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Am J Physiol Lung Cell Mol Physiol 291: L802-L810, 2006. First published April 21, 2006; doi:10.1152/ajplung.00104.2006
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BK channel beta1-subunit regulation of calcium handling and constriction in tracheal smooth muscle

Iurii Semenov, Bin Wang, Jeremiah T. Herlihy, and Robert Brenner

Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas

Submitted 22 March 2006 ; accepted in final form 18 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The large-conductance, Ca2+-activated K+ (BK) channels are regulators of voltage-dependent Ca2+ entry in many cell types. The BK channel accessory beta1-subunit promotes channel activation in smooth muscle and is required for proper tone in the vasculature and bladder. However, although BK channels have also been implicated in airway smooth muscle function, their regulation by the beta1-subunit has not been investigated. Utilizing the gene-targeted mice for the beta1-subunit gene, we have investigated the role of the beta1-subunit in tracheal smooth muscle. In mice with the beta1-subunit-knockout allele, BK channel activity was significantly reduced in excised tracheal smooth muscle patches and spontaneous BK currents were reduced in whole tracheal smooth muscle cells. Knockout of the beta1-subunit resulted in an increase in resting Ca2+ levels and an increase in the sustained component of Ca2+ influx after cholinergic signaling. Tracheal constriction studies demonstrate that the level of constriction is the same with knockout of the beta1-subunit and BK channel block with paxillin, indicating that BK channels contribute little to airway relaxation in the absence of the beta1-subunit. Utilizing nifedipine, we found that the increased constriction caused by knockout of the beta1-subunit could be accounted for by an increased recruitment of L-type voltage-dependent Ca2+ channels. These results indicate that the beta1-subunit is required in airway smooth muscle for control of voltage-dependent Ca2+ influx during rest and after cholinergic signaling in BK channels.

airway; knockout mice; gene-targeted mice

IN MANY SMOOTH MUSCLE CELLS, voltage-dependent Ca2+ channels are the major conduits for Ca2+-mediated constriction (21). K+ channels generally function to oppose constriction by hyperpolarizing membranes, which deactivates voltage-dependent Ca2+ channels and reduces Ca2+ influx. Among these, the large-conductance Ca2+-activated K+ (BK) channels are broadly expressed and have been studied in many cell types. These channels are composed of a pore-forming {alpha}-subunit and a tissue-specific beta-subunit. Binding of intracellular Ca2+ and depolarizing voltage gate BK channels to open. When open, BK channels have a very large outward K+ conductance (>200 pS) and, therefore, very effectively hyperpolarize the membrane. As such, they are important regulators of membrane voltage in a number of cell types (6, 15, 20).

BK channels bind Ca2+ at low affinity (~10 µM) (9, 28) and require colocalization with a Ca2+ source or the contribution of voltage to open the channel at physiological Ca2+ concentrations (8, 30, 31). In vascular smooth muscle, large voltage changes do not occur, and BK channels have an enhanced apparent Ca2+ sensitivity that is associated with a member of the accessory BK channel beta-subunit family (beta1- to beta4-subunits), the beta1-subunit (23, 42). The important role of the beta1-subunit was previously demonstrated by targeted gene knockout of the beta1-subunit locus in mice. Knockout mice demonstrated BK channels with reduced opening in vascular smooth muscle and increased vascular tone and hypertension (4, 37). Similarly, the beta1-subunit has been shown to have an important role was in regulating bladder and colon smooth muscle tone (16, 36).

