Modulation of BK channel activities by calcium-sensing receptor in rat bronchopulmonary sensory neurons

Respiratory Physiology & Neurobiology 203 (2014) 35–44

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Modulation of BK channel activities by calcium-sensing receptor in rat bronchopulmonary sensory neurons Zhanna V. Vysotskaya a , Charles R. Moss II a , Carolyn A. Gilbert a , Sabry A. Gabriel b , Qihai Gu a,∗ a b

Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA 31207, USA Department of Family Medicine, Mercer University School of Medicine and Medical Center of Central Georgia, Macon, GA 31207, USA

a r t i c l e

i n f o

Article history: Accepted 21 August 2014 Available online 1 September 2014 Keywords: Calcium-sensing receptor Large-conductance Ca2+ -activated potassium channel Bronchopulmonary sensory neuron Transient receptor potential vanilloid receptor 1

a b s t r a c t This study was carried out to investigate the expression of large-conductance Ca2+ -activated potassium (BK) channels and to explore the possible modulation of BK channel activities by calcium-sensing receptors (CaSR) in rat bronchopulmonary sensory neurons. The expression of BK channels was demonstrated by immunohistochemistry and RT-PCR. Results from whole-cell patch-clamp recordings demonstrated that activation of CaSR with its agonist spermine or NPS R-568 showed a dual regulating effect on BK channel activities: it potentiated BK currents in cells exhibiting low baseline BK activity while slightly inhibited BK currents in cells with high baseline BK activity. Blocking CaSR with its antagonist NPS 2143 significantly inhibited BK currents. Our results further showed that the modulation of BK currents by CaSR activation or blockade was completely abolished when the intracellular Ca2+ was chelated by BAPTA-AM. In summary, our data suggest that CaSR plays an integrative role in bronchopulmonary afferent signaling, at least partially through the regulation of BK channel activities. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Large-conductance Ca2+ -activated potassium (BK) channels are key regulators of cellular excitability (Stretton et al., 1992; Sah and Faber, 2002). They are characterized by large single-channel conductance, intrinsic voltage dependence, Ca2+ modulation, and blockade by charybdotoxin, iberiotoxin (IbTX) and paxilline (Stocker et al., 2004; Berkefeld et al., 2010). They are expressed in many different types of excitable cells and have significant physiological roles ranging from regulation of smooth muscle tone and microbial killing in leukocytes to modulation of neurotransmitter release and neuronal spike frequency adaptation (Oliver et al., 2006; Berkefeld et al., 2010), although variability in the biophysical properties and pharmacological sensitivity of these channels has been observed among various excitable tissues as well as within excitable cells (Dopico et al., 1999; Lee and Cui, 2010). The

∗ Corresponding author at: Division of Basic Medical Sciences, Mercer University School of Medicine, 1550 College Street, Macon, GA 31207, USA. Tel.: +1 478 301 4004; fax: +1 478 301 5489. E-mail address: GU [email protected] (Q. Gu). http://dx.doi.org/10.1016/j.resp.2014.08.014 1569-9048/© 2014 Elsevier B.V. All rights reserved.

main function of BK channels in the peripheral sensory neurons seems to shorten the action potential duration, enhance the rate of repolarization, and contribute to rapid afterhyperpolarization, which lead to reduced repetitive firing (Scholz et al., 1998; Zhang et al., 2003, 2010). However, the expression and properties of BK channels in bronchopulmonary sensory neurons are yet to be elucidated. Calcium-sensing receptor (CaSR), a G-protein-coupled receptor first isolated from bovine parathyroid, is known to play a key role in extracellular Ca2+ homeostasis (Brown et al., 1993; Brown and MacLeod, 2001). Although the involvement of CaSR in a variety of tissues and biological processes unrelated to Ca2+ balance in plasma has been increasingly recognized (Magno et al., 2011; Vyleta and Smith, 2011; Riccardi and Kemp, 2012), its function in peripheral sensory transmission is not well understood. A recent study from our laboratory (Gu et al., 2013) demonstrated the expression of CaSR in rat vagal bronchopulmonary sensory neurons. Our study further showed the functional interactions between CaSR and transient receptor potential vanilloid receptor 1 (TRPV1), a polymodal transducer that senses many endogenous and environmental stimuli in bronchopulmonary C-fiber afferents (Bessac and Jordt, 2008; Lee and Gu, 2009). In the present study, we aimed to

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investigate the functional expression of BK channels in rat bronchopulmonary sensory neurons, and to investigate the possible regulation of CaSR on BK channel activities in these neurons.

