P2Y receptor regulation of K2P channels that facilitate K⁺ secretion by human mammary epithelial cells

Abstract

The objective of this study was to determine the molecular identity of ion channels involved in K⁺ secretion by the mammary epithelium and to examine their regulation by purinoceptor agonists. Apical membrane voltage-clamp experiments were performed on human mammary epithelial cells where the basolateral membrane was exposed to the pore-forming antibiotic amphotericin B dissolved in a solution with intracellular-like ionic composition. Addition of the Na⁺ channel inhibitor benzamil reduced the basal current, consistent with inhibition of Na⁺ uptake across the apical membrane, whereas the K_Ca3.1 channel blocker TRAM-34 produced an increase in current resulting from inhibition of basal K⁺ efflux. Treatment with two-pore potassium (K2P) channel blockers quinidine, bupivacaine and a selective TASK1/TASK3 inhibitor (PK-THPP) all produced concentration-dependent inhibition of apical K⁺ efflux. qRT-PCR experiments detected mRNA expression for nine K2P channel subtypes. Western blot analysis of biotinylated apical membranes and confocal immunocytochemistry revealed that at least five K2P subtypes (TWIK1, TREK1, TREK2, TASK1, and TASK3) are expressed in the apical membrane. Apical UTP also increased the current, but pretreatment with the PKC inhibitor GF109203X blocked the response. Similarly, direct activation of PKC with phorbol 12-myristate 13-acetate produced a similar increase in current as observed with UTP. These results support the conclusion that the basal level of K⁺ secretion involves constitutive activity of apical K_Ca3.1 channels and multiple K2P channel subtypes. Apical UTP evoked a transient increase in K_Ca3.1 channel activity, but over time caused persistent inhibition of K2P channel function leading to an overall decrease in K⁺ secretion.

INTRODUCTION

Previous studies have shown that the post-partum Na⁺ concentration in human milk ranges between 5 and 8 mM whereas the K⁺ concentration can vary from 11 to 15 mM (25, 41, 49). The Na⁺ concentration continues to decrease with time after delivery, with the greatest reduction occurring within the first 90 days (41). The mean K⁺ concentration also decreases during the first 60 days, but then stabilizes and does not significantly change over the following 360 days of lactation (49). In contrast, colostrum, the fluid present during early lactation, is produced at a much lower volume and has a higher [Na⁺] and lower [K⁺] than in milk (28). Low Na⁺ and high K⁺ concentrations in milk relative to plasma have also been reported for mice, rabbits, guinea pigs, cattle, and horses, indicating that the concentrations of these electrolytes are tightly regulated and highly conserved among mammals (2, 4, 12, 37, 42). In humans, both Na⁺ and K⁺ concentrations exhibit circadian variations that correlate with changes in milk and plasma glucocorticoid levels (25). Moreover, administration of dexamethasone produced a significant decrease in Na⁺ concentration and increased K⁺ concentration in human milk, demonstrating a role for corticosteroids in the regulation of electrolyte composition. Similarly, prolactin secretion also affects ion composition by regulating the Na⁺/K⁺ concentration ratio in milk (37).