In airway smooth muscle, constriction generally occurs after receptor-coupled activation of inositol trisphosphate (IP3) receptors, which releases Ca2+ from internal Ca2+ stores. This has been termed pharmacomechanical coupling (41), in contrast to electromechanical coupling, which is mediated through voltage-dependent Ca2+ channels. The predominant role of pharmacomechanical coupling in airway smooth muscle is strongly supported by the fact that voltage-dependent Ca2+ channel blockers such as nifedipine only partially relax after cholinergically evoked constriction (14) and are poor bronchial dilators themselves (1, 25). Yet, there is considerable evidence that the control of membrane voltage by K+ channels is an important factor affecting the relative constriction of airway smooth muscle. For example, depolarization of airway smooth muscle with KCl causes constriction (47), and hyperpolarization with K+ channel agonists can relax airway smooth muscle (33). In addition, most of the effects of the most common acute treatments for asthma, the beta-adrenergic agonists, are due to activation of BK channels (24). Iberiotoxin, a highly specific BK channel blocker, reduces the bronchodilator effect of beta-adrenergic agonists (7). It has frequently been hypothesized that BK channels may serve an important role in controlling bronchial constriction and are potential pharmaceutical targets for regulation of airway smooth muscle constriction during asthma (17). Yet the role of K+ channels in general, and BK channels in particular, in Ca2+ signaling in airway smooth muscle is poorly understood. Here, we have utilized the beta1-subunit knockout to evaluate the role of the BK channel beta1-subunit in tracheal smooth muscle contraction during cholinergic signaling. We show that modulation by the beta1-subunit has effects on airway constriction and that these effects are mediated by regulation of voltage-dependent Ca2+ channels.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Tracheal constriction studies. BK channel beta1-subunit-knockout mice are congenic as a result of seven generations of inbreeding to the C57BL/6 line of Jackson Labs (strain C57BL/6J) and maintained as homozygous lines. Control animals used in these studies also were the background C57BL/6 mice strain from Jackson Labs. All animal procedures were reviewed and approved by the University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committee. For tracheal constriction studies, animals were deeply anesthetized with isoflurane and then killed by cervical dislocation. The trachea was quickly removed and then dissected clean of surrounding tissues in ice-cold normal physiological saline solution (PSS). The tracheal tube was cut below the pharynx and above the primary bronchus bifurcation. Two metal wires (attached to a force transducer and a micrometer) were threaded into the lumen of the trachea, and the trachea was placed in an oxygenated (95% O2-5% CO2, pH 7.35 at 37°C) organ bath. Resting tension was continuously readjusted to 1 g for 1 h and then challenged with high-K+ PSS solution (KCl substituted for NaCl on an equimolar basis) until a reproducible constriction response was obtained. Subsequent experimental challenges with drugs were normalized to the constriction response in the high-K+ PSS solution. In high-K+ PSS, the K+ reversal potential is depolarized; therefore, K+ currents are unlikely to play a role in controlling membrane voltage and tone. This was consistent with our finding of no significant difference in the K+-evoked constriction between wild-type and knockout mice (n = 14). Normal PSS consisted of (mM) 119 NaCl, 4.7 KCl, 2 CaCl2, 1.18 KH2PO4, 1.17 MgSO4·7H2O, 18 NaHCO3, 0.026 EDTA, 11 glucose, and 12.5 sucrose. For the high-K+ PSS, 56.7 mM NaCl was replaced with equimolar KCl and all other salts were unchanged.

Isolation of tracheal myocytes. The trachea was isolated as described above. The dorsal muscle layer was cut away from the hyaline cartilage rings and minced into ~1-mm pieces in Ca2+-free HEPES-buffered Krebs-BSA solution (140 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl, 10 mM HEPES, 10 mM glucose, and 1 mg/ml BSA fraction V, pH 7.3). After addition of 2.5 U/ml papain (MP Biomedicals) and 1 mg/ml dithiothreitol, the cells were dissociated at 37°C on a rocking platform for 20 min. The tissue was washed once with the Ca2+-free Krebs solution and digested with 12.5 U/ml of type VII collagenase (Sigma Chemical) for 10 min at 37°C. The tissue was washed three times in Ca2+-free Krebs-BSA solution and gently triturated to disburse single tracheal myocytes. Tracheal myocytes were stored on ice in Ca2+-free Krebs-BSA solution and used on the same day.

