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Am J Physiol Cell Physiol 293: C928-C937, 2007. First published June 20, 2007; doi:10.1152/ajpcell.00101.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

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

Eun A. Ko, Elyssa D. Burg, Oleksandr Platoshyn, Joseph Msefya, Amy L. Firth, and Jason X.-J. Yuan

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

Submitted 13 March 2007 ; accepted in final form 15 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice are useful animal models to study pathogenic mechanisms involved in pulmonary vascular disease. Altered expression and function of voltage-gated K+ (KV) channels in pulmonary artery smooth muscle cells (PASMCs) have been implicated in the development of pulmonary arterial hypertension. KV currents (IK(V)) in mouse PASMCs have not been comprehensively characterized. The main focus of this study was to determine the biophysical and pharmacological properties of IK(V) in freshly dissociated mouse PASMCs with the patch-clamp technique. Three distinct whole cell IK(V) were identified based on the kinetics of activation and inactivation: rapidly activating and noninactivating currents (in 58% of the cells tested), rapidly activating and slowly inactivating currents (23%), and slowly activating and noninactivating currents (17%). Of the cells that demonstrated the rapidly activating noninactivating current, 69% showed IK(V) inhibition with 4-aminopyridine (4-AP), while 31% were unaffected. Whole cell IK(V) were very sensitive to tetraethylammonium (TEA), as 1 mM TEA decreased the current amplitude by 32% while it took 10 mM 4-AP to decrease IK(V) by a similar amount (37%). Contribution of Ca2+-activated K+ (KCa) channels to whole cell IK(V) was minimal, as neither pharmacological inhibition with charybdotoxin or iberiotoxin nor perfusion with Ca2+-free solution had an effect on the whole cell IK(V). Steady-state activation and inactivation curves revealed a window K+ current between –40 and –10 mV with a peak at –31.5 mV. Single-channel recordings revealed large-, intermediate-, and small-amplitude currents, with an averaged slope conductance of 119.4 ± 2.7, 79.8 ± 2.8, 46.0 ± 2.2, and 23.6 ± 0.6 pS, respectively. These studies provide detailed electrophysiological and pharmacological profiles of the native KV currents in mouse PASMCs.

KV channels

K+ CHANNELS ARE FOUND IN VIRTUALLY all cell types examined, excitable and nonexcitable, including neurons, cardiomyocytes, skeletal and smooth muscle cells, and T lymphocytes (22, 26, 34, 47, 59). Activity of K+ channels in pulmonary artery smooth muscle cells (PASMCs) plays an important role in the regulation of membrane potential (Em) and thus excitation-contraction coupling in the pulmonary vasculature (29, 32). Activity of K+ channels is also involved in modulating proliferation and apoptosis in PASMCs (9, 24, 37). K+ channels can be divided into subfamilies based on their electrophysiological or biophysical properties (e.g., voltage dependence, Ca2+ dependence, or ATP sensitivity) and molecular topology (e.g., 6 transmembrane domain and 1-pore channels or 4 transmembrane domain and 2-pore channels). The voltage-gated K+ (KV) channel family is the most diverse subfamily of K+ channels expressed in various cell types including vascular smooth muscle cells (32). Functional KV channels are hetero- or homotetramers composed of four pore-forming {alpha}-subunits that associate with four cytoplasmic regulatory beta-subunits (11). The different combinations of {alpha}- and beta-subunits in the functional channel generate K+ currents with differing biophysical (e.g., activation and inactivation kinetics, current amplitude) and pharmacological (e.g., sensitivity to drugs) properties.

Recent advances in mouse genetics and physiology, including the development of transgenic or knockout mice, have made the mouse the model of choice in the study of the genetic basis of many cardiopulmonary diseases. Mice have been used as models to study not only normal physiological functions, such as respiratory control (14), but also pathogenic mechanisms involved in pulmonary vascular disease (19, 27, 55) and asthma (23, 30, 56). However, a comprehensive electrophysiological characterization of KV channels in mouse PASMCs has not been reported. Previous studies have used transgenic mice to study the effects of NADPH oxidase subunits (5) and hypoxia-inducible factor-1{alpha} mutants (41) to investigate effects on K+ currents; however, these studies predominantly focused on current amplitude and differences between wild-type and mutant mice. Understanding the basic electrophysiological and pharmacological properties of KV channels in mouse PASMCs is essential to define potential pathogenic roles of K+ channels in various mouse models of cardiopulmonary diseases. Therefore, this study aimed to investigate the electrophysiological and pharmacological properties of native KV currents (IK(V)) in freshly isolated mouse PASMCs with whole cell and single-cell patch-clamp techniques.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of PASMCs from mice. Freshly dissociated mouse PASMCs were used in this study to functionally characterize KV channels. Use of mice for the experiments presented in this study was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego. Briefly, male Balb C mice (7–8 wk) were killed by cervical dislocation in accordance with the IACUC-approved guidelines. Lungs were removed from the chest cavity and washed in normal Tyrode solution composed of (mM) 143 NaCl, 5.4 KCl, 0.33 NaH2PO4, 0.5 MgCl2, 1.8 CaCl2, 5.0 HEPES, and 16.6 glucose (adjusted with 2 M NaOH to pH 7.4). With the lung in normal Tyrode solution under a light microscope, intrapulmonary arteries (3rd- to 5th-order intralobar branches of <400-µm external diameter) were isolated by removing the surrounding parenchymal and connective tissues. Furthermore, the adventitia was carefully removed from the isolated arteries with fine forceps under a high-magnification microscope. The arteries were then cut open longitudinally and incubated for 20 min in Ca2+-free Tyrode solution (37°C) containing 2 mg/ml collagenase (Worthington Biochemical, Lakewood, NJ) and 1 mg/ml bovine serum albumin (BSA; Sigma, St. Louis, MO). After the arteries were washed in Ca2+-free Tyrode solution, the remaining smooth muscle was further digested for 9 min with 0.5 mg/ml elastase (Sigma) and 1 mg/ml BSA at 37°C. The digested tissue was then removed from the enzyme solution and placed in the storage medium containing (mM) 70 KOH, 50 L-glutamate, 55 KCl, 20 taurine, 20 KH2PO4, 3 MgCl2, 20 glucose, 10 HEPES, and 0.5 EGTA (pH = 7.4).