2. Methods The procedures described below were performed in compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals (Office of Laboratory Animal Welfare, Amended August, 2002) and U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. These procedures were also approved by the Mercer University Institutional Animal Care and Use Committee.

2.1. Retrograde labeling of vagal bronchopulmonary sensory neurons Sensory neurons innervating airways and lungs were identified by retrograde labeling using the fluorescent tracer 1,1 -dioctadecyl3,3,3 ,3 -tetramethylindocarbocyanine perchlorate (DiI), as described in our recent studies (Gu and Lin, 2010; Gu et al., 2013). Briefly, young Sprague–Dawley rats (80−120 g) were anesthetized with continuous inhalation of 2% isoflurane in oxygen. DiI (0.2 mg/ml for cell culture and 1 mg/ml for immunohistochemistry and RT-PCR, 50 ␮l in volume) was instilled into the lungs via surgically exposed trachea. Animals were kept undisturbed for 7–10 days until they were used for tissue harvest.

2.4. RT-PCR Cytoplasm of 10 individual bronchopulmonary (DiI labeled) nodose or jugular ganglion neurons was retrieved by aspiration through a patch pipette. The pipette content was expelled into a test tube in which RT-PCR was performed by using Titan One Tube RT-PCT Kit (Roche Applied Science, Indianapolis, IN). A nested PCR was then performed by using the first PCR product as the template. Primers for both the first and nested PCR amplifications were designed to cross exon/exon boundaries to distinguish amplified products from mRNA versus genomic DNA. Primer sequences and predicted product sizes are summarized in Table 1. Reverse transcription was performed at 50 ◦ C for 30 min. Cycling conditions for the first PCR were as the following: (1) initial denaturation at 94 ◦ C for 3 min; (2) 10 cycles of denaturation at 94 ◦ C for 30 s, annealing at 56 ◦ C for 30 s, elongation at 68 ◦ C for 1 min; (3) 25 cycles of denaturation at 94 ◦ C for 30 s, annealing at 56 ◦ C for 30 s, elongation at 68 ◦ C (45 s–4 min) cycle elongation of 5 s for each cycle; and (4) a prolonged elongation at 68 ◦ C for 7 min. Reaction products were subsequently maintained at 4 ◦ C until they were used as templates for nested reactions. The nested PCR was performed as the following by using AccuPrime Taq DNA Polymerase System (Invitrogen, Carlsbad, CA): an initial denaturation at 94 ◦ C for 3 min was followed by 30 cycles of denaturation at 94 ◦ C for 1 min, annealing at 56 ◦ C for 1 min and elongation at 68 ◦ C for 2 min. Reaction products were separated on 1.5% agarose gel in Tris–borate EDTA buffer and visualized with the GelStar stain (Lonza, Rockland, ME). 2.5. Whole-cell perforated patch-clamp recordings

2.2. Immunohistochemistry Rat nodose and jugular ganglia were harvested and standard immunohistochemistry was carried out as described previously (Gu et al., 2013). Briefly, the ganglion was cryosectioned at 12 ␮m, and tissue sections were incubated overnight at 4 ◦ C with rabbit polyclonal anti-BK antibody (1:100; anti-KCa 1.1, Alomone Labs, Jerusalem). The sections were then incubated with fluorescein isothiocyanate-labeled donkey anti-rabbit secondary IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature. Finally, the sections were visualized and photo captured using an inverted fluorescent microscope. Absorption control as instructed by the manufacturer and negative control with BK primary antibody omitted, were carried out to confirm the specificity of receptor-like immunoreactivity.

2.3. Isolation of nodose and jugular ganglion neurons Rat nodose and jugular ganglia were extracted, desheathed, and subject to tissue culture process, as described previously (Gu et al., 2013). Briefly, each ganglion was cut into ∼8 pieces, placed in a 0.08% type IV collagenase, and incubated for 60 min in 5% CO2 in air at 37 ◦ C. The ganglion suspension was centrifuged (150 × g, 5 min) and supernatant aspirated. The cell pellet was resuspended in 0.05% trypsin for 1 min and centrifuged (150 × g, 5 min); the pellet was then resuspended in a modified DMEM/F12 solution [supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 ␮g/ml streptomycin, and 100 ␮M minimum essential media nonessential amino acids] and gently triturated with a small-bore Pasteur pipette. Myelin debris was separated and discarded after centrifugation of the dispersed cell suspension (500 × g, 8 min) through a layer of 15% bovine serum albumin. The cell pellet was resuspended in the modified DMEM/F12 solution, plated onto poly-l-lysine-coated glass coverslips, and incubated overnight (5% CO2 in air at 37 ◦ C).