Measurement of BK channel activity. Isolated tracheal myocytes were attached to German glass coverslips (Bioindustrial Products, San Antonio, TX) for 30 min at room temperature. For inside-out excised patches, isotonic recording solutions consisted of 140 mM potassium methanesulfonate, 2 mM KCl, and 10 mM HEPES (pH 7.2). Internal (bath solution, cytosolic side of the membrane) solution included Ca2+ buffered with 5 mM hydroxyethyl-EDTA to give a final Ca2+ concentration of 7 µM, as predicted by the Maxchelator programs (32) and confirmed with a Ca2+-sensitive electrode. To reduce muscle contraction during patch clamping, the internal solution was supplemented with 20 µM cytochalasin D. External (pipette, external side of the membrane) solution was supplemented with 2 mM MgCl2. Patch-clamp pipettes were pulled to a resistance of 4–6 M{Omega}. Single channels were recorded using a patch-clamp amplifier (model EPC8, HEKA Electronics) and Pulse software and filtered at 3.3 kHz. Single-channel open probability and open channel dwell times were determined using the TAC analysis program (Bruxton). For recording of transient outward currents (whole cell mode), tracheal smooth muscle cells were plated in Krebs solution and voltage clamped with the amphotericin B perforated patch-clamp technique at 60-s time steps at a given voltage (34). Spontaneous outward currents were analyzed with a 10-pA threshold detection using the Minianalysis program (Synaptosoft Software, Leonia, NJ).

Fura 2 measurement of Ca2+. Cells were loaded with 1 µM fura 2-AM and 40 µM pluronic acid in HEPES-buffered Krebs solution at room temperature for 30 min in the dark. We found that longer periods of loading or loading at 37°C allowed loading of fura 2 into subcellular compartments. This was apparent as a larger baseline fura 2 ratio and a smaller response to cholinergic stimulation. Cells were washed free of fura 2-AM and placed in a perfusion chamber in a microscope (model TE2000S, Nikon) fitted with a phototube (Bialkali, IonOptix, Milton, MA) for assay of fluorescence intensity. Excitation wavelength was controlled with a high-speed filter changer (model DG5, Sutter Instrument, Novato, CA) using 100-ms windows for the 340- and 380-nm wavelength excitations and monitoring of the 510-nm emission at a frequency of 1 Hz during the experiment. For calibration of the fura 2 ratio to Ca2+ concentration, we used culture cells with beta-escin to make the membrane permeable to Ca2+ and EGTA-buffered Ca2+ calibration solutions purchased from Molecular Probes (Invitrogen, Carlsbad, CA).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Expression of beta1-subunit in airway smooth muscle. BK channel activity is readily detected in tracheal and bronchial smooth muscle cells by patch-clamp techniques (40). Biochemical copurification of the beta1-subunit from bovine tracheal smooth muscle in a 1:1 ratio with the pore-forming {alpha}-subunit has provided direct evidence for the accessory beta1-subunit (22, 23). We utilized the beta-galactosidase reporter that was gene targeted to the beta1-subunit translation initiation site in mice to directly determine the cell type that expresses the beta1-subunit gene in airway tissues. Reporter activity is detected in the trachea of gene-targeted animals (Fig. 1, A, C, and E) but not control animals that lack the reporter (Fig. 1, B and D). Figure 1A shows a low-magnification image of beta-galactosidase activity in the posterior wall of the trachea (region of muscle between hyaline cartilage rings). Consistent with previous reports showing that beta1-subunit gene expression is smooth muscle specific (4, 46), expression in the trachea is observed only in the smooth muscle layers (arrows in Fig. 1, C and E).


Figure 1
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  		Fig. 1. Large-conductance Ca2+-activated K+ (BK) channel beta1-subunit is expressed in airway smooth muscle tissues. Expression is assayed from a beta-galactosidase reporter gene targeted to the BK channel beta1-subunit locus in mice [knockout (KO) tissues]. A: filleted whole-mount KO trachea showing staining in tracheal smooth muscle. B: wild-type (WT) control. C: frozen section of KO trachea. D: frozen section of WT trachea. E: magnification of trachea showing staining in smooth muscle layer. Arrows, smooth muscle layers.