To create a suspension of single PASMCs, cells were dispersed by gentle trituration with a fire-polished glass pipette. Cells were used within 12 h and stored at 4°C until use. For electrophysiological recordings, two or three drops of cell suspension were placed onto a glass coverslip in the recording chamber on a Nikon inverted microscope and allowed to adhere to the coverslip for 30 min. Cells attached on the coverslip were then superfused with Tyrode solution for 10–20 min before the experiments started. All experiments were performed at room temperature (22–24°C).

Electrophysiological measurement. Whole cell and single-channel K+ currents were recorded with an Axopatch-1D amplifier and a DigiData 1322 interface (Molecular Devices, Sunnyvale, CA) and the patch-clamp technique (38). Borosilicate patch pipettes (3–4 M{Omega}), which were fabricated on a model P-97 electrode puller (Sutter Instrument, Novato, CA) and polished with a MF-63 microforge (Narashige Scientific Instruments Laboratories), were used to make high-resistance seals with the cell membrane for whole cell (and single channel) current recording. After break-in, the averaged series resistance (Rs) was 19.4 ± 6.5 M{Omega} (mean ± SD; n = 14). A high membrane resistance (Rm; 1.02 ± 0.04 G{Omega}) was recorded in most of the cells and the Rs-to-Rm ratio is close to 1.9%. Command voltage protocols and data acquisition were performed with pCLAMP 8.1 software (Molecular Devices). Currents were filtered at 1–2 kHz and digitized at 2–4 kHz.

The bath (extracellular) solution for whole cell recording was normal Tyrode solution containing (in mM) 143 NaCl, 5.4 KCl, 0.33 NaH2PO4, 0.5 MgCl2, 1.8 CaCl2, 5 HEPES, and 16.6 glucose (adjusted with 2 M NaOH to pH 7.4). In Ca2+-free bath solution, CaCl2 was replaced by equimolar MgCl2 to maintain osmolarity and 1 mM EGTA was added to chelate residual Ca2+. The pipette (intracellular) solution for whole cell current recording contained (in mM) 105 K-aspartate, 25 KCl, 5 NaCl, 1 MgCl2, 4 Mg-ATP, 10 BAPTA (a Ca2+ chelator), and 10 HEPES (adjusted with 2 M KOH to pH 7.3). Leakage currents and capacitative currents were subtracted with the P/4 subtraction protocol in the pCLAMP software.

For cell-attached recording of single-channel K+ currents, the pipette (extracellular) solution contained (in mM) 135 KCl, 10 HEPES, 1.2 MgCl2, 10 glucose, and 5 EGTA (adjusted to pH 7.2). Therefore, the K+ equilibrium potential (EK) for the membrane patch is close to zero [because pipette or extracellular K+ concentration ([K+]) {approx} intracellular [K+]] based on the Nernst equation: EK = 25·ln([K+]out/[K+]in), where "out" and "in" denote extracellular and intracellular concentrations, respectively. Given that the cells were superfused with normal Tyrode solution and possessed a resting Em, the actual potential applied to the cell-attached membrane patch was not exactly the same as the potential applied to the cell (Vpatch), which is equal to the inverse of the command potential (Vcomm) from the computer. The actual value can be calculated by the following equation: (–Vcomm) + Em = Em – Vcomm. Furthermore, because of the negative resting Em (which is approximately –30 mV in freshly dissociated mouse PASMCs), the reversal potential (Erev) for single K+ channels in the cell-attached membrane patch was not at the EK, but close to the potential determined by EK – Em. In other words, the K+ currents through the K+ channels on the cell-attached membrane patches reverse at the potential of –Em (because EK {approx} 0). The amplitude of single-channel K+ currents in cell-attached membrane patches was determined with FETCHAN and pSTAT analysis programs (Molecular Devices). Results of channel amplitudes are presented as a function of Vpatch; therefore, there is a rightward shift of the current-voltage (I-V) relationship curves.