Early in vitro flux experiments using primary mouse mammary epithelium grown on floating collagen gels mounted in Ussing chambers revealed net Na⁺ absorption and essentially no net K⁺ transport (based on ⁸⁶Rb⁺ tracer flux measurements) under basal conditions in the presence of insulin and cortisol (6, 7). The net Na⁺ flux was shown to completely account for the basal short-circuit current (I_sc), suggesting that the mechanism of Na⁺ absorption was electrogenic. This interpretation was supported by data showing that apical treatment with amiloride, an inhibitor of epithelial sodium channels (ENaC), reduced the I_sc. Stimulation with prolactin significantly increased net Na⁺ absorption by 3.7-fold but did not significantly increase K⁺ or Cl⁻ transport (6). More recent studies have shown that ENaC subunits and CFTR are expressed within the apical membrane of a murine mammary epithelial cell line (31EG4 cells), which possesses properties of both acinar and ductal epithelial cells (8). Fluid secretion across monolayers of 31EG4 cells was stimulated by increasing intracellular cAMP, resulting in activation of CFTR-dependent anion secretion or by inhibiting ENaC-dependent Na⁺ transport with amiloride. Fluid absorption was reported to be dependent on ENaC-mediated Na⁺ absorption and the coupled paracellular movement of Cl⁻. Measurements of I_sc in an immortalized bovine mammary epithelial cell line (BME-UV cells) showed that basal amiloride-sensitive Na⁺ absorption was very low; however, basolateral exposure to natural or synthetic glucocorticoids for 72 h significantly stimulated Na⁺ transport (40). This increase in Na⁺ absorption was associated with significant upregulation of β and γ ENaC mRNA expression with little change in the expression of the α subunit. Similarly, previous experiments with an immortalized human mammary epithelial cell line grown in the presence of a pituitary extract containing prolactin exhibited basal ENaC-dependent Na⁺ absorption (31). Exposure to hydrocortisone significantly upregulated the expression of α, β, and γ ENaC subunits and stimulated a nearly twofold increase in ENaC-dependent I_sc. Together the results from experiments with murine, bovine, and human mammary epithelial cell models indicate that the predominant electrogenic mechanism for transepithelial Na⁺ absorption involves apical ENaC and that this channel is subject to regulation by glucocorticoids and prolactin. Therefore, it is reasonable to speculate that the low Na⁺ concentration observed in milk is a consequence of ENaC-dependent Na⁺ absorption.

The ion transport function of the mammary epithelium was previously shown to be regulated by purinergic receptor agonists. Earlier studies in human MCF-7 breast tumor cells showed that exposure to ATP induced a concentration-dependent increase in intracellular calcium concentration ([Ca²⁺]_i) and an increase in ¹²⁵I efflux, a measure of anion channel activation (20). The rank order of potency for several purinergic agonists as well as the lack of effect of extracellular Ca²⁺ chelation on changes in [Ca²⁺]_i evoked by ATP indicated that P2Y receptors were responsible for the increase in [Ca²⁺]_i and anion channel activation. Furthermore, treatment with 4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid (DIDS), a known inhibitor of Ca²⁺-activated Cl⁻ channels, partially blocked ¹²⁵I efflux, suggesting that these channels were involved in Ca²⁺-dependent anion transport. P2Y₂ receptor agonists (UTP and ATP) also stimulated Ca²⁺-dependent Cl⁻ secretion and fluid secretion in 31EG4 cells (9). The receptors were localized on both apical and basolateral membranes and apical administration of DIDS partially blocked anion secretion evoked by either apical or basolateral addition of UTP or ATP. More recently, experiments with HC11 cells, an immortalized murine mammary epithelial cell line, revealed that exposure to prolactin and dexamethasone reduced TRPC3-mediated Ca²⁺ uptake, resulting in suppression of Ca²⁺ signaling following ATP stimulation (1). This effect was dependent on the endogenous expression of the long form of the prolactin receptor (PRLR-L). Interestingly, expression of the short form of PRLR, in the absence of prolactin and dexamethasone, also attenuated Ca²⁺ signaling by inhibiting TRPC3-dependent Ca²⁺ influx and increasing Ca²⁺ sequestration by stimulating Ca²⁺-ATPase 2 expression.

Basolateral stimulation of primary and immortalized human mammary epithelial cells produced an oscillating increase in the ENaC-dependent I_sc, indicating an increase in electrogenic Na⁺ absorption (31). This effect appeared to be dependent on an increase in driving force for apical Na⁺ uptake resulting from opening of basolateral clotrimazole and charybdotoxin (CTX)-sensitive K_Ca3.1 Ca²⁺-activated K⁺ channels and on CTX/clotrimazole-insensitive K⁺ channels of unknown molecular identity. Interestingly, apical administration of UTP evoked an increase in K⁺ secretion mediated by activation of K_Ca3.1 channels (38). The distinct ion transport-related responses to apical versus basolateral P2Y receptor stimulation appears to result from discrete compartmentalization of Ca²⁺ signaling in human mammary epithelial cells. These findings supported the conclusion that apical P2Y receptor stimulation could produce a transient increase in K⁺ secretion in human mammary epithelial cells and that K_Ca3.1 channels may contribute to the elevated levels of K⁺ observed in human milk.