 
Reduced opening of BK channels in beta1-subunit-knockout smooth muscle cells. The beta1-subunit confers an increased open probability and slow gating kinetics to BK channels expressed in heterologous expression systems (11, 27, 28). Knockout of the beta1-subunit causes a dramatic reduction in BK channel openings and faster gating kinetics of a number of smooth muscle tissues (4, 10, 36). However, BK channel {alpha}-subunits in different smooth muscle tissues undergo alternative splicing (26, 44, 45), and the functional properties conferred by the beta1-subunit can be dependent on the tissue-specific splice product of the {alpha}-subunit (12, 38). We investigated the functional consequences of the lack of beta1-subunit in tracheal smooth muscle. Figure 2A shows BK single-channel activity after excision in 7 µM Ca2+-buffered internal solution. At –40 and +40 mV, wild-type BK channels show increased activity compared with knockout BK channels. As shown by composite data in Fig. 2, B and C, wild-type BK channels at +40 mV have an approximately twofold increase in open probability and a twofold increase in open channel dwell time compared with BK channels lacking beta1-subunits: 0.83 ± 0.06 vs. 0.44 ± 0.1 open probability and 5 ± 1.7 vs. 2.7 ± 0.7 ms open channel dwell time. In tracheal smooth muscle, membrane potentials generally do not overshoot 0 mV, and cholinergic signaling alters membrane potentials within negative voltage ranges (19). At –40 mV, open probability in the beta1-subunit knockout was reduced >10-fold compared with wild-type cells: 2.4 ± 0.76e–2 vs. 0.17 ± 0.07e–2 (P < 0.05; Fig. 2B). These results indicate that BK channel opening is severely perturbed in the beta1-subunit-knockout at physiological voltages. The mechanism by which BK channels increase open probability is an increase in the open channel burst duration (30). As expected, channel burst durations are reduced in the beta1-subunit knockout compared with wild-type BK channels: 2.1 ± 0.6 vs. 0.54 ± 0.08 ms at –40 mV (P < 0.05; Fig. 2C).


Figure 2
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  		Fig. 2. KO of beta1-subunit reduces BK channel opening probability (Po). A: single-channel recording of BK channels from WT and KO tracheal smooth muscle cells assayed in excised patch, inside-out configuration (internal solution was 7 µM buffered Ca2+). C, closed; O, open. B: summary data showing Po. C: open dwell times for WT and KO. Scale bar, 1 s for –40 mV and 0.1 s for +40 mV. Values are means ± SE (n = 6 WT and 8 KO). *P < 0.05.

 
Physiological changes in BK channel activity can be assessed by measurement of spontaneous transient outward currents (STOCs) in nondialyzed cells by perforated patch-clamp recording (34). Transient outward currents are mediated by BK currents after Ca2+-induced Ca2+ release events ("Ca2+ sparks"), which colocalize with, and activate, BK channels (18). Gene knockout of the pore-forming {alpha}-subunit has been shown to eliminate transient outward currents in bladder and vascular smooth muscle (29, 39), whereas knockout of the beta1-subunit does not eliminate, but reduces the frequency of, spontaneous outward currents in vascular smooth muscle (4, 37). In tracheal smooth muscle, we see a similar effect. At –40 mV, BK current activity is significant in wild-type cells (6 of 6 cells at –40 mV; Fig. 3A), whereas beta1-subunit-knockout smooth muscle cells show no or very little activity (0 of 5 cells at –20 mV; Fig. 3B). Nevertheless, as in the single-channel measurements (Fig. 2B), given sufficient depolarization, beta1-subunit-knockout airway smooth muscle demonstrates spontaneous transient BK currents (Fig. 3A), although at a lower frequency. Composite data for frequency are shown in Fig. 3B: over the whole range of holding potentials, the knockout cells exhibited significantly lower transient BK currents: 487 ± 112 vs. 113 ± 29 STOCs/min at +20 mV (P < 0.01).


Figure 3
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  		Fig. 3. Spontaneous transient outward currents (STOCs) in isolated tracheal smooth muscle cells. A: spontaneous transient BK current in WT and KO airway smooth muscle cells. B: summary of STOC frequency. C: summary of STOC amplitude. D: summary of STOC area. Values are means ± SE; n = 6–8 cells for WT and beta1-subunit KO. At –40 mV, 7 of 7 KO cells showed no STOCs, whereas 3 of 8 WT cells showed STOCs. ***P < 0.001; **P < 0.01; *P < 0.05.