Chemicals. Tetraethylammonium (TEA), 4-aminopyridine (4-AP), charybdotoxin (ChTX), iberiotoxin (IbTX), and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) were all obtained from Sigma. TEA and 4-AP were directly dissolved in the bath solution on the day of use; the pH value was readjusted to 7.4 with KOH. ChTX, IbTX, and FCCP were dissolved in water to make stock solutions of 0.1–1 mM; aliquots of the stock solution were diluted 1:1,000 into the bath solution to final concentrations of 0.1–5 µM.

Statistics. Data analysis, curve fitting, and presentation were performed with pCLAMP 8.1 and SigmaPlot 2000 software. It should be noted that the n values given represent the number of individual cells patched; for each experiment these were derived from a minimum of three different mice. The results are expressed as means ± SE. For statistical analysis of the physiological data, we used the Student's t-test. Values of P < 0.05 were considered to indicate significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Morphology and passive membrane properties of mouse PASMCs. Freshly dissociated mouse PASMCs from intrapulmonary arteries were oblong or round (Fig. 1), unlike the long spindle shape of PASMCs from other species. Most of the cells further contracted with extracellular application of 50 mM K+ or 20 µM 5-HT, indicating that the passive biophysical properties of mouse PASMCs are similar to those of PASMCs from other species. Using the whole cell configuration, we measured the resting Em, Rm, and membrane capacitance (Cm) as shown in Fig. 2. Cm in mouse PASMCs was 9.42 ± 0.69 pF (n = 57); mean Rm in mouse PASMCs was 1.02 ± 0.04 G{Omega} (n = 49); and resting Em was –27.9 ± 0.9 mV (n = 51). The passive biophysical properties of mouse PASMCs appeared to be similar to those of rat PASMCs, although Em in freshly dissociated mouse PASMCs was less negative than that in freshly dissociated and primary cultured PASMCs from rats or humans (42, 57, 58).


Figure 1
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  		Fig. 1. Morphology of isolated pulmonary artery (PA; A) and freshly dissociated pulmonary artery smooth muscle cells (PASMCs; B) from mice. A: 3rd-5th orders of intrapulmonary arterial branches isolated from mice, external diameter <400 µm. B: freshly dissociated single PASMCs from the intrapulmonary arterial tissue shown in A. The cells are partially contracted because the bath solution contains 1.8 mM Ca2+.

 

Figure 2
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  		Fig. 2. Passive membrane properties of freshly dissociated mouse PASMCs. Histogram showing the distribution of membrane capacitance (Cm, n = 57 cells; A), membrane input resistance (Rm, n = 49 cells; B), and resting membrane potential (RMP, n = 51 cells; C). Peak values based on fitting curves for Cm, Rm, and RMP are 9.5625 pF, 1.0430 G{Omega}, and –26.8125 mV, respectively, while the averaged values (means ± SE) are 9.42 ± 0.69 pF, 1.02 ± 0.04 G{Omega}, and –27.94 ± 0.93 mV, respectively. Cells were isolated from 42–50 mice.

 
Classification of whole cell IK(V) based on activation and inactivation kinetics. Depolarizing the cells from a holding potential of –70 mV to a series of test potentials ranging from –60 to +60 mV elicited outward K+ currents. Three families of IK(V) could be distinguished based on their activation and inactivation kinetics (Fig. 3A) : 1) a rapidly activating and noninactivating IK(V) (n = 10; 58% of the cells tested), 2) a rapidly activating and slowly inactivating IK(V) (n = 4; 23%), and 3) a slowly activating and noninactivating IK(V) (n = 3; 17%). Most of the mouse PASMCs possessed the rapidly activating and noninactivating currents.


Figure 3
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  		Fig. 3. Kinetically distinct whole cell voltage-gated K+ (KV) channel currents in freshly dissociated mouse PASMCs. A: representative currents, elicited by depolarizing cells from a holding potential of –70 mV to a series of test potentials ranging from –60 to +60 mV in 20-mV increments, show 3 kinetically distinct currents: rapidly activating and noninactivating currents (left), rapidly activating and slowly inactivating currents (center), and slowly activating and noninactivating currents (right). B and C: current-voltage (I-V; B) and conductance-voltage (G-V; C) relationship curves of the rapidly activating and noninactivating, rapidly activating and slowly inactivating, and slowly activating and noninactivating KV currents. Currents were measured at the steady state (i.e., at ~500–550 ms). For the I-V curves of the rapidly activating and slowly inactivating currents (center), the amplitudes of both transient currents (Itr, measured at 20–50 ms) and steady-state currents (Iss) against the test potentials are shown (B and C). G was calculated from the equation: G = I/(V – EK), where I is the steady-state current, V is the test potential, and EK is the equilibrium potential for K+. D: time constants of current activation ({tau}act) for the rapidly activating and noninactivating (left), rapidly activating and slowly inactivating (center), and slowly activating and noninactivating (right) currents, elicited by various test potentials ranging from –20 to +60 mV. Data are means ± SE. Cells were isolated from 3–8 mice.

