constriction in tracheal smooth muscle

Semenov, et al. 1

BK Channel β1 Subunit Regulation of Calcium Handling and

Constriction in Tracheal Smooth Muscle

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

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

San Antonio, Texas 78229.

Running Title: BK Channel β1 and Airway

Correspondence to:

Robert Brenner, Ph.D.Department of PhysiologyUniversity of Texas Health Science Center San Antonio7703 Floyd Curl DriveSan Antonio, Texas 78229Phone: (210)-567-4343Fax: (210)-567-4410E-mail: [email protected],

of 36Articles in PresS. Am J Physiol Lung Cell Mol Physiol (April 21, 2006). doi:10.1152/ajplung.00104.2006

Copyright © 2006 by the American Physiological Society.

Semenov, et al. 2

ABSTRACT

The large conductance, calcium-activated (BK-type) potassium channels are

regulators of voltage-dependent calcium entry in many cell types. The BK channel

accessory β1 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 β1 subunit had

not been investigated. We have investigated the role of the β1 subunit in tracheal smooth

muscle by utilizing the gene targeted mice for the β1 subunit gene. Mice containing the

β1 knockout allele had significantly reduced BK channel activity in excised tracheal

smooth muscle patches, and had reduced spontaneous BK currents in whole tracheal

smooth muscle cells. The consequence of the β1 knockout was an increase in resting

calcium levels, and an increase in the sustained component of calcium influx following

cholinergic signaling. Tracheal constriction studies demonstrate that the β1 knockout

have the relatively same level of constriction as compared to BK channel block with

paxilline, indicating that BK channels contribute little to airway relaxation in the absence

of the β1 subunit. Utilizing nifedipine, we found that the increase constriction caused by

the β1 knockout could be accounted for by an increased recruitment of L-type voltage-

dependent calcium channels. These results indicate that the β1 subunit is required in

airway smooth muscle for BK channels to control voltage-dependent calcium influx

during rest and following cholinergic signaling.

KEYWORDS: BK Channels, Airway, β1 Subunit

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INTRODUCTION

In many smooth muscle cells, voltage-dependent calcium channels are the major

conduits for calcium mediating constriction (21). Potassium channels generally function

to oppose constriction by hyperpolarizing membranes, which deactivates voltage-

dependent calcium channels and reduces calcium influx. Among these, the large-

conductance (BK-type) calcium activated potassium 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 calcium and

depolarizing voltage gate BK channels to open. When open, BK channels have a very

large outward potassium conductance (>200 pS) and are therefore very effective in

hyperpolarizing the membrane. As such, they are important regulators of membrane

voltage in a number of cell types (6, 15, 20).

BK channels bind calcium at low affinity (around 10 µM) (9, 28) and require

colocalization with a calcium source or the contribution of voltage to open the channel at

physiological calcium concentrations (8, 30, 31). In vascular smooth muscle, large

voltage changes do not occur, and BK channels have an enhanced apparent calcium

sensitivity that is conferred by association with the one member of the accessory BK

channel beta subunit family (β1−β4 subunits), the β1 subunit (23, 42). Previously, the

important role of the β1 subunit was demonstrated utilizing targeted gene knockout of the

β1 locus in mice. Knockout mice demonstrated BK channels with reduced opening in

vascular smooth muscle, and increased vascular tone and essential hypertension (4, 37).

Similarly, the β1 subunit has been shown to have an important role was in regulating

bladder and colon smooth muscle tone (16, 36).

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Semenov, et al. 4

In airway smooth muscle (ASM) constriction generally occurs following receptor-

coupled activation of IP3 receptors, which releases calcium from internal calcium stores.

This has been termed pharmacomechanical coupling (41), in contrast to the

electromechanical coupling that is mediated through voltage-dependent calcium

channels. The predominant role of pharmacomechanical coupling in ASM is strongly

supported by the fact that voltage-dependent calcium channel blockers such as nifedipine

only partially relax following cholinergic evoked constriction (14) and are poor bronchial

dilators themselves (1, 25). Yet, there is considerable evidence that the control of

membrane voltage by potassium channels is an important factor affecting the relative

constriction of ASM. For example, depolarization of ASM with potassium chloride

causes constriction (47) and hyperpolarization with potassium channel agonists can relax

ASM (33). In addition, the most common acute treatments for asthma, the β–adrenergic

agonists confer much of their effects through activation of large conductance calcium-

activated (BK-type) potassium channels (24). Iberiotoxin, a highly specific BK channel

blocker reduces the bronchial dilatory effect of β-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 regulating

ASM constriction during asthma (17). Yet the role of potassium channels in general, and

BK channels specifically in calcium signaling in ASM is poorly understood. Here, we

have utilized the β1 knockout to evaluate the role of BK channel β1 subunit in tracheal

constriction smooth muscle contraction during cholinergic signaling. We show that

modulation by the β1 subunit does have effects on airway constriction, and these effects

are mediated by regulation of voltage-dependent calcium channels.