 
The effects of the beta1-subunit knockout on STOC amplitudes and areas (Fig. 3, C and D) were less dramatic than the effects on frequency (Fig. 3B). Given that the STOC amplitude should represent the number of channels opening after a spark event (50), these data suggest that beta1-subunit knockout causes a somewhat smaller recruitment of BK channels during a Ca2+ spark event (Fig. 3C). In addition, wild-type tracheal BK channels appear to have a tendency toward more sustained opening, as indicated by a greater STOC area (Fig. 3D). This is consistent with the longer open dwell times of wild-type BK channels (Fig. 2C). In summary, these results indicate that BK channel activity is significantly greater in wild-type than in beta1-subunit-knockout trachea and, therefore, would be expected to play a greater role at physiological voltages in controlling Ca2+ influx and constriction.

Knockout of the beta1-subunit increases resting Ca2+ and cholinergically evoked Ca2+ release. In airway smooth muscle, Ca2+ release via IP3 receptors initiates contraction. After Ca2+ release, contraction is maintained by Ca2+ influx channels (3). Acutely isolated tracheal smooth muscle cells were loaded with fura 2 for determination of the influence of the beta1-subunit on Ca2+ influx. In tracheal smooth muscle, we found an ~50 nM increase in resting Ca2+ levels in the beta1-subunit knockout: 177 ± 11 vs. 124 ± 0.8 nM (P < 0.01; Fig. 4A, inset). This indicates that BK channels contribute to regulation of basal Ca2+.


Figure 4
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  		Fig. 4. Carbachol (CCh)-induced Ca2+ transients in WT and KO tracheal smooth muscle cells. A: fura 2-measured Ca2+ transients from WT ({circ}) and KO (bullet) tracheal smooth muscle cells. Left inset: enlargement of non-baseline-subtracted resting Ca2+ (note nM concentration on left and right inset y-axes). Large trace and right inset KO traces are baseline subtracted to adjust KO to the same baseline as WT. Components of Ca2+ are labeled as follows: resting Ca2+ (A), transient phase (B), early sustained phase (C), and late sustained phase (D). B: fura 2-measured Ca2+ transients from KO tracheal smooth muscle cells with oscillatory Ca2+ changes. C: summary data comparing average values for different components in WT and KO. Significant difference is seen at resting Ca2+ (component A, **P = 0.0004, n = 28 WT and 24 KO) and during sustained phase (component D, P = 0.0062, n = 14 WT and 12 KO). Sustained Ca2+ phases (components C and D) were measured at 15–17 and 77–80 s, respectively, after addition of carbachol. Cells with oscillatory Ca2+ transients were excluded from these analysis.

 
During cholinergically evoked excitation, Ca2+ increases from an early phase, which is mainly contributed by Ca2+ release from the sarcoplasmic reticulum (Ca2+ transient; Fig. 4A, component B), to a late sustained phase, which underlies sustained Ca2+ influx [Fig. 4A, components C (early sustained) and D (late sustained)]. Because the beta1-subunit knockouts have an increased baseline Ca2+, we quantified the relative Ca2+ increases (subtracted from resting Ca2+) at different phases of the Ca2+ transient to determine the changes in evoked Ca2+. In addition to the peak transient, we averaged the sustained component 15–17 s (component C) and 77–80 s (component D) after addition of carbachol (see METHODS) in anticipation that the beta1-subunit knockout may have different effects on the components of the Ca2+ transients. The major effect of the beta1-subunit knockout is an increase in the late sustained component of the Ca2+ influx (component D, 131 ± 16 and 77.2 ± 12 nM Ca2+ in knockout and wild-type cells, respectively, P < 0.005), with no significant effect on the Ca2+ transient and early sustained component (components B and C). In addition, although there was no statistical difference between early sustained components (component C), we saw a greater tendency for Ca2+ oscillations in the beta1-subunit knockout than in wild-type cells (12 of 26 vs. 3 of 29 cells; Fig. 4B). Because very few wild-type cells exhibited Ca2+ oscillations, it would not be meaningful to quantify Ca2+ differences between knockout oscillating cells and wild-type cells. Therefore, the oscillating cells were not included in further analysis.