The objective of the present study was to investigate the molecular mechanisms that underlie the constitutive secretion of K⁺ by human mammary epithelial cells. Earlier ⁸⁶Rb flux experiments using cell fragments consisting of cytoplasm encapsulated by the apical membrane of epithelial cells from goats’ milk revealed the existence of Ba²⁺ and quinine-sensitive K⁺ channels (45). Since Ba²⁺ and quinine are blockers of K2P channels, we investigated the hypothesis that K2P channels expressed within the apical membrane of human mammary epithelial cells are responsible for basal K⁺ secretion coupled to ENaC-dependent Na⁺ absorption. Our results demonstrated that these cells express several K2P channel subtypes within the apical membrane and some of these channels are inhibited following purinergic receptor stimulation.

MATERIALS AND METHODS

Materials.

Uridine triphosphate (UTP), benzamil hydrochloride, 1-[(2-chlorophenyl) diphenylmethyl]-1H -pyrazole (TRAM-34), quinidine, amphotericin B, phorbol 12-myristate 13-acetate (PMA), and 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO). Paraformaldehyde (16%) was obtained from VWR (Radnor, PA), and GF109203X and PK-THPP were purchased from Tocris (Bristol, UK). Methyl β-cyclodextrin (MβCD) was procured from Santa Cruz (Dallas, TX) while bupivacaine was obtained from Cayman Chemical (Ann Arbor, MI).

Cell culture.

Human mammary epithelial cells were immortalized following transfection with the catalytic subunit of human telomerase gene as previously described (26). These cells (passage number 38–50) were cultured in mammary epithelial basal medium (MEGM) with growth factor supplements (Lonza, Basel, Switzerland). Cell monolayers grown on 12 mm, 0.4 μm pore size Snapwell polyester membranes (Corning Life Sciences, Lowell, MA) were incubated at 37°C in a humidified atmosphere of 5% CO₂ in air.

Epithelial voltage-clamp experiments.

Transepithelial resistance was measured with Ag-AgCl₂ chopstick electrodes connected to an epithelial voltohmmeter (EVOM) [World Precision Instruments (WPI), Sarasota, FL]. Epithelial monolayers at their maximum resistance (200–250 Ω·cm²) during 9–14 days after seeding were mounted in Ussing chambers and bathed on both apical and basolateral sides with standard saline solution containing (in mM): 130 NaCl, 5 KCl, 1.5 CaCl₂, 1 MgCl₂, 20 NaHCO₃, 0.3 NaH₂PO₄, and 1.3 Na₂HPO₄, pH 7.4 and maintained at 37°C with 95% O₂-5% CO₂ gassing. Measurements of short-circuit current (I_sc) were performed using DVC1000 epithelial voltage clamps (WPI). Data were acquired using a Digidata 1322 data acquisition system (Axon Instruments/Molecular Devices, Union City, CA) connected to a Dell Optiplex 760 microcomputer and analyzed using Axoscope software (Axon Instruments/Molecular Devices). The reference electrode was located in the apical bathing solution for both I_sc and apical membrane current measurements. For apical membrane current experiments, the basolateral membrane was permeabilized with amphotericin B (15 μM) and bathed with KMeSO₄ saline solution (in mM: 120 K-MeSO₄, 10 NaCl, 20 KHCO₃, 1 CaCl₂, 1 MgCl₂, 1 Na₂HPO₄, pH 7.4) while the apical membrane was bathed with standard saline solution. Partitioning of amphotericin B into the basolateral membrane was associated with a time-dependent increase in current, which reached steady state within ~15 min. This current was blocked by treatment with 5 μM benzamil (Fig. 1, C and D), reflecting movement of Na⁺ from the apical solution into the intracellular compartment. In contrast, addition of TRAM-34, an inhibitor of K_Ca3.1 channels, produced a sustained increase in current resulting from inhibition of basal K⁺ secretion (Fig. 1, C and D). Basolateral permeabilization by amphotericin B was also indicated by the persistent effects of luminal UTP on apical membrane current, which was not observed in measurements of I_sc.