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Semenov, et al. 5

METHODS

Tracheal Constriction Studies

The BK channel β1 subunit knockout mice are congenic by 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. For tracheal constriction studies, animals were

deeply anesthetized with isoflurane and then sacrificed with cervical dislocation. Trachea

were quickly removed and then dissected clean of surrounding tissues in ice-cold normal

physiological saline solution (PSS) according to compositions given below. The tracheal

tube was cut below the pharynx and above the primary bronchus bifurcation. Two metal

wires (attached to a force transducer and micrometer) were threaded into the lumen of the

trachea and the trachea was placed into an oxygenated (95% oxygen, 5% CO2, pH =

7.35), at 37oC organ bath. Resting tension was continuously readjusted to 1 gram for one

hour and then challenged with high potassium 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 potassium PSS solution. In high potassium PSS, the potassium reversal potential is

depolarized and therefore potassium currents are unlikely to play a role in controlling

membrane voltage and tone. This was consistent with our finding that there was no

significant difference in the potassium evoked constriction between wild type and

knockout mice (N= 14). Normal PSS used was (mM) 119 NaCl, 4.7 KCl, 2 CaCl2, 1.18

KH2PO4, 1.17 MgSO4*7H20, 18 NaHCO3, 0.026 EDTA, 11 glucose, and 12.5 sucrose.

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Semenov, et al. 6

The high potassium PSS substituted 56.7 mM NaCl with equimolar KCl, all other salts

were unchanged.

Tracheal Myocytes Isolation

Tracheas were isolated as described above. The dorsal muscle layer was cut away

from the hyaline cartiladge rings and minced into approximately 1 mm pieces in calcium-

free Hepes buffered Krebs/BSA solution (in mM: 140 NaCl, 4.7 KCl, 1.13 MgCl, 10

Hepes, 10 glucose, 1 mg/ml bovine serum albumin fraction V, pH 7.3). After 2.5

units/ml papain (MP Biomedicals) and 1 mg/ml dithiothreitol were added to cells, cells

were dissociated at 37oC on a rocking platform for 20 minutes. The tissue was washed

once with the calcium-free Krebs solution and digested with 12.5 units/ml of type VII

collagenase (Sigma Chemical) for 10 minutes at 37oC. The tissue was washed 3X in

calcium-free Krebs, BSA and gently triturated to disburse single tracheal myocytes.

Tracheal myocytes were stored on ice in calcium-free Krebs/BSA and used the same day.

Measurement of BK Channel Activity.

Isolated tracheal myocytes were attached to german glass coverslips

(Bioindustrial Products, San Antonio, Texas) for 30 minutes at room temperature. For

inside/out excised patches, isotonic recording solutions consisted of 140 mM K-

Methanesulfonate, 2 mM KCl and 10 mM Hepes (pH 7.2). Internal solutions (bath

solution, cytosolic side of membrane) included calcium buffered with 5 mM HEDTA to

give a final calcium concentrations of 7 µM as predicted by the Maxchelator programs

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Semenov, et al. 7

(32), and confirmed with a calcium sensitive electrode. To reduce contraction of muscle

during patch clamping the internal solution was supplemented with 20 micromolar

cytochalasin D. External (pipet, external side of membrane) solution was supplemented

with 2 mM MgCl2. Patch clamp pipets were pulled to resistance of 4 to 6 megaohm.

Single channels were recorded using a HEKA electronics EPC8 patch clamp amplifier

and Pulse software and filtered at 3.3 KHz. Single channel open probability and open

channel dwell times was determined using the TAC analysis program (Bruxton Corp).

For recording of transient outward currents (whole cell mode), tracheal smooth muscle

cells were plated in Krebs solution and voltage clamped with the amphoterecin B

perforated patch clamp technique for 60 seconds 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 calcium.