Because a major function of K+ channels is control of membrane voltage, a reasonable assumption is that increased Ca2+ in the beta1-subunit knockouts arises from depolarization and recruitment of voltage-sensitive Ca2+ channels. Therefore, we utilized the L-type voltage-dependent Ca2+ channel blocker nifedipine (1 µM) to study the contribution of these channels to each component of cholinergically evoked Ca2+ transients. Indeed, nifedipine caused a substantial drop in all components of the Ca2+ transient (Fig. 5, A and B) in wild-type and knockout cells. Figure 5C shows that the average response of all components is similar between wild-type and knockout muscle blocked with nifedipine. Particularly important was the observation that nifedipine eliminated the difference in the late sustained Ca2+ influx (component D) between wild-type and beta1-subunit-knockout cells (P = 0.22, wild-type vs. knockout; Figs. 4B and 5C). This can also be seen when the relative differences in Ca2+ evoked by carbachol before and after block with nifedipine are measured (Fig. 5, A and B; data are paired with the same tracheal cell). Consistent with these results, knockouts show a larger nifedipine-sensitive late sustained component than wild-type trachea cells: 48 ± 5 vs. 120 ± 33 nM (P < 0.02; Fig. 5D). A similar result was obtained with perfusion of 0 extracellular Ca2+, achieved with addition of the Ca2+ chelator EGTA at 1 mM (Fig. 6, A and B). As in nifedipine experiments, the significant difference between wild-type and knockout Ca2+ transients during the late sustained component in 2 mM Ca2+ solutions (Fig. 4B) was eliminated with 0 Ca2+: 46 ± 13 and 41 ± 10 nM in wild-type and knockout, respectively (Fig. 6C). The largest change in Ca2+ mediated by the beta1-subunit is seen during the late sustained component: 43 ± 11 and 93 ± 16 nM in wild-type and knockout, respectively (Fig. 6D). In summary, these results indicate that increased Ca2+ in the knockout trachea can be accounted for by increased activation of voltage-dependent Ca2+ channels. Also, although the beta1-subunit knockout showed an increased frequency of cells with Ca2+ oscillations, block of voltage-dependent Ca2+ channels or use of 0 Ca2+ solutions did not eliminate Ca2+ oscillations (data not shown).


Figure 5
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  		Fig. 5. Effect of nifedipine on carbachol-induced Ca2+ transients. A and B: traces of carbachol-induced Ca2+ transients before ({circ}) and after nifedipine block of voltage-gated Ca2+ channels (bullet) for WT and beta1-subunit-KO cells. C: summary data comparing average values for different components in WT (open bars) and KO (gray bars) during Ca2+ channel block with nifedipine. No significant difference is seen with any component. D: summary of relative change of different components calculated as Ca2+ concentration after nifedipine subtracted from Ca2+ concentration before nifedipine ({Delta}[Ca2+]). Values are means ± SE from paired experiments (n = 13 WT and 11 KO). *P = 0.025.

 

Figure 6
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  		Fig. 6. Effect of Ca2+ removal on carbachol-induced Ca2+ transients. A and B: traces of carbachol-induced Ca2+ transients before ({circ}) and after (bullet) addition of 0 Ca2+, 1 mM EGTA-Krebs solution for WT and beta1-subunit KO cells. C: summary data comparing average values for different components in WT and KO during cholinergically evoked Ca2+ in 0 external Ca2+. No significant difference is seen at any component. D: summary of relative change of different components after removal of extracellular Ca2+. Values are means ± SE (n = 14 WT and 12 KO). **P = 0.028.

 
BK channel beta1-subunit knockout increases tracheal constriction. Figure 7A shows cholinergically evoked constrictions normalized to tracheal responses in high-K+ conditions. Consistent with the expectation that BK channels control airway constriction, the beta1-subunit-knockout mice have a larger carbachol-induced constriction than the wild-type animals. Summary dose-response curves (Fig. 7B) indicate that the main effect of the beta1-subunit knockout is an increase in the maximal response, rather than a shift in the dose-response, curve. Although some knockouts showed Ca2+ oscillations, we did not see oscillation in carbachol-induced constrictions.