 
I-V and conductance-voltage (G-V) relationship curves of the outward currents (measured at steady state) show that the currents were activated at approximately –40 mV (Fig. 3, B and C). For the rapidly activating and slowly inactivating currents, the I-V and G-V curves indicate that the transient currents (Itr) and the steady-state currents (Iss) had a similar activation threshold (around –40 mV). The activation time constant, {tau}act, was measured by fitting a monoexponential curve to the rising phase of each type of current and is shown over the voltage range from –20 to +60 mV for each family of currents. For the rapidly activating and noninactivating IK(V), {tau}act was significantly decreased (or activation rate was significantly accelerated) when test potentials were changed from –20 to +60 mV (Fig. 3D, left). However, for the rapidly activating and slowly inactivating IK(V) (Fig. 3D, center) and the slowly activating and noninactivating IK(V) (Fig. 3D, right), the activation kinetics were not significantly changed when test potentials became more positive. These data suggest that the kinetically distinct whole cell IK(V) in different cells may arise from different KV channels and different cells may express functionally distinct KV channels.

Window currents. We also determined the steady-state activation and voltage-dependent inactivation kinetics of IK(V). The inactivation pulse protocol was as follows: fifteen 1-s prepulses to potentials between –120 and +20 mV in steps of 10 mV were given before a 100-ms step depolarization from –70 to +40 mV (n = 10). A representative current recording is shown in Fig. 4A. Steady-state activation was determined from tail currents with the following pulse protocol: tail currents were assessed at a repolarizing potential of –40 mV (100 ms) after channels were activated to test potentials between –60 and +40 mV in 10-mV increments (300-ms voltage steps were applied every 5 s). A representative current trace is shown in Fig. 4B.


Figure 4
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  		Fig. 4. Steady-state voltage dependence of inactivation and activation of whole cell KV currents in freshly dissociated mouse PASMCs. A: representative family of currents, elicited by a double-pulse protocol, to construct steady-state inactivation curve. The cell was held at –70 mV and stepped to a series of conditioning pulses (1 s) ranging from –120 to +20 mV before depolarization to +60 mV for 100 ms. Inactivation was plotted as the normalized current (I/Imax) to the test potential of (+60 mV) as a function of the conditioning potentials (–120 to +20 mV). B: representative family of superimposed currents, elicited by depolarization from a holding potential of –70 mV to test potentials (300 ms) ranging from –60 to +40 mV (in 10-mV increments) followed by repolarization to –40 mV (to record tail currents). Inset: magnified tail currents. C: averaged inactivation points (gray circles) from 10 cells represent normalized outward currents during test depolarization to +60 mV that are plotted against the conditioning potentials (–120 to +20 mV). Averaged activation points (black circles) from 10 cells represent normalized tail currents elicited by a repolarization step to –40 mV after depolarization steps (from –60 to +40 mV in 10-mV increments). Data are expressed as means ± SE. Cells were isolated from 7-9 mice. Smooth curves through inactivation and activation points are the best fit generated by the computer. D: magnified fitting curves for inactivation and activation points (as shown in C) to illustrate the window currents (shaded area) between –45 and –10 mV; the maximal window current appeared to be at –31.5 mV (indicated by arrow).

 
The normalized activation curve (n = 10 cells) indicates that the channels were activated at –40 mV, which is more negative than the measured resting Em (–29 mV) in mouse PASMCs, and the channel activation maximized at approximately +10 mV (Fig. 4C). The normalized inactivation curve (n = 10 cells) indicates that the channels were inactivated at potentials from –80 to –70 mV and the maximal inactivation occurred at –10 mV (Fig. 4C). The voltage dependence of activation and inactivation was well described by a Boltzmann distribution with a voltage of half-maximal activation of –25 mV and a voltage of half-maximal inactivation of –50 mV (Fig. 4C). The overlap between the activation and inactivation steady-state curves revealed a voltage "window" between –40 and –10 mV, in which the channels were partially activated and incompletely inactivated (Fig. 4D). The peak window currents were at –31.5 mV (Fig. 4D), a potential very close to the measured resting Em in these cells. These data suggest that the KV currents in freshly dissociated mouse PASMCs are active under resting conditions and may significantly contribute to the regulation of the resting Em.

Effects of 4-AP, TEA, ChTX, and IbTX on whole cell IK(V) in mouse PASMCs. The pharmacological properties of the KV channels, especially in response to the classic K+ channel blockers TEA and 4-AP, were also assessed in mouse PASMCs. Whole cell IK(V) was not sensitive to a low concentration (1 mM) of 4-AP (data not shown) but was sensitive to high concentrations of 4-AP. Extracellular application of 10 mM 4-AP, for example, reduced the currents by 37% at +60 mV (Fig. 5A; n = 12 cells). After 10 min of washout with normal Tyrode solution, IK(V) was only partially recovered. The currents, however, appeared to be very sensitive to TEA (Fig. 5B). At +60 mV, 1 mM TEA markedly reduced the amplitude of IK(V) by 32% (n = 4), while IK(V) reductions by 5 and 10 mM TEA were similar [56% (n = 4) and 55% (n = 8), respectively] (Fig. 5Bd). The inhibitory effect of TEA was independent of test potentials and fully reversible after washout (Fig. 5B, c and d).