Cells were loaded with 1 µM Fura-2 AM ester and 40 micromolar pluronic acid in

Hepes buffered Krebs solution. The loading was at room temperature for 30 minutes in

the dark. We found that loading for longer incubation times or at 37oC allowed the Fura-2

to load into subcellular compartments. This was apparent as a larger baseline Fura-2 ratio

and smaller response to cholinergic stimulation. Cells were washed free of Fura-2 AM

and placed into perfusion chamber in a Nikon TE2000S (Nikon USA) microscope fitted

with an Ion Optix Bialkali phototube (IonOptix Corporation, Milton, MA) to assay

fluorescence intensity. Excitation wavelength was controlled with a Sutter Instruments

DG5 high speed filter changer (Sutter Instrument Company, Novato, CA) using 100 ms

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Semenov, et al. 8

windows for the 340 and 380 wavelength excitations, and monitoring the 510 nM

emission at a frequency of 1 Hz during the experiment. The Fura-2 ratio was calibrated to

calcium concentration using culture cells with β-escin to make the membrane permeable

to calcium, and EGTA buffered calcium calibration solutions purchased from Molecular

Probes (Invitrogen Corporation, Carlsbad, CA).

RESULTS

β1 Subunit Expression in Airway Smooth Muscle

BK channel activity is readily detected in both tracheal and bronchial smooth

muscle cells utilizing patch clamp techniques (40). Direct evidence for the accessory β1

subunit has been shown by biochemical copurification of the β1 subunit from bovine

tracheal smooth muscle in a 1:1 ratio with the pore-forming alpha subunit (22, 23). We

utilizing the β-galactosidase reporter that was gene targeted to the β1 subunit translation

initiation site in mice to directly determine the cell type that expresses the β1 gene in

airway tissues. Reporter activity is detected in trachea in the gene targeted animals

(Figure 1A, 1C, 1E), but not in control animals that lack the reporter (Figure 1B, 1D)

Figure 1A shows low magnification picture of β−galactosidase activity in the posterior

wall of the trachea (region of muscle between hyaline cartiledge rings). Consistent with

previous reports showing that β1 gene expression is smooth muscle-specific (4, 46),

expression in trachea is observed only in the smooth muscle layers (arrows in Figure 1C

and 1E).

BK Channels have a reduced opening in β1 knockout smooth muscle cells.

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The β1 subunit confers an increased open probability and slow gating kinetics to

BK channels expressed in heterologous expression systems (11, 27, 28). The knockout of

the β1 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 channels alpha

subunits in different smooth muscle tissues undergo alternative splicing (26, 44, 45) and

the functional properties conferred by the β1 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 β1 subunit in tracheal smooth muscle. Figure 2A shows BK

single channel activity after excision in 7 micromolar calcium buffered internal solution.

At both –40 and +40 mV, wild type BK channels show increased activity over knockout

BK channels. As composite data in Figure 2B and Figure 2C show, wild type BK

channels at +40 mV have an approximately two fold increase in open probability and two

fold increase in open channel dwell time, respectively, as compared to BK channels

lacking β1 subunits (WT Po = 0.83 ± 0.06 vs. KO Po = 0.44 ± 0.1; WT open dwell time =

5 ± 1.7 ms vs. KO dwell time = 2.7 ± 0.7 ms). In tracheal smooth muscle, membrane

potentials generally do not overshoot 0 mV, and cholinergic signaling alters membrane

potentials within negative voltages ranges (19). At –40 mV we see a greater than 10 fold

reduction in open probability in the β1 knockout as compared to wild type cells (Figure

2B, WT = 2.4e-2 ± 0.76e-2 vs. KO = 0.17e-2 ± 0.07e-2, P < 0.05). These results indicate

that BK channel opening is severely perturbed in the β1 knockout at physiological

voltages. The mechanism by which BK channels increases open probability is by

increasing the open channel burst duration (30). As expected, channel burst durations in

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the β1 knockout are reduced as compared to wild type BK channels (Figure 2C, -40 mV

WT = 2.1 ± 0.6 ms vs. KO = 0.54 ± 0.08 ms, P < 0.05).

Physiological changes in BK channel activity can be assessed by measuring

spontaneous transient outward currents (STOC) in non-dialyzed cells using perforated

patch clamp recording (34). Transient outward currents are mediated by BK currents

following calcium-induced calcium release events (so called “calcium sparks”), that

colocalize with, and activate, BK channels (18). Gene knockout of the pore-forming α

subunit has been shown to eliminate transient outward currents in bladder and vascular

smooth muscle (29, 39). Whereas in the knockout of the β1 subunit, spontaneous outward

currents are not absent but reduced in frequency in vascular smooth muscle (4, 37). In

tracheal smooth muscle, we see a similar effect. Figure 3A shows that at -40 mV, wild

type cells (lowest trace) have significant BK current activity (seen in 6/6 cells at –40

mV), whereas β1 knockout smooth muscle cells (upper trace) show no (0/5 cells) or very

little activity (-20 mV, see Figure 3B). Nevertheless, as in the single channel

measurements (Figure 2B), given sufficient depolarization β1 knockout airway smooth

muscle demonstrate spontaneous transient BK currents (Figure 3A), albeit at a lower

frequency. Figure 3B depicts composite data for frequency; over the whole range of

holding potentials the knockout cells exhibited significantly lower transient BK currents

(Figure 3B, WT = 487 ± 112 STOC/min vs. KO = 113 ± 29 STOC/min at +20 mV, P <

0.01).