Fig. 1.

Effects of two-pore potassium (K2P) channel blockers on the basal short-circuit current (I_sc)_c of immortalized human mammary epithelial cells. A: representative tracing showing the I_sc response to apical administration of the K2P channel blocker bupivacaine (200 μM) followed by apical UTP (10 μM). The remaining I_sc was inhibited by apical addition of the Na⁺ channel blocker benzamil (5 μM). B: average changes in the I_sc response to the K2P channel blockers quinidine and bupivacaine followed by stimulation with apical UTP (n = 6). *P < 0.05, significantly different (unpaired two-tailed t-test) from zero (no change in I_sc). Initial mean ± SE values for current and resistance were: 6.9 ± 1.7 μA; 210.8 ± 14.6 Ω·cm² (bupivacaine, n = 6); 6.2 ± 2.1 μA; 215.6 ± 38.9 Ω·cm² (quinidine, n = 6). C: representative tracing of apical membrane current showing the effect of the K_Ca3.1 channel blocker TRAM-34 (10 μM, apical) followed by apical addition of benzamil. D: average changes in the apical membrane current after 10 μM TRAM-34 (apical) followed by benzamil (n = 10). *P < 0.05, significantly different (unpaired two-tailed t-test) from zero (no change in I_sc). Initial mean ± SE values for current and resistance were 6.4 ± 1.7 μA; 219.8 ± 21.0 Ω·cm² (TRAM-34, n = 10).

Western blotting.

Apical membrane protein was harvested from cells grown on six-well plates using the Pierce cell surface protein isolation kit (Thermo-Fisher Scientific, Waltham, MA) and quantified with the Qubit Protein Assay Kit (Thermo-Fisher Scientific). Apical membrane protein (20 µg/ml) was heated to 70°C for 10 min in NuPAGE LDS sample buffer before loading onto NuPAGE 4–12% Bis-Tris gels with Chameleon Duo-prestained protein ladder (Li-Cor Biosciences, Lincoln, NE). Proteins were separated by electrophoresis using 3-(N-morpholino)-propanesulfonic acid (MOPS)/sodium dodecyl sulfate (SDS) buffer (200 V, 35 min) followed by transfer to a nitrocellulose membrane (Thermo-Fisher Scientific) in NuPAGE transfer buffer. Blotted membranes were blocked in Odyssey blocking buffer (Li-Cor Biosciences) for 1 h at room temperature. Membranes were incubated overnight at 4°C with primary antibodies diluted in Odyssey blocking buffer containing 0.1% Tween20 followed by a 1 h incubation at room temperature with IR Dye secondary antibodies (Li-Cor Biosciences) diluted in Odyssey blocking buffer. Proteins were visualized using an Odyssey CLx imager (Li-Cor Biosciences). The primary antibodies used were goat anti-TWIK1 (1:200) (Santa Cruz), rabbit anti-TREK1 (1:200), rabbit anti-TREK2 (1:200), rabbit anti-TASK1 (1:200) (Alomone Laboratories, Jerusalem, Israel), and rabbit anti-TASK3 (1:200) (Abcam, Cambridge, UK).

Immunocytochemistry.