Figure 5
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  		Fig. 5. Inhibitory effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on whole cell IK(V) in freshly dissociated mouse PASMCs. A and B: representative currents (a), elicited by depolarization from a holding potential of –70 mV to a series of test potentials ranging from –60 to +60 mV (in 20-mV increments), in cells before (Control) and during extracellular application of 10 mM 4-AP (A) or 10 mM TEA (B). The 4-AP (A)- or TEA (B)-sensitive currents shown in b were constructed by subtracting the currents recorded during treatment with 10 mM 4-AP or 10 mM TEA from the currents recorded under control conditions. c: I-V relationship curves for the currents recorded before (Control), during (4-AP or TEA), and after (Wash) treatment with 4-AP or TEA. Data are expressed as means ± SE (n = 5–10 cells). d: Concentration-dependent inhibition of currents by TEA at 1, 5, and 10 mM. There is no significant difference in TEA-mediated inhibition in currents elicited by different test potentials (0 to +60 mV) (d). Data shown are means ± SE. Cells represent data from 3–10 mice.

 
The rapidly activating and noninactivating current could be further divided into two subgroups based on pharmacological response to 4-AP. As shown in Fig. 6, ~69% of the cells contained a rapidly activating and noninactivating IK(V) that was sensitive to 4-AP (10 mM), while 31% of the cells exhibited a rapidly activating and noninactivating IK(V) that was unaffected by 4-AP. The 4-AP-sensitive and -insensitive IK(V), which share similar kinetics of activation and inactivation, suggest that 1) different KV channel {alpha}- and beta-subunits can form heterotetrameric channels that are functionally indistinguishable, 2) the heterotetrameric (or homotetrameric) channels that are functionally identical may have different binding affinity to drugs and intracellular modulators, and 3) the functionally identical channels may contain different chaperones that may confer different sensitivity to drugs.


Figure 6
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  		Fig. 6. Comparison of whole cell KV currents in cells that have different sensitivity to 4-AP. Representative currents (top), elicited by depolarization from a holding potential of –70 mV to a test potential of +60 mV, and {tau}act (mean ± SE) of the currents (bottom) in cells before (Control) and during (4-AP) extracellular application of 10 mM 4-AP are shown. The inability of 4-AP to reduce the rapidly activating and noninactivating currents in some PASMCs appears to correlate with the negligible effect of 4-AP on {tau}act (A). In PASMCs, in which 4-AP significantly reduces the current amplitude, 4-AP also significantly increases {tau}act or decreases the current activation (B and C). ***P < 0.001 vs. Control. Four or five mice were used for these experiments.

 
In addition to current amplitude, we also measured activation kinetics or {tau}act by fitting a single exponential curve to the rising component of the current traces at +60 mV. In cells whose amplitude of IK(V) was not affected by 4-AP, {tau}act was not significantly altered by extracellular application of 4-AP (Fig. 6A, bottom). However, for cells in which 4-AP decreased the amplitude of IK(V), 4-AP (10 mM) increased {tau}act of the rapidly activating and noninactivating currents from 14 to 46 ms (n = 5; P < 0.001) (Fig. 6B, bottom) and increased {tau}act of the rapidly activating and slowly inactivating currents from 5 to 57 ms (n = 4; P < 0.001) (Fig. 6C, bottom). These data indicate that the 4-AP-mediated decrease in IK(V) may at least partially result from its inhibitory effect on channel activation.

To verify that the whole cell currents we were recording were KV currents and were not contaminated with Ca2+-activated K+ (KCa) currents, we tested the effect of ChTX (100 nM), a blocker of intermediate- and large-conductance KCa channels, and IbTX (100 nM), a selective blocker for the large-conductance KCa channels (or maxiK channels), on whole cell outward K+ currents. As shown in Fig. 7, neither extracellular application of ChTX (100 nM) or IbTX (100 nM) nor removal of extracellular Ca2+ (by replacing CaCl2 with MgCl2 and adding 1 mM EGTA in the bath solution) affected the amplitude of whole cell currents (n = 4–6 cells). These data indicate that the whole cell outward currents measured in mouse PASMCs superfused with normal Tyrode solution and dialyzed with Ca2+-free (with 1 mM EGTA) and 5 mM ATP-containing solution are predominantly KV currents.


Figure 7
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  		Fig. 7. Effects of charybdotoxin (ChTX), iberiotoxin (IbTX), and removal of extracellular Ca2+ on whole cell IK(V) in freshly dissociated mouse PASMCs. A–C, a: representative currents elicited by depolarization from a holding potential of –70 mV to a series of test potentials ranging from –60 to +60 mV (in 20-mV increments), in cells before (Control) and during extracellular application of 100 nM ChTX (A) or 100 nM IbTX (B) or superfusion with Ca2+-free bath solution (0 Ca; C). b: I-V relationship curves for the currents recorded before (Control), during (ChTX, IbTX, or 0 Ca) and after (Wash) treatment of ChTX or IbTX or removal of extracellular Ca2+ (0 Ca). Data are expressed as means ± SE (n = 5–10 cells from 3–7 mice).