The effects of the β1 knockout on STOC amplitudes and areas (Figure 3C and

3D) were less dramatic than that seen with frequency (Figure 3B). Given that the STOC

amplitude should represents the number of channels opening following a spark event

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(50), these data suggest that the β1 knockout causes a somewhat smaller recruitment of

BK channels during a calcium spark event (Figure 3C). In addition, wild type tracheal

BK channels appear to have a tendency towards more sustained opening as indicated by a

greater STOC area (Figure 3D). This is consistent with the longer open dwell times of

wild type BK channels (Figure 2C). In summary, these results indicate that BK channel

activity is significantly greater in wild type trachea relative to β1 knockout trachea, and

therefore would be expected to play a greater role at physiological voltages in controlling

calcium influx and constriction.

Knockout of the β1 subunit increases resting calcium and cholinergic evoked

calcium release.

In airway smooth muscle, calcium release via IP3 receptors initiates contraction.

Following calcium release, contraction is maintained by calcium influx channels (3).

Acutely isolated tracheal smooth muscle cells were loaded with Fura-2 to determine the

influence of the β1 subunit on calcium influx. We found that tracheal smooth muscle

have an approximately 50 nM increase in resting calcium levels in the β1 knockout (Inset

A in Figure 4A, 177 ± 11 nM in KO vs. 124 ± 0.8 nM in WT, P < 0.01). This indicates

that BK channels contribute to regulation of basal calcium.

During cholinergic evoked excitation, calcium increases from an early phase that

is mainly contributed by calcium release from the SR (calcium transient, B in Figure 4A)

and late sustained components that underlie sustained calcium influx (C, early sustained,

and D, late sustained, in Figure 4A). Because the β1 knockouts have an increased

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baseline calcium, we quantified the relative calcium increases (subtracted from resting

calcium) at different phases of the calcium transient to determine the changes in evoked

calcium. Example traces where baseline calcium is normalized is shown in Figure 4A and

data is summarized in Figure 4B. In addition to the peak transient, we averaged the

sustained component between 15-17 seconds (component C) and 77-80 seconds

(component D) after carbachol addition (see methods for more detailed description) in

anticipation that the β1 knockout may have different effects of the components of the

calcium transients. The major effect of the β1 knockout is to increase the late sustained

component of the calcium influx (component D, KO = 131 ± 16 nM [Ca2+] vs. WT =

77.2 nM ± 12 nM [Ca2+], P < 0.005), with no significant effect on the calcium transient

and early sustained component (Figure 4A and 4C). In addition, although there was no

statistical difference between early sustained components (component C), we saw a

tendency for the β1 knockout to exhibit calcium oscillations much more frequently than

wild type cells (example in Figure 4B, Oscillations in KO = 12/26 cells vs. WT = 3/29

cells). Because very few wild type cells exhibited calcium oscillations, it would not be

meaningful to quantify calcium differences between knockout oscillating cells and wild

type cells. Therefore the oscillating cells were not included in further analysis.

Since a major function of potassium channels is to control membrane voltage, a

reasonable assumption is that increased calcium in the β1 knockouts arises from

depolarization and recruitment of voltage sensitive calcium channels. To these ends, we

utilized the L-type voltage-dependent calcium channel blocker, nifedipine (1 µM), to

observe the contribution of these channels to each component of cholinergic evoked

calcium transients. Indeed, nifedipine caused a substantial drop in all components of the

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calcium transient (Figure 5A and 5B) in both wild type and knockout cells. Figure 5C

summary shows that the average response of all components are similar between wild

type and knockout muscle blocked with nifedipine. Particularly important was the

observation that nifedipine eliminated the difference in the late sustained calcium influx

(component D) observed between wild type and β1 knockout cells (summary in Figure