Cell monolayers grown on Snapwell membranes were fixed in 4% paraformaldehyde and permeabilized using 0.3% Triton-X. After nonspecific blocking with 3% bovine serum albumin (BSA; Sigma Aldrich Chemicals) in phosphate-buffered saline solution (PBS; Thermo-Fisher Scientific), the cells were incubated with the primary antibody diluted in 3% BSA solution overnight at 4°C. After rinsing with PBS, cells were incubated with secondary antibody for 1 h at room temperature. The primary antibodies used for immunocytochemistry along with their source, dilution, and company where they were purchased are listed in Table 1. The Alexa Fluor-conjugated secondary antibodies (dilution; 1:250) that were used included Alexa 488 chicken anti-rabbit IgG, Alexa 568 donkey anti-mouse IgG, and Alexa 647 donkey anti-goat IgG (Thermo-Fisher). 4′,6-Diamidino-2-phenylindole (DAPI) was used to label the nuclei. Monolayers were mounted on microscope slides and imaging was performed using an Olympus FV1000 confocal microscope.

Table 1.

Primary antibodies used for Western blot and immunocytochemistry experiments

Target Protein	Source	Dilution	Company	Catalog
CFTR	Mouse	1:150	Abcam, Cambridge, UK	Ab2784
ENaCα	Rabbit	1:150	Abcam, Cambridge, UK	Ab65710
ENaCγ	Goat	1:150	Abcam, Cambridge, UK	Ab115272
KCa3.1	Goat	1:150	Santa Cruz, Dallas, TX	SC-27080
α1-Na+-K+-ATPase	Mouse	1:150	Abcam, Cambridge, UK	Ab7671
α2-Na+-K+ ATPase	Rabbit	1:150	Abcam, Cambridge, UK	Ab166888
P2Y2	Rabbit	1:100	Santa Cruz, Dallas, TX	SC-20124
P2Y4	Goat	1:100	Santa Cruz, Dallas, TX	SC-17634
P2Y6	Goat	1:50	Santa Cruz, Dallas, TX	SC-15215
TWIK1	Goat	1:100	Santa Cruz, Dallas, TX	SC-11481
TREK1	Rabbit	1:100	Alomone Laboratories, Jerusalem, Israel	APC-047
TREK2	Rabbit	1:100	Alomone Laboratories, Jerusalem, Israel	APC-055
TASK1	Rabbit	1:100	Alomone Laboratories, Jerusalem, Israel	APC-024
TASK3	Rabbit	1:100	Abcam, Cambridge, UK	Ab85289

Semiquantitative RT-PCR.

Total RNA was extracted using the RNeasy Mini Kit from Qiagen (Hilden, Germany). RNA was quantified by spectrophotometry with a ratio of A260/A280 (1.8–2.0). Total RNA was then reverse-transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). cDNA (1 µg), and TaqMan PCR probes (TWIK 1–2, TREK 1–2, TASK 1–5, GAPDH; Thermo Fisher Scientific) were used to determine relative expression by quantitative RT-PCR on an Applied Biosystems 7300 Real-Time qPCR system. Relative expression was analyzed using the comparative method (2^−Δct), which normalizes the threshold cycle (C_T) of the target gene to an internal reference gene (GAPDH).

Statistics.

All data are reported as means ± SE, and n is the number of monolayers used in each experiment. Significant differences between control and treatment conditions in each experiment were analyzed by using unpaired, two-tailed t-tests or by an analysis of variance (ANOVA) followed by Bonferroni’s posttest (Prism 6.0, GraphPad Software, San Diego, CA), and P < 0.05 was considered significant. The concentration-response data presented in Fig. 2B were fit by nonlinear regression using a three parameter logistic function where apical current (I_a) =

$$\left[\frac{I_{\text{a}\left(\text{max}\right)}}{1+{10}^{\left({\text{log\hspace{0.17em}EC}}_{\text{50}}-x\right)}}\right]$$

using Prism 6.0. The EC₅₀ values provided estimates of blocker concentrations that produced half-maximal inhibition of the current.

Fig. 2.