 
Identification of different KV channels based on single-channel conductance. Single-channel K+ currents (iK) were recorded from mouse PASMCs with the cell-attached configuration. When the membrane patch was held at +80 or +100 mV, we detected multiple different-amplitude currents (Fig. 8, A and B). On the basis of current amplitude at +80 mV, the single-channel currents can be classified as a large-amplitude current (6–7 pA), two intermediate-amplitude currents (4–5 pA and 2–3 pA), and a small-amplitude current (~1 pA) (Figs. 8 and 9). The slope conductance (g) of the unitary outward K+ currents was calculated from the i-V relationship curves. The calculated slope conductances for the large-, intermediate-, and small-amplitude currents are 119.4 ± 2.7, 79.8 ± 2.8, 46.0 ± 2.2, and 23.6 ± 0.6 pS, respectively (Fig. 9).


Figure 8
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  		Fig. 8. Single-channel K+ currents in cell-attached membrane patches of freshly dissociated mouse PASMCs. A: representative record showing multiple-amplitude currents recorded in a cell-attached patch at the applied membrane voltage (Eapp) of +80 mV. There are at least 4 different-amplitude currents: a small-amplitude current (a), two intermediate-amplitude currents (b and c), and a large-amplitude current (d). C indicates the channel closed state. B: representative record showing multiple-amplitude currents recorded in a cell-attached patch at +120 mV. Three different-amplitude currents are observed in these patches, a large-amplitude current (1) and 2 intermediate-amplitude currents (2 and 3). C: representative currents (left) in a cell-attached membrane patch, elicited by depolarizing the patch to test potentials (Vpatch) from +60 to +120 mV, and the I-V relationship curve (right) of the large-amplitude current. The slope conductance of the unitary current is 115 pS. D: representative currents (left) in a cell-attached patch, elicited by Vpatch from +60 to +100 mV, and the I-V curve (right) of the intermediate-amplitude current. The slope conductance of the unitary current is 46 pS. n = 7–15 cells from 5–10 mice.

 

Figure 9
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  		Fig. 9. Single-channel K+ currents in cell-attached membrane patches of freshly dissociated mouse PASMCs. A: representative current records in a cell-attached membrane patch at various test potentials (Vpatch) of +60, +80, and +100 mV. Two different-amplitude currents, an intermediate-amplitude current (a) and a small-amplitude current (b), are recorded in this patch. C indicates the current level when the channels are closed. B: I-V relationship curves constructed from the 2 different-amplitude currents shown in A. The slope conductances for the intermediate- and small-amplitude currents are 76 and 23 pS, respectively. C: I-V curves constructed from averaged currents recorded in 5 cells (data are means ± SE). The calculated slope conductance is 21 pS. D: calculated slope conductances (means ± SE) from groups of cells in which various amplitude (large, intermediate-1, intermediate-2, and small) of single-channel currents were recorded. Numbers in parentheses denote the number of cells tested; n = 5–12 from 4–8 mice.

 
The "large-conductance" (>110 pS) current was reminiscent of the intermediate-conductance KCa current (IK) but seemed to be different from the large-conductance KCa current or the MaxiK (or BK) current of ~200 pS recorded in rat and human PASMCs (3, 25, 36, 39, 60). Increasing cytoplasmic [Ca2+] ([Ca2+]cyt) by FCCP, a protonophore and mitochondrial uncoupler that increases [Ca2+]cyt by mobilizing Ca2+ from mitochondria and intracellular organelles (60), significantly increased the steady-state open probability of the 119-pS unitary currents, iK(Ca) (Fig. 10, A–C). Similar to the currents previously reported in rat PASMCs (60), brief (<3 min) application of FCCP (20 µM) increased the open probability of iKCa by mobilizing Ca2+ from mitochondria and sarcoplasmic or endoplasmic reticulum (Fig. 10, A and C), whereas longer (>15 min) treatment with FCCP reversibly reduced whole cell IK(V) by potentially inhibiting mitochondrial oxidative phosphorylation and ATP production (Fig. 10B). These data indicate that both KV and IK channels are functionally expressed in mouse PASMCs. However, the activity of KV channels is important in regulating the resting Em, while the activity of IK channels may be involved in membrane repolarization when [Ca2+]cyt is increased.


Figure 10
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  		Fig. 10. Effects of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on large-conductance K+ currents in cell-attached membrane patch and whole cell KV currents in mouse PASMCs. A: representative unitary K+ currents in a cell-attached membrane patch at test potentials (Vpatch) of +60, +80, +100 and +120 mV before (Control) and after (FCCP) treatment of the cell with 20 µM FCCP. B: I-V relationship curve of the currents shown in A indicates a slope conductance of 111 pS. C: in a different cell, the lower dose of FCCP (4 µM) caused a similar augmenting effect on the large-conductance current in cell-attached patch; the effect was also reversible. D: inhibitory effect of prolonged treatment with FCCP on whole cell KV currents. Currents were recorded in PASMCs before (Control), during (FCCP), and after (Washout) 20-min extracellular application of 20 µM FCCP. These data indicate that FCCP, by increasing cytoplasmic [Ca2+], increases the large-conductance K+ channel activity in cell-attached patches (as shown in A and C). However, FCCP, potentially by inhibiting mitochondrial oxidative phosphorylation, decreases the whole cell KV currents in PASMCs dialyzed with Ca2+-free and 10 mM EGTA-containing pipette solution (as shown in D). n = 4–7 from 3–5 mice.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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The expression and proper functioning of KV channels in PASMCs are essential to the regulation of pulmonary vascular tone and play a major role in pathological states. Previous studies have reported that the development of sustained pulmonary vasoconstriction and vascular remodeling in pulmonary hypertension are related to dysfunction and downregulation of K+ channels in PASMCs of humans and animals (46, 57, 61, 62). At least two types of K+ channels, voltage-gated K+ (KV) channels and Ca2+-activated K+ (KCa) channels, are known to be involved in the regulation of pulmonary vascular tone and PASMC proliferation and apoptosis in various animal models (12, 13, 17, 24, 37). While mice are used widely as models for various diseases, little is known about the biophysical properties and molecular basis of K+ channels in mouse PASMCs. Therefore, we fully characterized the electrophysiological and pharmacological properties of KV and KCa channels in freshly dissociated mouse PASMCs; this study provides a foundation for future studies on pathogenic roles of ion channels using knockout and transgenic mice.