4B, nifedipine and Figure 5C, P = 0.22 WT vs. KO). This can also be seen when

measuring the relative difference in calcium evoked by carbachol before and after block

with nifedipine (Figure 5A and 5B, data are paired with the same tracheal cell). The

relative changes are summarized in panel 5D. Consistent with the above results,

knockouts show a larger nifedipine sensitive late sustained component than wild type

trachea cells (Figure 5D, WT = 48 nM ± 5 vs. KO = 120 nM ± 33, P < 0.02). A similar

result was obtained with perfusion of zero extracellular calcium (with 1 mM EGTA

calcium chelator) (Figure 6A and 6B). As in nifedipine experiments, the significant

difference between wild type and knockout calcium transients during late sustained

component seen in 2 mM calcium solutions (Figure 4B) was eliminated with zero

calcium (Figure 6C, WT = 46 nM ± 13 vs. KO = 41 nM ± 10). As well, the largest

change in calcium mediated by the β1 subunit is seen during the late sustained

component (Figure 6D, WT = 43 nM ± 11 vs. KO = 93 nM ± 16 ). In summary, these

results indicate that increased calcium in the knockout trachea can be accounted for by

increased activation of voltage-dependent calcium channels. It was also observed that

although the β1 knockout showed an increased frequency of cells showing calcium

oscillations, block of voltage-dependent calcium channels or 0 calcium solutions did not

eliminate calcium oscillations from cells (data not shown).

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Semenov, et al. 14

BK Channel β1 Knockout Causes Increased Constriction of Trachea

Figure 7A shows cholinergic evoked constrictions normalized to trachea

responses in high potassium. Consistent with the expectation that BK channels control

airway constriction, the data shows that the β1 knockouts have a larger carbachol-

induced constriction than the wild type mice. Summary dose-response curves (Figure 7B)

indicate that the main effect of the β1 knockout is an increase in the maximal response,

rather than a shift in the dose-response curve. Of note, although some knockouts showed

calcium oscillations, we did not see oscillation in carbachol-induced constrictions.

The increased constriction of the β1 knockouts is accounted for by increased

contribution of voltage-dependent calcium channels during sustained contractions.

The most likely role of BK potassium channels is to oppose membrane

depolarization during agonist induced constriction. A reasonable hypothesis is that

membrane depolarization recruits voltage-dependent calcium channels and increased

calcium influx and constriction. Consistent with this hypothesis, we found that the β1

knockouts relax to the same relative constriction as wild type mice (Figure 7A, right

panel, Figure 7B dose-response + nifedipine). This indicates that the increased

constriction of the knockouts is largely accounted for by increased calcium influx through

L-type voltage-dependent calcium channels.

It is fairly well established that two sources of calcium underlie tracheal

constriction; an early transient component that is dependent on agonist-induced calcium

release from ER calcium stores and a subsequent sustained component that is dependent

on calcium influx from plasma membrane channels. Consistent with the nifedipine

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Semenov, et al. 15

experiments above (Figure 5), the β1 knockouts show the greatest effect on constriction

during the sustained component where voltage-dependent calcium channels are expected

to play a role. This is shown in representative trace in Figure 7A. Nifedipine block of

voltage-dependent calcium channels brings constriction during the sustained component

to similar levels between the knockout and wild-type (Figure 7A and 7B).

In vascular smooth muscle, block of BK channels in wild type cells shows similar

levels of constriction as β1 knockouts (4). This indicates that the absence of the β1

subunit renders BK channels nonfunctional in controlling constriction. In tracheal smooth

muscle, we see a similar effect. Figure 7D and 7E shows that the KO trachea had no

significant difference in cholinergic evoked constriction in KO in the presence or absence

of paxilline block, indicating BK channel activity do not significantly contribute to

relaxation in the absence of the β1 subunit. In contrast, wild type trachea show a

significant difference with and without paxilline block (Figure 7C and 7E).

DISCUSSION

Airway smooth muscle presents a medically important model for studying the role

of ion channels during receptor-coupled activation of calcium transients. Unlike vascular

smooth muscle, where voltage-dependent calcium channel are a major calcium conduit

mediating contraction, the role of voltage-dependent calcium channels and other calcium

influx pathways in airway smooth muscle is poorly defined. As such, pharmaceuticals

targeted to ion channels in airway have not been utilized to treat asthma.