Whole-cell perforated patch-clamp recordings were carried out in selected neurons labeled with DiI, as described previously (Gu et al., 2013). The extracellular solution (ECS) consisted of the following (in mM): 140 choline Cl, 3 KCl, 1 MgCl2 , 1.8 CaCl2 , 10 HEPES and 10 glucose; pH was adjusted to 7.4 with Tris-base. The pipette solution contained (in mM): 108 choline Cl, 40 KCl, 1 MgCl2 , 0.1 EGTA and 10 HEPES; pH was adjusted to 7.2 with Tris-base. All chemical agents at their respective concentrations (except for BAPTA-AM which was applied intracellularly) were applied by a pressuredriven drug delivery system (VC3 8, ALA Scientific Instruments, Westbury, NY); the tip of which was positioned about 200 ␮m upstream of the recording cell. The series resistance was usually in the range of 6−10 M and was compensated up to 80%. 2.6. Chemicals NPS R-568 and NPS 2143 were purchased from Tocris Bioscience (Ellisville, MI). Paxilline and IbTX were obtained from Alomone Labs. All other chemicals were purchased from Sigma–Aldrich (St. Louis, MO). Stock solutions of paxilline (1 mM), IbTX (10 ␮M), NPS R-568 (10 mM), NPS 2143 (50 mM) and BAPTA-AM (100 mM) were prepared in dimethyl sulfoxide. A stock solution of capsaicin (1 mM) was prepared in 1% Tween 80, 1% ethanol, and 98% saline; that of spermine (0.2 M) in distilled water. Aliquots of these stock solutions were kept at −80 ◦ C. Chemicals at desired concentrations were then prepared daily by dilution with ECS before experiments except for BAPTA-AM which was diluted with pipette solution. No detectable effect of the vehicles of these chemical agents was found in our preliminary experiments. 2.7. Statistical analysis Initial analysis of patch-clamp data was performed with Clampfit 10 (Molecular Devices, Sunnyvale, CA), with subsequent statistical analysis performed with SigmaPlot 12 (Systat Software,

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Table 1 PCR primer sequences. Receptor

Accession no.

Primers for the first PCR

Size, bp

Primers for the nested PCR

Size, bp

CaSR Sense Anti-sense

NM 016996

5 -ccatcactcctgctctgtca-3 5 -ggcttcctctcggaacttct-3

829

5 -ctcccttcttcccaacatga-3 5 -gtgcggaggaaggatttgta-3

303

BK Sense Anti-sense

NM 031828

5 -agagaatgagccgagcatgt-3 5 -tccttgtcctgaagcgaagt-3

715

5 -tgaggaactcacccaacaca-3 5 -ggggaagttgtgcagtgttt-3

368

TRPV1 Sense Anti-sense

NM 031982

5 -agcgagttcaaagacccaga-3 5 -gttcaagggttccacgagaa-3

822

5 -tggaggtggcagataacaca-3 5 -ttaggggtctcactgctgct-3

349

CaSR, calcium-sensing receptor; BK, large-conductance Ca2+ -activated potassium channel; TRPV1, transient potential receptor vanilloid receptor 1.

Fig. 1. Expression of BK channels in rat bronchopulmonary sensory neurons. Left panel: BK receptor immunohistochemistry in a cryosection (12 ␮m) of nodose ganglion; middle panel: DiI labeling identifies the sensory neurons innervating airways and lung structures; right panel: merged image. Scale bar, 50 ␮m.

San Jose, CA). The current–voltage (I–V) curves for BK channels in individual neurons were generated by calculating the peak outward currents at each test potential and normalized to the cell capacitance. Although neurons from nodose and jugular ganglia were isolated and studied separately, data from neurons of these two different origins were pooled for group analysis because no difference was found between responses of the neurons obtained from these two ganglia in our study. Data are presented as mean ± SEM. Unless mentioned otherwise, differences between the means were tested for significance using two-way repeated-measure ANOVA. When the ANOVA showed a significant interaction, pair-wise comparisons were made with the Student–Newman–Keuls post hoc analysis. A value of P < 0.05 was considered to be significant.