In the present study, three types of KV currents are distinguished based on activation and inactivation kinetics in mouse PASMCs: 1) a rapidly activating and noninactivating current, 2) a rapidly activating and slowly inactivating current, and 3) a slowly activating and noninactivating current. However, the rapidly activating and noninactivating current, which is present in most of the cells studied, can be further divided into two subcategories based on pharmacological response to 4-AP. About two-thirds of the cells with the rapidly activating and noninactivating current showed IK(V) inhibition with 1 mM 4-AP, while in one-third of the cells the currents were unaffected by 4-AP. Extracellular application of TEA, however, reversibly decreased all current types in all cells tested. Identification of the molecular origin of these differences will require further experimental work.

Previous studies reported that whole cell I(K(V) appears as a rapidly activating and slowly decaying delayed rectifier with no transient component in freshly isolated rat PASMCs (35, 43, 45, 50). The current is only slightly attenuated by 3–10 mM TEA (35, 43, 45) but markedly blocked by 4-AP (18, 43, 45, 58). In our experiments, 1 mM 4-AP had no effect on whole cell I(K(V) in freshly dissociated mouse PASMCs and 5 mM 4-AP had a modest inhibitory effect (9.9% inhibition). Only 10 mM 4-AP had a significant inhibitory effect on the amplitude of whole cell I(K(V), which was associated with a significant increase in the time constant of current activation ({tau}act), indicating that the high dose of 4-AP decreases IK(V) at least partially by decelerating the channel activation. Such an effect of 10 mM 4-AP has been previously observed (41); however, a significant reduction in IK(V) amplitude at 1 mM 4-AP has been shown in perforated-patch mode when cells were perfused with solution bubbled at a PO2 of 120 mmHg (5). In contrast to the effect of 4-AP, TEA showed significant inhibitory effects on whole cell IK(V) with concentrations as low as 1 mM. This is also in contrast to other vascular smooth muscle cells, such as rabbit portal vein and pulmonary arterial smooth muscle cells, in which 4-AP appears to be a more potent blocker of IK(V) and TEA is less effective (7, 33, 51). In general, KV channels from other types of smooth muscle cells demonstrate a higher sensitivity to 4-AP, although the degree of inhibition varies. However, whole cell IK(V) in mouse gallbladder smooth muscle cells are relatively insensitive to 4-AP (21). At +50 mV, 1 and 10 mM 4-AP inhibit K+ currents by 17.4% and 35.4%, respectively. In isolated smooth muscle cells from the longitudinal layer of dog colon, 5 mM 4-AP caused only a 20% reduction of currents (10). However, the K+ currents in mouse gallbladder smooth muscle cells and canine colonic smooth muscle cells were more sensitive to TEA (21, 48). Taken together with the results from this study, it is possible that functional KV channels, which predominantly contribute to the whole cell IK(V) in freshly dissociated mouse PASMCs, are formed by KV channel {alpha} (and beta)-subunits that are less sensitive to 4-AP but more sensitive to TEA. A previous study in mouse PASMC did show ~50% decrease in the current at +50 mV on addition of 5 mM TEA as apposed to a ~25% inhibition of the same current by 1 mM 4-AP, which supports our finding that the currents are more sensitive to TEA (5).

The low dose of TEA (e.g., ≤1 mM) has been demonstrated to be a potent blocker of the large-conductance KCa channels in vascular smooth muscle cells (32). The marked inhibition of whole cell outward K+ currents in mouse PASMCs, however, does not necessarily indicate that the voltage-dependent outward K+ currents we recorded were KCa currents because 1) the low dose of TEA also blocks KV channels (48) and 2) the whole cell outward K+ currents in mouse PASMCs were not affected by either ChTX or IbTX, both selective KCa channel blockers in vascular smooth muscle cells (32, 49), or removal of extracellular Ca2+. These data also indicate that, although mouse PASMCs express functional KCa channels, intracellular dialysis with Ca2+-free and EGTA-containing pipette solution significantly minimizes the contribution of the large (and/or intermediate)-conductance KCa channels to the whole cell K+ currents. The KCa channels in mouse PASMCs may be more sensitive to intracellular [Ca2+] than similar Ca2+-sensitive K+ channels in PASMCs from other species (e.g., humans, rats, rabbits, and dogs) (3, 6, 8, 28, 58).