Figure 7
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  		Fig. 7. Greater cholinergically evoked contraction of trachea in beta1-subunit KO cells. A: carbachol-evoked contraction before and after 1 µM nifedipine. Contraction traces are normalized to high-K+-induced contractions. B: cumulative dose-response curve to carbachol. Last dose (3 x 10–5 carbachol) is repeated as previous dose with 1 µM nifedipine. *P < 0.05 for 2 x 10–7 to 1 x 10–5 carbachol. Values are means ± SE (n = 14). C: effect of BK channel block with 5 µM paxillin in wild-type trachea. D: constriction to 1 µM carbachol without (dark trace) and with 5 µM paxillin block of BK channels in WT and KO tracheas. E: summary data for C and D.

 
Increased constriction of beta1-subunit knockouts is accounted for by increased contribution of voltage-dependent Ca2+ channels during sustained contractions. The most likely role of BK channels is opposition of membrane depolarization during agonist-induced constriction. A reasonable hypothesis is that membrane depolarization recruits voltage-dependent Ca2+ channels and increases Ca2+ influx and constriction. Consistent with this hypothesis, we found that the beta1-subunit-knockout mice relax to the same relative constriction as wild-type animals when treated with nifedipine [Fig. 7A, right, and Fig. 7B (dose-response + nifedipine)]. This indicates that the increased constriction of the knockout mice is largely accounted for by increased Ca2+ influx through L-type voltage-dependent Ca2+ channels.

It is fairly well established that two sources of Ca2+ underlie tracheal constriction: an early transient component, which is dependent on agonist-induced Ca2+ release from endoplasmic reticulum Ca2+ stores, and a subsequent sustained component, which is dependent on Ca2+ influx from plasma membrane channels. Consistent with the nifedipine experiments described above (Fig. 5), the beta1-subunit knockouts show the greatest effect on constriction during the sustained component, where voltage-dependent Ca2+ channels are expected to play a role (Fig. 7A). Nifedipine block of voltage-dependent Ca2+ channels results in similar constriction during the sustained component in knockout and wild-type cells (Fig. 7, A and B).

In vascular smooth muscle, block of BK channels results in similar levels of constriction in wild-type and beta1-subunit-knockout cells (4). This indicates that, without the beta1-subunit, BK channels cannot control constriction. In tracheal smooth muscle, the effect was similar. There was no significant difference in cholinergically evoked constriction in knockout trachea in the presence or absence of paxilline block (Fig. 7, D and E), indicating that BK channel activity does not significantly contribute to relaxation in the absence of the beta1-subunit. In contrast, wild-type trachea shows a significant difference with and without paxilline block (Fig. 7, C and E).


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Airway smooth muscle presents a medically important model for studying the role of ion channels during receptor-coupled activation of Ca2+ transients. In contrast to vascular smooth muscle, where the voltage-dependent Ca2+ channel is a major Ca2+ conduit mediating contraction, the role of voltage-dependent Ca2+ channels and other Ca2+ influx pathways in airway smooth muscle is poorly defined. Therefore, pharmaceuticals targeted to ion channels in the airway have not been utilized to treat asthma.

These data demonstrate that BK channels in beta1-subunit-knockout airway smooth muscle exhibit a large reduction in channel openings as indicated by single-channel activity and STOC frequency. As a consequence, the BK channel contribution to relaxation is perturbed sufficiently that pharmacological block of BK channels shows that same relative constriction as the beta1-subunit-knockout trachea (Fig. 7, C–E). This is similar to vascular smooth muscle, in which iberiotoxin-induced block of wild-type vessels induced constriction but had no further constrictive effect on beta1-subunit-knockout vessels (4). This is in contrast to bladder smooth muscle, where the beta1-subunit knockout shows a partial effect on constriction compared with iberiotoxin block (36). A distinction between tonic smooth muscles, such as vascular and tracheal muscle, and bladder smooth muscle, which is phasic in excitation, is that large depolarizations in bladder smooth muscle are likely to complement BK activation in the beta1-subunit knockout. Indeed, we see much smaller differences in single-channel and STOC activity of wild-type and knockout cells at depolarizing voltages.