Two-pore potassium (K2P) channel blockers increase apical membrane current. A: representative traces showing the effects of PK-THPP (5 μM), bupivacaine (100 μM), and quinidine (100 μM) on apical membrane current. B: concentration-response relationships for PK-THPP (●), bupivacaine (♦), and quinidine (▲). Half-maximal inhibitory concentrations were calculated by fitting these data with a three-parameter logistic function in Prism 6.0. Initial mean ± SE values for current and resistance were 6.8 ± 1.7 μA; 225.8 ± 12.1 Ω·cm² (PK-THPP, n = 6), 6.2 ± 2.1 μA; 254.2 ± 34.2 Ω·cm² (bupivacaine, n = 8); 6.4 ± 1.3 μA; 243.3 ± 21.5 Ω·cm² (quinidine, n = 8).

RESULTS

Our previous studies showed that apical addition of UTP (10 μM) produced a transient decrease in I_sc, which was due to activation of K⁺ secretion mediated by intermediate-conductance Ca²⁺-activated K⁺ channels (K_Ca3.1) in the apical membrane (38). In the present study, administration of the K2P channel blocker bupivacaine (200 μM) to the apical solution produced an immediate increase in I_sc with a peak response of 1.5 ± 0.4 μA (n = 6), which gradually decreased to baseline as shown in Fig. 1A. Subsequent apical addition of UTP induced a small decrease in I_sc of 1.2 ± 0.2 μA (n = 6). The remaining current was abolished by apical addition of the epithelial Na⁺ channel blocker benzamil (5 μM). A similar increase in the I_sc response was observed with 200 μM quinidine, another K2P channel blocker (0.7 ± 0.3 μA, n = 4) followed by a small decrease in current after treatment with UTP (0.5 ± 0.1 μA, n = 6) as shown in Fig. 1B. Experiments with amphotericin B-permeabilized monolayers showed that pretreatment with 10 μM TRAM-34, a K_Ca3.1 channel blocker, produced a small sustained increase in apical membrane current, consistent with inhibition of basal K⁺ efflux (2.0 ± 0.6 μA, n = 10). Subsequent apical addition of benzamil resulted in a decrease in current which correlated with inhibition of Na⁺ uptake (13.0 ± 1.3 μA, n = 10) as shown in Fig. 1, C and D.

The effects of bupivacaine, quinidine, and a known TASK1/TASK3 inhibitor (13) (PK-THPP) on apical membrane current are shown in Fig. 2A. Each of these K2P channel blockers produced concentration-dependent increases in current consistent with inhibition of basal K⁺ efflux across the apical membrane (Fig. 2B). Analyses of concentration-response curves revealed half-maximal inhibitory concentrations of 19.2 ± 0.9 (n = 8), 27.1 ± 1.1 (n = 8), and 1.1 ± 2.0 (n = 6) μM for bupivacaine, quinidine, and PK-THPP, respectively. Thus PK-THPP was ~20–30 times more potent than either quinidine or bupivacaine. The values obtained with human mammary epithelial cells differed from results using cloned human TASK1 channels expressed in oocytes (IC₅₀ = 170 μM and 300 μM for quinidine and bupivacaine, respectively) and previously measured PK-THPP IC₅₀ values for human TASK1 (35 nM) and TASK3 (300 nM) channels expressed in HEK293 cells where channel activity was measured using a voltage-sensitive dye (12, 46). These differences may be due to apical expression of multiple K2P channels in human mammary epithelial cells where, presumably, subtypes other than TASK1 and TASK3 contribute to the effects of these blockers. Hence, comparisons to cloned channels expressed in heterologous expression systems should be interpreted accordingly.

The expression and subcellular localization of K_Ca3.1 and ENaC subunits were examined by immunocytochemistry and confocal imaging. As shown in Fig. 3, ENaCα, ENaCγ, and K_Ca3.1 appeared to be associated with the apical membrane. In contrast, immunoreactivity of the α₁ subunit (ATP1A1) of the Na⁺-K⁺ ATPase was localized at or near the basolateral membrane. However, the α₂ (ATP1A2) subunit antibody labeled the cytoplasm as well as the basolateral membrane.

Fig. 3.