These data demonstrate that BK channels in β1 knockout airway smooth muscle

exhibit a large reduction in channel openings as indicated by single channel activity and

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STOC frequency. As a consequence, BK channel contribution to relaxation is perturbed

to a sufficient extent that pharmacological block of BK channels shows that same relative

constriction as the β1 knockout trachea (Figure 7C-E). This is similar to vascular smooth

muscle wherein iberiotoxin block of wild type vessels induced constriction, but had no

further constrictive effect on β1 knockout vessels (4). This is in contrast to bladder

smooth muscle where the β1 knockout shows a partial effect on constriction as compared

to iberiotoxin block (36). A distinction between tonic smooth muscles such as vascular

and trachea muscle, as compared to bladder smooth muscle that is phasic in excitation, is

that large depolarizations occur in bladder that are likely to complement BK activation in

the β1 knockout. Indeed, we see much smaller differences in single channel and STOC

activity of WT and KO at depolarizing voltages.

Tao et al (43) demonstrated that cholinergic evoked constriction in bovine airway

is largely independent of voltage-activated calcium channels unless sarcoplasmic

reticulum calcium stores are depleted or BK potassium channels are blocked. In the latter

case, the mode of contraction coupling switches to an excitation, voltage-dependent

coupling. Our findings utilizing the β1 knockout in mouse trachea indicate that there is

more of a partial contribution, rather than a switch in coupling between pharmaco- and

excitation evoked contraction. In wild type mice, voltage-dependent calcium channels

appear to contribute approximately 17 % of the carbachol induced contraction at

saturating carbachol doses (Figure 7B). In the absence of the β1 subunit, BK channel

activity decreases and the contribution of voltage-dependent calcium channels increase to

approximately 30% of the constriction (Figure 7B). This is a smaller contribution than β1

subunits appear to have in aortic smooth muscle. In those studies, maximal contraction is

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increased approximately 65% by the β1 knockout (37). Thus, BK potassium channels in

tracheal smooth muscle make a significant, but smaller contribution, to agonist induced

relaxation than in vascular muscle.

The calcium pathways downstream of voltage-dependent calcium channels that

are regulated by BK channels are likely to be complex. The consequence of the β1

knockout was to increase resting calcium levels and increases in the sustained component

of calcium influx during cholinergic evoked signaling. These were translated to increased

cholinergic evoked constriction. A conclusion that may be drawn is that the regulation of

membrane voltage by the BK channel is important, albeit less apparent, until these

channels are blocked or by deletion of the β1 subunit. The hyperpolarizing activity of BK

channels is likely to hold membrane voltage during cholinergic contractions below the

operational voltages where voltage-dependent calcium channels and excitation-

contraction coupling contribute to airway constriction (Bourreau 1993). This may

reconcile the fact that voltage-dependent calcium channels are regarded to provide a

minority of the calcium for contraction (Janssen 2002), yet the potassium channels would

have an important role such as is observed during β-adrenergic mediated relaxation.

An interesting finding was that the β1 knockout mice had a high frequency of

cells showing oscillatory calcium changes following cholinergic activation (Figure 4B).

However we did not see a similar effect on constriction, which were all tonic in response

to high potassium or cholinergic evoked constriction. Thus, it is as yet unclear how the

β1 knockout-induced calcium oscillations influence the muscle contractile properties in

the trachea. Other examples exist where perturbing BK channels promotes oscillations in

trachea. Oscillatory contractions have been described for guinea pig tracheal smooth

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muscle cells treated with the BK channel blockers charybdotoxin and iberiotoxin (48,

49). However, these studies did not investigate calcium changes that may underlie the

oscillatory contractions. More recent studies in bronchioles have observed slow

oscillatory calcium transients in response to depolarization with potassium (35). This may

be considered analogous to BK channel block, or the β1 knockout since blocking of

potassium channels presumably depolarizes the cell. However, slow calcium oscillations

were correlated with twitching rather than constriction of airway (35).

The fact that the calcium oscillations are not affected by nifedipine or 0 external

calcium suggests that the β1 subunit has other effects in addition to their actions on

membrane voltage or on calcium influx through plasmallema channels. In this regard, the

increase in resting calcium concentration observed in the β1 knockout could play a role.

Increases in global calcium have been shown to promote SR calcium loading and thereby

sustain calcium release (5). As well, increases in cytosolic calcium concentrations and SR

calcium stores promote IP3 receptor activation and calcium oscillations (2, 13). Indeed,

the mechanism by which knockout of the β1 subunit causes calcium oscillations and

perhaps affects other currents requires further study.

In summary, these findings show that tracheal smooth muscle require the

accessory β1 subunit to promote normal BK channel activation. Despite the predominant

role of agonist induced signaling in trachea, BK channels have a significant role in

controling resting calcium and cholinergic evoked calcium influx contributed by voltage-

dependent calcium channels.