3. Results

two different animals. Control reactions that did not contain RNA template showed no amplification products (n = 3). 3.3. Functional characterization of BK channels in rat vagal bronchopulmonary sensory neurons The total K+ current is evoked by the following voltage protocol: from a holding potential of −70 mV, neurons were depolarized from −100 to 80 mV in 15 mV increments at 5-s intervals, following a prepulse at 0 mV for 200 ms. After establishing a stable I–V relationship for total K+ current, one of the two selective BK channel blockers IbTX or paxilline was applied to each neuron for at least 2 min when current stabilization in the presence of the blocker was achieved. The difference in K+ current between before and after application of a blocker was considered the BK current. The use of IbTX and paxilline precludes cross contamination of BK currents with other

3.1. Detection of BK receptor protein in rat vagal bronchopulmonary sensory neurons The immunoreactivity for BK was detected in vagal sensory (nodose and jugular ganglion) neurons, including those innervating the airways and lung structures as identified by DiI labeling (Fig. 1). There was no fluorescent staining for BK in these neurons in the absence of the primary antibody, or in the study of absorption control (not shown). 3.2. Detection of CaSR, BK and TRPV1 receptor mRNAs in rat vagal bronchopulmonary sensory neurons RT-PCR and the nested PCR were carried out using the cytoplasm harvested from ten DiI labeled nodose or jugular ganglion neurons to determine the presence of CaSR, BK and TRPV1 receptor mRNAs. As shown in Fig. 2, our results showed that bronchopulmonary sensory neurons from both nodose and jugular ganglia contain mRNAs for the three ion channels. These results were confirmed in three separate trials using the neurons collected from

Fig. 2. Presence of CaSR, BK and TRPV1 mRNAs in rat bronchopulmonary sensory neurons. Cytoplasm of 10 individual bronchopulmonary (DiI labeled) nodose or jugular neurons was collected into single PCR tubes. One-step RT-PCR and the nested PCR were carried out to detect the presence of the transcripts for CaSR, BK and TRPV1 receptors. M, DNA molecular weight marker.

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Fig. 3. Functional expression of BK channels in rat bronchopulmonary sensory neurons. (A−C) Representative traces from one cell showing total K+ currents, IbTX (100 nM)resistant currents, and IbTX-sensitive BK currents (subtract B from A), respectively. (D−F) Representative traces from another cell showing total K+ currents, paxilline (1 ␮M)-resistant currents, and paxilline-sensitive BK currents (subtract E from D), respectively. (G) Voltage protocol showing neurons were voltage-clamped at −70 mV and depolarized from −100 to 80 mV in 15 mV increments following a pre-pulse at 0 mV for 200 ms.

Ca2+ -dependent currents in our neurons (Zhang et al., 2010). At their generally used concentration at 100 nM and 1 ␮M for IbTX (n = 11) and paxilline (n = 68), respectively, paxilline appeared to be a more potent BK blocker in our neurons (P < 0.05) (Fig. 3), and therefore was used to separate the BK current in the rest of our study series. Recordings of BK currents using paxilline were obtained from 81 bronchopulmonary sensory neurons, out of which 68 neurons (84.0%) showed paxillin-sensitive BK currents. The amplitude of BK currents varied in different neurons. Based on the current density elicited by 80 mV voltage step, we arbitrarily categorized BK currents into two groups: low or high baseline BK current when the maximal current density was smaller or greater than 50 pA/pF, respectively, in individual neurons (Table 2). In 68 neurons tested with paxilline-sensitive BK currents, 38 neurons had low current density (37.8 ± 3.7 pA/pF) and 30 neurons had high current density (142.9 ± 15.8 pA/pF) (P < 0.05). While the resting membrane potentials of these two groups of neurons were almost identical, neurons with high BK current density were relatively smaller in size: the whole-cell capacitances were 28.2 ± 2.1 and 21.1 ± 1.6 pF for low current density and high current density groups, respectively (P < 0.05). At the end of BK current recordings, we tested the sensitivity of these neurons to 1 ␮M capsaicin, a specific agonist for TRPV1. Our results showed that 61 out of 68 (89.7%) BKexpressing neurons also responded to capsaicin, whereas none of the paxilline-insensitive neurons (13 out of 81 neurons) showed capsaicin sensitivity, indicating the co-expression of BK and TRPV1 channels in the majority (61 out of 81) of bronchopulmonary sensory neurons. 3.4. Activation of CaSR with spermine or NPS R-568 dually regulates BK channel activity in bronchopulmonary sensory neurons Pretreatment with spermine (3 mM, 60 s) at a concentration recently shown to strongly activate CaSR in bronchopulmonary sensory neurons (Gu et al., 2013), did not significantly alter the total K+ currents or paxilline-resistant currents in neurons with either low (Fig. 4) or high baseline BK current density (Fig. 5). However, spermine showed distinct effects on paxilline-sensitive BK currents in two groups of neurons with different BK current densities. In neurons with low BK current density, presence of spermine significantly enhanced BK currents in the voltage range of 20−80 mV (P < 0.05, n = 10) (Fig. 4); whereas in neurons with high BK current density, spermine appeared inhibiting BK currents although no significant difference was detected statistically (P > 0.05, n = 9) (Fig. 5).