Confocal immunocytochemistry images showing subcellular localization of α (green) and γ (red) epithelial sodium channel (ENaC) subunits, K_Ca3.1 (red), α1 (red) and α₂ (green) subunits of the Na⁺-K⁺ ATPase (ATP1) in immortalized human mammary epithelial cells. Nuclei (blue) were labeled with DAPI. Z-stack projections show localization of ENaCα, ENaCγ, and K_Ca3.1 in the apical region, and expression of α₁ and α₂ subunits of the Na-K ATPase associated with the basolateral membrane (blm) and cytoplasm.

The effect of apical P2Y receptor stimulation with UTP on apical membrane current is presented in Fig. 4. Apical addition of 10 μM UTP evoked a biphasic change in current. UTP initially produced a transient decrease consistent with stimulation of K⁺ secretion followed by a slow and sustained increase in current reflecting inhibition of apical K⁺ efflux (Fig. 4A). Pretreatment with the Ca²⁺-chelating agent BAPTA-AM (50 μM) for 15 min completely abolished the UTP-dependent decrease in current and significantly reduced the increase in current by 36% (Fig. 4B). This result indicated that both stimulatory and a portion of the inhibitory effect of UTP on apical membrane K⁺ transport were dependent on intracellular [Ca²⁺]. The possible involvement of K2P channels in UTP-inhibited K⁺ secretion was subsequently investigated by pretreating permeabilized monolayers with K2P channel blockers followed by stimulation with apical UTP. As shown in Fig. 4C, apical addition of bupivacaine (100 μM), quinidine (100 μM), or PK-THPP (5 μM) all increased the apical current by 10.2 ± 1.0 μA (n = 9), 14.5 ± 1.1 μA (n = 8), and 12.1 ± 1.6 μA (n = 9), respectively. Pretreatment with the same concentrations of bupivacaine or quinidine reduced most of the inhibitory effect of UTP on apical membrane current, whereas pretreatment with PK-THPP completely abolished the UTP response.

Fig. 4.

Apical stimulation with UTP inhibits two-pore potassium (K2P) channel-dependent current. A: representative tracing of the change in apical current following apical UTP (10 μM) stimulation. Note that the initial decrease in current indicates stimulation of K⁺ secretion whereas the sustained increase in current reflects inhibition of K⁺ secretion. B: average changes in the apical membrane current following stimulation with apical UTP alone or after pretreatment with 50 μM BAPTA-AM (n = 6) and following a 2 h treatment with 10 mM methyl β-cyclodextrin (MβCD; n = 5). Initial mean ± SE values for current and resistances were 6.2 ± 1.3 μA; 232.2 ± 33.7 Ω·cm² (UTP, n = 8); 6.5 ± 1.1 μA; 228.0 ± 19.2 Ω·cm² (BAPTA-AM + UTP, n = 6); 4.3 ± 2.8 μA; 229.7 ± 12.8 Ω·cm² (MβCD + UTP, n = 5). *P < 0.05, significant differences between UTP and BAPTA-AM treatment conditions and MβCD + UTP (inhibition of K⁺ secretion) using a one-way ANOVA with Bonferroni’s multiple-comparisons posttest. C: average changes in apical current following treatment with bupivacaine (Bupiv), quinidine (Quin), or PK-THPP alone or with UTP after pretreatment with these K2P channel blockers. Initial mean ± SE values for current and resistances were 4.8 ± 2.6 μA; 221.8 ± 33.7 Ω·cm² (bupivacaine, n = 9); 4.6 ± 1.7 μA; 224.5 ± 9.8 Ω·cm² (quinidine, n = 8); 5.3 ± 2.2 μA; 230.5 ± 7.7 Ω·cm² (PK-THPP, n = 9). *P < 0.0001, significant differences using unpaired, two-tailed t-tests.