ACKNOWLEDGEMENTS

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This work was supported by the Sandler Program for Asthma Research to R.B.

and J.T.H., and a National American Heart Association grant 0335007N to R.B. We

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

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FIGURE LEGENDS

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Semenov, et al. 26

Figure 1. The BK Channel β1 subunit is expressed in airway smooth muscle tissues.

Expression is assayed from a β-galactosidase reporter gene targeted to the BK channel

beta1 locus in mice (KO tissues). A. Filleted whole-mount KO trachea showing staining

in trachea smooth muscle. B. Wild type control. C. Frozen section of trachea in KO. D.

Frozen section of trachea in wild type tissue E. Magnification of trachea showing staining

in the smooth muscle layer. Arrows indicate smooth muscle layers.

Figure 2. Knockout of the β1 subunit reduces BK channel opening probability. A.

Single channel recording of BK channels from wild type and knockout trachea smooth

muscle cells assayed in excised patch, inside/out configuration (Internal solution was 7

µM buffered calcium). B. Summary data showing Po and C. open dwell times for wild

type (WT, open column) and knockout (KO, shaded column). Scale bar indicates 1

second for –40 mV traces, and 0.1 second for +40 mV traces. N= 6 WT, 8 KO. Heights

of columns and vertical lines represent means ± sem. Single asterisk indicate difference

of P < 0.05.

Figure 3. Spontaneous transient outward currents in isolated tracheal smooth

muscle cells. A. Examples of spontaneous transient BK current in wild type and

knockout airway smooth muscle cells. B. Summary of STOC frequency. C. Summary of

STOC amplitude. D. Summary of STOC area. Data is for N= 6-8 cells for WT and β1

KO. At –40 mV KOs did not show any STOCs in 7/7 cells whereas WT showed STOCS

in 3/8 cells. Data are mean ± sem. Triple asterisk represent P < 0.001, double asterisk P

difference < 0.01 and single asterisk represent P < 0.05.

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Semenov, et al. 27

Figure 4. Carbachol-induced calcium transients in wild type and knockout tracheal

smooth muscle cells. A. Example of Fura-2 measured calcium transients from wild type

(open circles) and knockout (filled circles) tracheal smooth muscle cells. Left inset shows

an enlargement of resting calcium that is not baseline subtracted (Note left and right

insets Y-scale is in nM). Large trace and right inset knockout traces are baseline

subtracted to adjust knockout to the same baseline as wild type. Components of calcium

are labeled A, resting calcium, B transient phase, C early sustained phase, D late

sustained phase. B. Example of Fura-2 measured calcium transients from knockout

tracheal smooth muscle cells that has oscillatory calcium changes. C. Summary data

comparing average values for different components in wild type (open columns) and

knockout (gray columns). Significant difference is seen at resting calcium (P = 0.0004,

N= 28 WT, 24 KO), during the sustained component D (P = 0.0062, N = 14 WT, 12 KO).

Sustained calcium components C and D were measured from 15-17 seconds, and 77-80

seconds, respectively, after addition of carbachol. Cells with oscillatory calcium

transients were excluded from these analysis.

Figure 5. Effect of nifedipine on carbachol-induced calcium transients. Example

traces of carbachol induced calcium transients before (open circle) and after nifedipine

block of voltage-gated calcium channels (closed circles) for A, Wild type cell, and B, β1

knockout cell. C. Summary data comparing average values for different components in

wild type (open boxes) and knockout (gray boxes) during calcium channel block with

nifedipine. No significant difference is seen at any component. D. Summary of relative

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Semenov, et al. 28

change of different components calculated as calcium after nifedipine subtracted from

before nifedipine (Δ[Ca2+]). Results are from paired experiments. Asterisk indicates

significance of P = 0.025. N = 13 WT, 11 KO.

Figure 6. Effect of removing calcium on carbachol-induced calcium transients.

Example traces of carbachol induced calcium transients before (open circle) and after

addition of 0 calcium (closed circles), 1 mM EGTA Krebs solution for A, Wild type cell,

and B, β1 knockout cell. C. Summary data comparing average values for different

components in wild type (open column) and knockout (gray column) during cholinergic

evoked calcium in 0 external calcium. No significant difference is seen at any

component. D. Summary of relative change of different components following

extracellular calcium removal. Asterisk indicates significance of P = 0.028. N = 14 WT,

12 KO.

Figure 7. β1 knockouts have greater cholinergic-evoked contraction of trachea. A.