Since the high concentration of spermine has been shown to increase the pH rather markedly when dissolved in the ECS (Gu et al., 2013) and the alkalosis pH may potentially increase the agonist sensitivity of CaSR (Quinn et al., 2004), in separate group of neurons, we used a specific agonist NPS R-568 (0.5 ␮M, 60 s) to activate CaSR. Similarly as shown in the spermine study, NPS R568 dually regulates BK channel activities in bronchopulmonary sensory neurons: it significantly potentiated BK currents in neurons exhibiting low baseline BK current density (P < 0.05, n = 17) (Fig. 6), whereas inhibited those currents in neurons with high BK current density (P < 0.05, n = 11) (Fig. 7). 3.5. Blockade of CaSR with NPS 2143 inhibited BK channel activity in bronchopulmonary sensory neurons Ca2+ is known as the principal physiological ligand of CaSR. Considering the 1.8 mM Ca2+ contained in our ECS could tonically activate CaSR, we investigated the BK channel activities in the presence of CaSR antagonist NPS 2143. Pretreatment with NPS 2143 (10 ␮M, 60 s) significantly decreased total K+ currents in the voltage range of 5−80 mV (P < 0.05, n = 10) which seemed mainly due to the significant inhibition of BK currents (P < 0.05, n = 10) since paxilline-resistant currents were not affected significantly by NPS 2143 (P > 0.05, n = 10) (Fig. 8). This inhibition of BK currents by NPS 2143 was equally effective in neurons with either low (n = 5) or high (n = 5) baseline BK current densities (P > 0.05). In a separate group of neurons, we investigated whether blockade of CaSR can abolish the regulation of BK currents by CaSR activation. Indeed, our results showed that pretreatment with a mixture of CaSR agonist NPS R-568 (0.5 ␮M) and antagonist NPS 2143 (1 ␮M) did not significantly alter the BK currents in broncopulmonary sensory neurons with either low (n = 5) or high (n = 3) baseline BK current densities (P > 0.05, n = 8) (Fig. 9). 3.6. Regulation of BK channel activity by CaSR is abolished by Ca2+ chelator BAPTA-AM Since CaSR activation is known to cause intracellular Ca2+ mobilization in a variety of cell types (Hofer and Brown, 2003), we next tested whether abolishing the increase in intracellular Ca2+ affected the regulation of BK currents by CaSR. BAPTA-AM, the membranepermeable form of Ca2+ chelator BAPTA (Vyleta and Smith, 2011), was added to the pipette solution (35 ␮M) of patch-clamp recordings. The BK currents obtained in the presence of BAPTA-AM (n = 22) were then compared to the control ones without this Ca2+ chelator (n = 57). Our results showed that intracellular application of BAPTA-AM significantly inhibited BK currents (P < 0.01; Fig. 10A).

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Fig. 4. CaSR agonist spermine potentiated BK currents in cells with low baseline BK current density in rat bronchopulmonary sensory neurons. (A−F) Representative traces of total K+ currents, paxilline (1 ␮M)-resistant currents, and paxilline-sensitive BK currents elicited by the voltage protocol (G) without (A−C) or with (D−F) application of spermine (3 mM, 60 s). All traces were recorded from a single cell. (H) I–V curves showing the current densities of total K+ currents and paxilline (Pax)-resistant currents, before and after spermine application. (I) I–V curves showing that spermine markedly potentiated the BK current density. *Significant difference in current density between before and after spermine (P < 0.05, n = 10).

