in similar conditions (69).

Unlike the mainly heterotetrameric KV channels, Maxi-KCa channels are mainly homomeric tetramers composed of the pore-forming {alpha}-subunits and auxiliary {beta}-subunits (67). KCa {alpha}-subunits differ from those of KV channels in that they have an extra S0 segment that interacts with the regulatory {beta}-subunits, and the carboxy-terminal domain contains Ca2+ binding sites (Fig. 9A) (35). Human KCa channel {alpha}-subunits that encode maxi-KCa, IKCa, and SKCa channels have been cloned and characterized in vascular smooth muscle cells (41). However, unlike KV channels, the diversity of KCa {alpha}-subunits is very limited with only two slo and four SK genes currently identified (15). Functional maxi-KCa channels in human PASMC are formed from slo-{alpha}1 subunits (Fig. 9A). This is supported by similarities in voltage dependence, Ca2+ sensitivity, pharmacological properties, and single channel conductance between cloned slo-{alpha}1 genes and native Maxi-KCa channels (15). Another slo gene, slo-{alpha}3, is expressed only in rat testis and does not exhibit Ca2+ sensitivity (15); it was not tested in our assays.

SK genes comprise the SKCa (SK1–3) and IKCa (SK4) KCa channels. Their structure (Fig. 9B) is similar to that of Maxi-KCa channel {alpha}-subunits, except for the omission of the S0 domain, and the long cytoplasmic (as opposed to extracellular) amino terminal. We identified all but SK1 in human PASMC by RT-PCR (Fig. 9B). We have also provided evidence that a rise in [Ca2+]cyt due to CPA-induced Ca2+ mobilization from the sarcoplasmic reticulum activates a low-conductance (49 pS) KCa channel (Fig. 5C). The latter's conductance resembles that of cloned SK4 channels (30–40 pS) (41) more than of cloned SK1–3 (9–10 pS) (15). This discrepancy raises the possibility that SK genes may assemble as heteromers to form functional native channels with intermediary properties (much like KV channels).

The two-transmembrane segment KCa {beta}-subunits, all of which were identified in human PASMC (Fig. 9B), interact with the amino terminus of KCa slo {alpha}-subunits to upregulate KCa channel activity. The multiple {beta}-isoforms modulate KCa channels differently, possibly accounting for the diversity of KCa channels' activity. For example, association of {beta}1- or {beta}2-subunits with human slo channels 1) increases the voltage sensitivity of KCa channels (shift of the half-maximal activation voltage to more negative potentials) (67), 2) enhances the Ca2+ sensitivity of KCa channels (10, 18), 3) promotes KCa channel inactivation in some cases (68), and 4) confers high affinity binding of charybdotoxin to the {alpha}/{beta}-complex (36). Paradoxically, neuronal {beta}4-subunits can render KCa channels resistant to both charybdotoxin and iberiotoxin (36).

Because KCa channel activation serves an important role as a feedback modulator of vascular tone when [Ca2+]cyt becomes elevated (40), Ca2+ spark activity may play an important role in the regulation of vascular tone. A recent study by Brenner et al. (10) showed that {beta}1-subunit deletion in murine cerebral artery smooth muscle cells leads to a decrease in the Ca2+ sensitivity of Maxi-KCa channels and a reduction in the functional coupling of Ca2+ sparks to Maxi-KCa activation, culminating in increased arterial tone and blood pressure. Because Ca2+ sparks have been measured in rat PASMC (56), it is possible that similar mechanisms may play a role in controlling human pulmonary arterial tone. It is therefore important to have extensive knowledge of the properties and molecular identity KCa channels in human PASMC to develop therapies against pulmonary hypertension.

Contribution of cation channels to the regulation of Em and [Ca2+]cyt in human PASMC. Excitable and quiescent cells both possess a relatively negative resting Em that is close to the EK. Em has been demonstrated to control electrical excitability (e.g., generation and propagation of action potentials) (39), muscle contraction (63), apoptosis (22, 76), and gene expression (58, 59). From the latter functions, it is apparent that the mechanisms controlling Em and [Ca2+]cyt are interrelated.

Membrane depolarization elevates [Ca2+]cyt mainly by activating sarcolemmal voltage-dependent Ca2+ channels (VDCC) (39, 81) and the reverse-mode Na+/Ca2+ exchanger (9). Although the activation of Na+ [energy of Na+ activation (ENa) ~+66 mV] and Ca2+ (ECa ~+122 mV) channels tends to depolarize cells and enhance [Ca2+]cyt, K+ channel activation hyperpolarizes the membrane and decreases sarcolemmal Ca2+ influx. Because of their voltage and/or Ca2+ dependence, K+ channels are key elements in the maintenance of Em at near resting levels. Our previous studies demonstrated that inhibition of KV channels with 4-AP induced membrane depolarization and increased [Ca2+]cyt by opening VDCC in PASMC (79, 81). An increase in [Ca2+]cyt is believed to play an important role in stimulating cell growth by activating protein kinases and transcription factors that are essential for the progression of cell cycle (13, 27, 58, 59). The observations from the present study suggest that activity of KV channels in human PASMC also may play an important role in modulating pulmonary vascular contractility and remodeling by regulating Em and [Ca2+]cyt. Indeed, both KCa and KV channels have been identified as feedback modulators of myogenic tone and agonist-induced vascular tone in systemic (29, 39, 40) and pulmonary arteries (4, 25, 48, 60).