Example of carbachol evoked contraction before and after 1 µM nifedipine. Contraction

traces are normalized to high potassium induced contractions. B. Cumulative dose

response curve to carbachol. Last dose (3x10-5 carbachol) is repeated as previous dose

with 1 µM nifedipine). P < 0.05 for 2x10-7 to 1x10-5 carbachol. N = 14 wild type, 14

knockout. Effect of BK channel block with paxilline (5 µM) in wild type, C, Example of

constriction to 1 µM carbachol without (dark trace) and with 5 µM paxilline block of BK

channels in WT and D, β1 knockout tracheas. E. Summary data for C and D.

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Semenov, et al. 29

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A

B C D E

Figure 1

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WT KO-40 mV

+40 mV

20 pA0.1 s1 s

oc

oc

A

Figure 2

Ope

n D

wel

l Tim

e (m

s)

0

0.5

1.0

1.5

2.0

2.5

WT KO

-40 mV-40 mV

0

0.01

0.02

0.03

WT KO 0

0.2

0.4

0.6

0.8

1.0+40 mV

WT KO

Ope

n P

roba

bilit

y

B C

**

01

23

456

7+40 mV

WT KO

of 36

100 pA10 s

KO (α alone)

WT (α +β1)

-20 mV

0 mV

0 mV +20 mV

A

WT KO

Figure 3

0

5

10

15

20

25

30

35

-40 -20 0 +20Holding Potential (mV)

ST

OC

Am

plitu

de (

pA)

C

* ****

0

100

200

300

400

500

600


ST

OC

Fre

quen

cy (

ST

OC

/min

)

B

***

**

**

0

100

200

300

400

500


ST

OC

Are

a (

pA-m

s)

D

*

1.0 s100 pA

-20 mV-40 mV

-40 mV

of 36

Figure 4

0

40

80

120

160

200 **

A Resting

[Ca

2+] (

nM)

WT KO

0.5

**

C Early Sustained

2 Ca2+B Transient

C

[Ca

2+] (

µM

)

100

200

300

400

1.0

1.5

2.0

[Ca

2+] (

nM)

Nifedipine

D Late Sustained

CCh 10µMCCh 10µM

0 50 100 150 200

1.0

2.0

1.5

0.5

KnockoutCa2+ Oscillations

B

Time (seconds)

[Ca

2+ ](

µM

)

200150100500

Wash300

250

200

150

100

50170160150140130

Time (seconds)

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

[C

a2+

](µ

M)

A300

250

200

150

100

50

5040

AD

A

B

C

D

of 36

WT KO

[Ca

2+] (

nM)

0.5

C Early Sustained

D Late Sustained

B Transient

[Ca

2+] (

µM

)

100

200

300

400

1.0

1.5

2.0

-0.2

-0.4

-0.6

-0.8

-1.0

Δ[C

a2+

] (µ

M)

B Transient

-50

-100

-150

-200Δ

[Ca

2+] (

nM)

C Early Sustained

D Late Sustained

*

C D

Figure 5

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

200150100500

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

200150100500

A[C

a2+

](µ

M)

[Ca

2+ ](

µM

)

B

Time (seconds)Time (seconds)

WT 10µM CCh WT 10µM CCh + Nifedipine

KO 10µM CCh KO 10µM CCh + Nifedipine

of 36

WT KO

[Ca

2+] (

nM)

C Early Sustained

D Late Sustained

B Transient

[Ca

2+] (

µM

)

100

200

300

400

-0.2

-0.6

-1.0

-1.4

Δ[C

a2+

] (µ

M)

B Transient

-40

-80

Δ[C

a2+

] (nM

)C Early

Sustained D Late

Sustained

**

C D

Figure 6

0.2

0.4

0.6

0.8

1.0

A B

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

200150100500

[Ca

2+ ](

µM

)

Time (seconds)

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

200150100500

[Ca

2+ ](

µM

)

Time (seconds)

WT 10µM CCh WT 10µM CCh + 0 Ca2+

KO 10µM CCh KO 10µM CCh + 0 Ca2+

-120

of 36

-4-5-6-7-80

50

100

150

200

250

log [Carbachol] (M)

WTKO

+Nifedipine

KO

WT

0 10 20 30 40 50 60 70

200

150

100

50

0

Time (min)

A B1 µM CCh

1 µM CCh+ 1 µM Nifedipine

KO

WTWTKO

Figure 7%

K+

con

stric

tion 200

150

100

50

020151050

CCh 1µM

WT

WT+PX

Time (min)

CCh 1µM200

150

100

50

020151050

KO

KO+PX

Time (min)

200

150

100

50

0WT WT+PX KO KO+PX

C D E*

% K

+ c

onst

rictio

n

% K

+ c

onst

rictio

n

% K

+ c

onst

rictio

n

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constriction in tracheal smooth muscle

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