Fig. 5. Effect of spermine on BK currents in cells with high baseline BK current density in rat bronchopulmonary sensory neurons. (A−F) Representative traces of total K+ currents, paxilline-resistant currents, and paxilline-sensitive BK currents elicited by the voltage protocol (G) without (A−C) or with (D−F) application of spermine (3 mM, 60 s). (H) I–V curves showing the current densities of total K+ currents and paxilline-resistant currents, before and after spermine application. (I) I–V curves showing that spermine did not significantly affect BK current density (P > 0.05, n = 9).

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Table 2 Characteristics of paxilline-sensitive BK currents in rat bronchopulmonary sensory neurons. Groups of BK currents

No. of neurons

Low BK current density High BK current density

38 30

*

Resting membrane potential (mV) −60.1 ± 0.7 −60.9 ± 1.1

Capacitance (pF) 28.2 ± 2.1 21.1 ± 1.6*

Current density (pA/pF) 37.8 ± 3.7 142.9 ± 15.8*

No. of capsaicinsensitive neurons 33 28

P < 0.05, significant difference between the corresponding values in two BK current groups; unpaired Student’s t-tests.

In addition, BAPTA-AM completely abolished the modulation of BK currents by either CaSR agonist NPS R-568 (P > 0.05, n = 13; Fig. 10B) or CaSR antagonist NPS 2143 (P > 0.05, n = 9; Fig. 10C). 4. Discussion BK channels are expressed in primary afferents of most, if not all internal organs and are an intriguing target for pharmacological manipulation of visceral sensation (Li et al., 2011). Results from our immunohistochemical and RT-PCR studies demonstrated the expression of BK receptor protein and mRNA, respectively, in rat vagal bronchopulmonary sensory neurons. Using specific BK channel blockers IbTX and paxilline in the patch clamp experiments, we further demonstrated the functional expression of BK channels in these sensory neurons. Although our multiple-cell RT-PCR study did not provide the direct evidence of co-expression of BK and TRPV1 channels in individual neurons, our patch-clamp recordings showed that these two channels indeed co-expressed in ∼75% (61 out of 81) of bronchopulmonary sensory neurons tested, and TRPV1-expressing neurons exclusively exhibited BK sensitivity (61 out of 61), whereas only 10% (7 out of 68) of BK-expressing neurons were without TRPV1 sensitivity. These findings are in general agreement with a recent study by Li et al. (2011) which showed a preferential expression of BK channels in unmyelinated vagal afferent neurons in rats, and suggest that these channels make a functional contribution to the neurophysiological properties of

TRPV1-expressing C-fiber or high-threshold A␦-fiber vagal afferent neurons. BK channels have a large unitary conductance and activate in response to membrane depolarization and binding of intracellular Ca2+ and Mg2+ (Lee and Cui, 2010). They are involved in the control of neuronal excitability by accelerating afterpolarization, shorting the action potential duration, enhancing the repolarization and mediating spike-frequency adaptation. Consequently their activation leads to a negative-feedback regulation of membrane excitability, calcium influx, and neurotransmitter release (Scholz et al., 1998; Zhang et al., 2003; Gu et al., 2007; Cao et al., 2012). In the present study, our results showed that blocking CaSR with its antagonist NPS 2143 significantly inhibited BK currents. This finding seems to suggest that CaSR is tonically activated, interacts with BK channels and affects the excitability of vagal bronchopulmonary sensory neurons. The abolition of this tonic effect results in the inhibition of BK channel activity and therefore increased neuronal excitability. This hypothesis is in agreement with findings from our recent study (Gu et al., 2013) which showed that inhibition of CaSR with NPS 2143 significantly potentiated the capsaicin-evoked TRPV1 responses and presumably leads to an increased neuronal excitability. In that study, our results also demonstrated that activation of CaSR with spermine or NPS R-568 markedly inhibited TRPV1 currents. Results from our present study, however, showed that the same concentration of spermine (3 mM) or NPS R-568 (0.5 ␮M) affected BK currents differently

Fig. 6. CaSR agonist NPS R-568 enhanced BK currents in cells with low baseline BK current density in rat bronchopulmonary sensory neurons. (A−F) Representative traces of total K+ currents, paxilline-resistant currents, and paxilline-sensitive BK currents elicited by the voltage protocol (G) without (A−C) or with (D−F) application of NPS R-568 (R568; 0.5 ␮M, 60 s). (H) I–V curves showing the current densities of total K+ currents and paxilline-resistant currents, before and after NPS R-568 application. (I) I–V curves showing that NPS R-568 markedly potentiated the BK current density. *Significant difference in current density between before and after NPS R-568 (P < 0.05, n = 17).