K+ channels: roles in pulmonary vasoconstriction and vascular remodeling. Pulmonary vasoconstriction and vascular remodeling (e.g., medial hypertrophy due to smooth muscle cell proliferation and migration) greatly contribute to the elevated PVR in patients with pulmonary hypertension (64). Because both Em and Ca2+ are recognized as important modulators of pulmonary vascular tone and PASMC growth, it is plausible that ion channels also play a role in these processes, particularly those ion channels that regulate and can be regulated by Em and Ca2+. Indeed, dysfunctional and downregulated KV channels in PASMC have been implicated in the development of PPH (79, 83) and hypoxia-mediated pulmonary vasoconstriction (16, 17, 47, 53, 61).

Acute hypoxia inhibits K+ currents in cell lines transiently transfected with KV1.2, KV1.5, Kv1.2/Kv1.5, Kv2.1, Kv2.1/Kv9.3, KV3.1, KV3.3, or KV4.2 (2, 3, 16, 17, 20, 26, 33, 37, 42, 44, 45, 49, 52–54, 65, 73, 74, 80, 83, 84), suggesting that these KV channel {alpha}-subunits are sensitive to changes in O2 tension. Coexpression of KV channel {beta}-subunits, such as those we identified in human PASMC (KV{beta}1.1, KV{beta}2.1), with KV {alpha}-subunits confers the redox and O2 sensitivity to KV channel {alpha}-subunits, indicating that {beta}-subunits may be the target (or act as an O2 sensor) for acute hypoxia to inhibit KV channel activity (2, 3, 16, 17, 20, 26, 33, 37, 42, 44, 45, 49, 50, 52–54, 65, 73, 74, 80, 83, 84). In addition, chronic hypoxia (~2–3 days) can reduce KV channel activity by downregulating mRNA and protein expression of KV channel {alpha}-subunits, such as KV1.1, KV1.2, KV1.5, KV2.1, KV4.3, and KV9.3 in PASMC. The inhibitory effect of chronic hypoxia on KV channel expression is selective to {alpha}-subunits in PASMC because it has little effect on {beta}-subunits and is specific to PASMC because chronic hypoxia negligibly affects {alpha}-subunit expression in systemic (e.g., mesenteric and aortic) arterial smooth muscle cells (2, 3, 16, 17, 20, 26, 33, 37, 42, 44, 45, 49, 52–54, 65, 73, 74, 80, 83, 84).

In PASMC from patients with PPH, membrane depolarization and decreased IK(V) are associated with downregulation of mRNA expression of KV1.2, KV1.4, and KV1.5, the KV channel {alpha}-subunits that participate in forming functional channels in human PASMC (2, 3, 16, 17, 20, 26, 33, 37, 42, 44, 45, 49, 52–54, 65, 73, 74, 80, 83, 84). Inhibition of K+ channel activity and/or downregulation of K+ channel gene expression both contribute to reducing IK(V) and causing membrane depolarization. The resultant activation of VDCC and reverse-mode Na+/Ca2+ exchangers would lead to a rise in [Ca2+]cyt, triggering pulmonary vasoconstriction and stimulating PASMC proliferation (9, 16, 29, 39, 51). In contrast, in vitro overexpression of KV1.5 gene into human PASMC not only induces membrane hyperpolarization but also accelerates the apoptotic volume decrease and enhances apoptosis in PASMC (11). In vivo transfer of the KV1.5 gene to lung tissues in rats indeed inhibits hypoxia-induced pulmonary hypertension (2, 3, 16, 17, 20, 26, 33, 37, 42, 44, 45, 49, 52–54, 65, 73, 74, 80, 83, 84).

Summary. We have identified voltage-dependent K+ channels in human PASMC by electrophysiological and molecular biological techniques. Multiple isoforms of KV and KCa channels' pore-forming {alpha}-subunits, as well as their regulatory {beta}-subunits, are present in human PASMC. The currents generated by these K+ channels are similar to those previously characterized in systemic arterial myocytes and PASMC from other species. Furthermore, the diversity of K+ channel {alpha}-subunit tetramers is evident from the variety of macroscopic and unitary currents we observed. Our observations suggest that the activity of multiple K+ channels is essential to the regulation of Em and [Ca2+]cyt in human PASMC, both of which are important modulators of pulmonary vasoconstriction and vascular remodeling.


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This work was supported by National Heart, Lung, and Blood Institute Grants HL-64945, HL-54043, HL-66012, HL-69758, and HL-66941.


   ACKNOWLEDGMENTS
 
We thank A. Nicholson for technical assistance and N. Ottschytsch for providing primer sequences for the RT-PCR experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. X.-J. Yuan, Div. of Pulmonary and Critical Care Medicine, UCSD Medical Center, 200 W. Arbor Dr., San Diego, CA 92103-8382 (E-mail: xiyuan{at}ucsd.edu).

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

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


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