In the steady state, the whole cell current through KV channels depends on the balance between channel activation and inactivation. The slope factor of the channel activation curve indicates the sensitivity of channels to membrane potential (32). Activation curves for KV channels in coronary and cerebral arterial smooth muscle cells have slope factors of 9 and 11 mV, respectively, with the midpoint of the activation curve at –6 and –9 mV (40, 51). Based on the activation and inactivation curves, our data show that 1) the midpoint of activation of IK(V) is approximately –20.5 mV; 2) the midpoint of inactivation of IK(V) is approximately –52.25 mV; and 3) the "voltage window" for IK(V) (or the voltage range in which KV channels are not completely inactivated and can be partially activated) is between –40 and –10 mV (the peak window current occurs at –31.5 mV). These data also suggest that whole cell IK(V) in freshly dissociated mouse PASMCs are kinetically and pharmacologically different from those characterized in freshly dissociated and primary cultured PASMCs from rats (43, 44, 58), dogs (1), and humans (38).

Based on the mouse genome, there are more than 55 KV channel genes that encode the pore-forming {alpha}-subunits and the regulatory beta-subunits. Functional KV channels are homo- or heterotetramers composed of various {alpha}- and beta-subunits in native cells. Different homo- or heterotetrameric KV channels are believed to have different single-channel conductances. In vascular smooth muscle cells, various single-channel conductances have been reported for KV channels and can be broadly divided into three groups with symmetrical K+ gradient in cell-attached configuration: 1) small-conductance channels with slope conductances <15 pS, 2) intermediate-conductance channels with slope conductances ranging from 20 to 70 pS, and 3) large-conductance channels with slope conductances >100 pS (2, 36, 39, 54).

Small-conductance KV channels are present in rabbit basilar artery (5.5 pS; Ref. 40), porcine coronary artery (7.3 pS; Ref.52), and rabbit portal vein (5 and 8 pS; Ref.7) smooth muscle cells. However, an intermediate-conductance single channel has been reported in rabbit coronary artery smooth muscle (70 pS, with symmetrical K+; Ref.20) and in canine renal artery smooth muscle cells (57 pS; Ref. 16). In addition, a large-conductance (200–250 pS) KCa current (or maxiK or BK current) has been observed in many cell types including coronary arterial smooth muscle cells (20, 31), cerebral arterial smooth muscle cells (15, 53), and PASMCs (4, 25, 36, 38, 60). In freshly dissociated mouse PASMCs, all large-, intermediate-, and small-amplitude single-channel currents (iK) were observed in cell-attached membrane patches during sustained depolarization to positive potentials. The slope conductance (100–125 pS) of the large-amplitude single-channel iK, which is sensitive to intracellular [Ca2+], is, however, much smaller than the conductance for the maxiK channels (i.e., 200–250 pS).

In the present study, our data demonstrated a broad spectrum of single-channel conductance in mouse PASMCs, which can be categorized into four groups: 1) a large-conductance single-channel current with a slope conductance of 119 ± 3 pS, 2) two intermediate-conductance single-channel currents with slope conductances of 80 ± 3 and 46 ± 2 pS, respectively, and 3) a small-conductance single-channel current with a slope conductance of 24 ± 1 pS. The large-conductance (>100 pS) single-channel current in mouse PASMCs is sensitive to an increase in [Ca2+]cyt. Brief application of FCCP, a mitochondrial uncoupler that transiently increases [Ca2+]cyt by inducing Ca2+ mobilization from intracellular organelles (60), significantly and reversibly increased the steady-state open probability of the large-conductance iK recorded in cell-attached patches. These results suggest that the 119-pS single-channel iK in mouse PASMCs shares similar biophysical properties with the intermediate-conductance KCa currents characterized in rat and human PASMCs, although the conductance (100–125 pS) is much smaller than the conductance of the maxiK channels (200–250 pS) in other vascular smooth muscle cells (24, 36, 38).

It is increasingly clear that the KV and KCa channel characteristics in PASMCs vary greatly across species and even in different circulations within the same species. Differences in our data from the previously published data in mouse PASMCs may arise from the differences in experimental protocol, for example, composition of the intracellular solution, differences in patching method (perforated patch vs. whole cell), and isolation procedure or artery size. However, as our data are compiled from patching over 50 cells from more than 40 different mice, it provides the most extensive characterization of the K+ currents in mouse PASMCs. Further work will be essential to identify the molecular identity of the kinetically distinct KV channels in mouse PASMCs.

In summary, the present work provides an extensive functional characterization of the native KV and KCa channels in pulmonary vascular smooth muscle cells from mice. The different biophysical and pharmacological properties of KV and KCa channels in mouse PASMCs (in comparison to PASMCs from other species) provide an important basis for using mice as a disease model to study the pathogenic role of ion channels in the development of pulmonary vascular disease.


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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-064945, HL-054043, and HL-066012.


   ACKNOWLEDGMENTS
 
We thank Dr. Carmelle V. Remillard, Ann Nicholson, and Dr. Minlin Xu for their generous assistance.


   FOOTNOTES
 

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

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


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