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Fig. 7. NPS R-568 suppressed BK currents in cells with high baseline BK current density in rat bronchopulmonary sensory neurons. (A−F) Representative traces of total K+ currents, paxilline-resistant currents, and paxilline-sensitive BK currents elicited by the voltage protocol (G) without (A−C) or with (D−F) application of NPS R-568 (0.5 ␮M, 60 s). (H) I–V curves showing the current densities of total K+ currents and paxilline-resistant currents, before and after NPS R-568 application. (I) I–V curves showing that NPS R-568 inhibited BK current density. *Significant difference in current density between before and after NPS R-568 (P < 0.05, n = 11).

in two different groups of bronchopulmonary sensory neurons: a marked potentiation in cells with the low baseline BK activity, and a slight suppression in cells exhibiting the high baseline BK activity; both of which are effectively abolished by CaSR antagonist

NPS 2143 indicating that the dual effects are indeed a specific action of CaSR activation (Fig. 9). Although the capacitances of above mentioned two groups of neurons are significantly different (Table 2), the vast majority (61 out of 68) of these BK-expressing

Fig. 8. Blockade of CaSR with NPS 2143 inhibited BK currents in rat bronchopulmonary sensory neurons. (A−F) Representative traces of total K+ currents, paxilline-resistant currents, and paxilline-sensitive BK currents elicited by the voltage protocol (G) without (A−C) or with (D−F) application of NPS 2143 (10 ␮M, 60 s). (H) I–V curves showing the current densities of total K+ currents and paxilline-resistant currents, before and after NPS 2143 application. (I) I–V curves showing the BK current densities before and after NPS 2143. *Significant difference in corresponding current densities between before and after NPS 2143 (P < 0.01, n = 10).

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Fig. 9. Pretreatment with a mixture of CaSR agonist and antagonist did not alter BK currents in rat bronchopulmonary sensory neurons. (A−F) Representative traces of total K+ currents, paxilline-resistant currents, and paxilline-sensitive BK currents elicited by the voltage protocol (G) without (A−C) or with (D−F) 1 min pretreatment with a mixture of NPS R-568 (0.5 ␮M) and NPS 2143 (1 ␮M). (H) I–V curves showing the current densities of total K+ currents and paxilline-resistant currents, before and after the application of NPS R-568 and NPS 2143 (P > 0.05, n = 8). (I) I–V curves showing the BK current densities before and after NPS R-568 and NPS 2143 (P > 0.05, n = 8).

neurons are indeed sensitive to capsaicin and presumably give rise to bronchopulmonary C-fibers or high-threshold A␦-fibers. We believe that the difference in baseline BK current density reflects the different expression and/or sensitivity of BK channels in these bronchopulmonary sensory neurons, however, the physiological significance of these two subsets of neurons responding differently to CaSR activation remains to be illustrated. BK channels are known to be colocalized to certain Ca2+ influx sources, such as voltage-activated Ca2+ channels (Marrion and Tavalin, 1998; Berkefeld et al., 2010; Müller et al., 2010), NMDA receptors (Isaacson and Murphy, 2001), and ryanodine receptors (Beurg et al., 2005). Results from our present study suggest that CaSR is likely to be another such Ca2+ influx source that BK is functionally related to. This assumption is supported by a recent study by Heyeraas et al. (2008) which showed that dental pulpal blood flow as measured by laser-Doppler flowmetry was significantly increased by the stimulation of CaSR with 5 mM Ca2+ or NPS R-467, and the blood flow increase was completely inhibited by BK channel

blocker IbTX, indicating a potential functional interaction between CaSR and BK in tooth dental pulp. It is known that CaSR couples to multiple G proteins and is linked to numerous intracellular pathways (Brown and MacLeod, 2001; Magno et al., 2011); these include the activation of protein kinase C (PKC) and intracellular Ca2+ mobilization resulted from the stimulation of phosphoinositide-specific phospholipase C (PLC) (Hofer and Brown, 2003; Awumey et al., 2007; Tharmalingam et al., 2011), as well as the inhibition of adenylate cyclase activity and cAMP accumulation (Gerbino et al., 2005; Mamillapalli et al., 2008). On the other hand, BK channels are known to be potentially regulated by several serine/threonine kinases, such as protein kinase A (PKA), protein kinase G (PKG) and PKC (Schubert and Nelson, 2001). For example in smooth muscle, PKA and PKG predominantly activate BK channels by increasing the voltage and Ca2+ sensitivity of the channel, whereas PKC exerts opposite effects (Schubert and Nelson, 2001). A recent study by Zhou et al. (2010) demonstrated that PKC via the unconditional and conditional phosphorylation of the BK channel not only decreases