Tao et al. (43) demonstrated that cholinergically evoked constriction in bovine airway is largely independent of voltage-activated Ca2+ channels unless sarcoplasmic reticulum Ca2+ stores are depleted or BK channels are blocked. In the latter case, the mode of contraction coupling switches to an excitation, voltage-dependent coupling. Our findings utilizing the beta1-subunit knockout in mouse trachea indicate more of a partial contribution, rather than a switch in coupling, between pharmacologically evoked and excitation-evoked contraction. In wild-type mice, voltage-dependent Ca2+ channels appear to contribute ~17% of the carbachol-induced contraction at saturating carbachol doses (Fig. 7B). In the absence of the beta1-subunit, BK channel activity decreases and the contribution of voltage-dependent Ca2+ channels increases to ~30% of the constriction (Fig. 7B), which is smaller than the contribution of beta1-subunits in aortic smooth muscle. Pluger et al. (37) reported that maximal contraction is increased ~65% by the beta1-subunit knockout. Thus BK channels make a significant, but smaller, contribution to agonist-induced relaxation in tracheal smooth muscle than in vascular muscle.

The Ca2+ pathways downstream of voltage-dependent Ca2+ channels that are regulated by BK channels are likely to be complex. The beta1-subunit knockout increased resting Ca2+ levels and increased the sustained component of Ca2+ influx during cholinergic evoked signaling. These consequences were translated to increased cholinergically evoked constriction. A conclusion that may be drawn is that the regulation of membrane voltage by the BK channel is important, although less apparent, until these channels are blocked by deletion of the beta1-subunit. The hyperpolarizing activity of BK channels is likely to hold membrane voltage during cholinergic contractions below the operational voltages at which voltage-dependent Ca2+ channels and excitation-contraction coupling contribute to airway constriction (3). This may reconcile the fact that voltage-dependent Ca2+ channels are regarded to provide a minority of the Ca2+ for contraction (19), yet the K+ channels would have an important role such as that observed during beta-adrenergic-mediated relaxation.

An interesting finding was the high frequency of oscillatory Ca2+ changes after cholinergic activation in cells from beta1-subunit-knockout mice (Fig. 4B). However, we did not see a similar effect on constriction, which was entirely tonic in response to high K+ or cholinergically evoked constriction. Thus it is unclear how the beta1-subunit knockout-induced Ca2+ oscillations influence the muscle contractile properties in the trachea. In other examples, perturbing BK channels promote oscillations in the trachea. Oscillatory contractions have been described for guinea pig tracheal smooth muscle cells treated with the BK channel blockers charybdotoxin and iberiotoxin (48, 49). However, these studies did not investigate Ca2+ changes that may underlie the oscillatory contractions. In more recent studies in bronchioles, slow oscillatory Ca2+ transients have been observed in response to depolarization with K+ (35). This may be considered analogous to BK channel block or the beta1-subunit knockout, because blocking of K+ channels presumably depolarizes the cell. However, slow Ca2+ oscillations were correlated with twitching, rather than constriction, of the airway (35).

Because the Ca2+ oscillations are not affected by nifedipine or 0 external Ca2+, the beta1-subunit may have other effects in addition to their actions on membrane voltage or Ca2+ influx through plasmalemma channels. The increase in resting Ca2+ concentration in the beta1-subunit knockout could play a role. Increases in global Ca2+ have been shown to promote sarcoplasmic reticulum Ca2+ loading and, thereby, sustain Ca2+ release (5). As well, increases in cytosolic Ca2+ concentrations and sarcoplasmic reticulum Ca2+ stores promote IP3 receptor activation and Ca2+ oscillations (2, 13). Indeed, the mechanism by which knockout of the beta1-subunit causes Ca2+ oscillations and perhaps affects other currents requires further study.

In summary, these findings show that tracheal smooth muscle requires the accessory beta1-subunit for promotion of normal BK channel activation. Despite the predominant role of agonist-induced signaling in the trachea, BK channels have a significant role in controlling resting Ca2+ and cholinergically evoked Ca2+ influx contributed by voltage-dependent Ca2+ channels.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by the Sandler Program for Asthma Research (R. Brenner and J. T. Herlihy) and American Heart Association Grant 0335007N (R. Brenner).


   ACKNOWLEDGMENTS
 
We thank Dr. Qing H. Chen and David Petrik for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Brenner, Dept. of Physiology, Univ. of Texas Health Science Center San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229 (e-mail: brennerr{at}uthscsa.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.


   REFERENCES
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