Fig. 10. Regulation of BK currents by CaSR was abolished by Ca2+ chelator BAPTA-AM in rat bronchopulmonary sensory neurons. (A) Intracellular application of BAPTA-AM significantly inhibited BK current density (P < 0.01). BK currents were measured and compared between two different groups of neurons, in absence (n = 57) and presence (n = 22) of 35 ␮M BAPTA-AM in pipette solution. (B) CaSR agonist NPS R-568 did not significantly affect the BK current density in the presence of intracellular BAPTA-AM (P > 0.05, n = 13). (C) CaSR antagonist NPS 2143 failed to modulate BK currents in the presence of BAPTA-AM (P > 0.05, n = 9).

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channel open probability but also determines the regulation of the channel by PKA and PKG, in both recombinant channels and native BK channels from tracheal smooth muscle. Our results demonstrated that intracellular application of BAPTA-AM decreased BK channel function and completely abolished the regulation by either activation or blockade of CaSR, suggesting that the modulation of BK channel activities by CaSR is likely mediated through an alteration in the concentration of the intracellular Ca2+ . Although PLC–PKC intracellular cascade is known to be present in bronchopulmonary sensory neurons (Gu and Lee, 2012), whether activation of this intracellular pathway and the subsequent Ca2+ mobilization are fully responsible for the regulation of BK channel activities by CaSR shown in our present study remains to be determined. Vagal bronchopulmonary afferents are the primary communication pathways between the respiratory tract and central nervous system. The information sent from these afferent terminals in the form of action potentials is further integrated in the brainstem, and ultimately leads to sensations such as urge to cough and dyspnea, as well as various reflex outputs such as changes in breathing pattern, heart rate and blood pressure, cough, airway constriction, mucus secretion, mucosal edema and inflammatory cell chemotaxis (Barnes, 2001; Lee and Pisarri, 2001; Taylor-Clark and Undem, 2006). Together, these respiratory sensations and reflex responses serve important roles in maintaining physiological homeostasis and in defending the pulmonary system for the protection of its vital function of gas exchange (Coleridge and Coleridge, 1994; Bessac and Jordt, 2008). It has been known that the sensitivity of the afferent nerves to certain stimuli (e.g., cigarette smoke, airway acidification, etc.) and the pattern of action potential generation are not static properties, but rather can be regulated by physiological and disease conditions (Spina et al., 1998; Taylor-Clark and Undem, 2006; Gu and Lee, 2011). Although the concentration of Ca2+ is maintained near constant in the blood, local changes in Ca2+ occur physiologically due to the alterations in cellular activity, ion transport, disease, diet, or other processes (Brown and MacLeod, 2001; Bouschet and Henley, 2005; Gu et al., 2013). The ability of CaSR to orchestrate the neuronal excitability in the face of local changes in Ca2+ could be a physiological compensatory mechanism critical to maintain bronchopulmonary afferent functions. In addition, CaSR is known to recognize a variety of amazingly diverse substances such as other di- and tri-valent cations (e.g., Mg2+ , Gd3+ ), polyamines (e.g., spermine, spermidine), l-amino acids (e.g., phenylalanine, tryptophan) and polypeptides (e.g., polyarginine, polylysine) (Brown and MacLeod, 2001; Magno et al., 2011). The fluctuation of these CaSR activators other than Ca2+ may also serve as the extracellular signals triggering the integrative function of CaSR, and therefore regulate the excitability of vagal bronchopulmonary afferents. In summary, our study demonstrated the expression of BK and its coexpression with CaSR and TRPV1 channels in rat bronchopulmonary sensory neurons. Our results also showed that the BK channel activity is dynamically regulated by CaSR, an effect completely abolished by chelation of intracellular Ca2+ . Our data seem to suggest that CaSR plays an integrative role in bronchopulmonary afferent signaling, at least partially through the regulation of BK channel activities.

Acknowledgements The authors thank Timothy Kim, Phat Vu and Robin Jacob for their technical assistance. The authors also thank Dr. Hong-Zhen Hu from Washington University School of Medicine for his constructive comments and suggestions. This work was supported by grants from the American Heart Association, MEDCEN Community Health Foundation and Mercer University School of Medicine.

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