To address the question of whether apical P2Y receptor regulation of K2P channel activity depended on associations with lipid raft domains, the effect of UTP on apical current was tested in the presence of the lipid raft disruptor compound methyl-β-cyclodextrin (MβCD). As shown in Fig. 4B, pretreatment of monolayers with 10 mM MβCD for 2 h did not affect the decrease in apical membrane current induced by UTP, but significantly reduced the increase in current by more than 80%. It appears likely that P2Y₂ and/or P2Y₄ receptors were responsible for mediating the inhibitory effect of UTP since no response to apical UDP (50 μM; an activator of P2Y₆ receptors) on electrogenic Na⁺ or K⁺ transport was observed (data not shown). Moreover, immunocytochemistry experiments revealed that P2Y₂, P2Y₄, and P2Y₆ receptors were associated with the apical region of the epithelium (Fig. 5).

Fig. 5.

Confocal immunocytochemistry images showing localization of purinergic receptor P2Y₂ (P2Y₂-R; green) with γ-epithelial sodium channel (γENaC; red), and CFTR as apical membrane reference proteins and P2Y₄-R (green) and P2Y₆-R (green) receptors in or near the apical membrane of human mammary epithelial cells. Merged images show labeling overlap in yellow. Z-stack projections indicate localization of P2Y₂, P2Y₄, and P2Y₆ receptors with ENaC subunits and CFTR at or near the apical membrane. Nuclei (blue) were labeled with DAPI.

Data shown in Fig. 6, A and B, reveal that apical pretreatment with the PKC activator PMA (2 μM) for 10 min produced an increase in current and significantly reduced any further change in the steady-state current evoked by subsequent addition of UTP. In contrast, preincubation for 20 min with GF109203X (2 μM), a pan-selective PKC inhibitor, significantly blocked the inhibitory effect of UTP (Fig. 6B). However, neither PMA nor GF109203X altered the decrease in apical membrane current evoked by UTP. These results suggest that UTP-sensitive increases in apical membrane current were dependent on PKC activation.

Fig. 6.

Effects of protein kinase C activation and two-pore potassium (K2P) channel blockers on the UTP-dependent apical membrane current of human mammary epithelial cells. A: representative apical membrane current tracing showing the effect of the pan-selective PKC activator PMA (2 μM) added to the apical solution followed by apical UTP (10 μM). B: histogram showing the stimulatory and inhibitory effects of apical UTP on K⁺ secretion, the effect of apical UTP after pretreatment with apical PMA (2 μM) and the effect of apical UTP after pretreatment with the protein kinase C inhibitor GF109203x (2 μM). Initial mean ± SE values for current and resistances were 6.2 ± 1.3 μA; 232.2 ± 33.7 Ω·cm² (UTP, n = 8); 6.0 ± 2.6 μA; 238.0 ± 11.6 Ω·cm² (PMA, n = 8); 4.2 ± 2.2 μA; 221.3 ± 7.3 Ω·cm² (GF109203x, n = 8). *P < 0.05 and †P < 0.01, significant differences by ANOVA followed by Bonferroni’s multiple comparisons posttest.

The identity of K2P channels expressed by human mammary epithelial cells was examined using qRT-PCR, Western blotting, and immunocytochemistry. The relative mRNA expression of nine K2P channel subtypes including TWIK1, TWIK2, TREK1, TREK2, TASK1, TASK2, TASK3, TASK4, and TASK5 was measured and is reported in Fig. 7A. Western blots of proteins obtained from biotinylated apical membranes identified four K2P channel subtypes that were previously shown to be regulated by PKC along with TWIK1, which exhibited the highest level of mRNA expression compared with the other K2P channels (Fig. 7B). Results with the anti-TWIK1 antibody showed labeling of three proteins between ~47 and ~55 kDa. The same antibody previously identified TWIK1 in cultures of cerebellar granule neurons, appearing as a broad band of labeling ranging between ~45 and 55 kDa (11). In contrast, a single band was observed for TASK1 with a molecular mass of ~50 KDa, consistent with results from an earlier study using the same antibody to detect the channel in murine heart and brain (35). Knockout of TASK1 in these mice eliminated detection of the protein with this antibody, confirming its specificity for TASK1. Anti-TASK3 antibody labeled two proteins at ~49 and ~57 kDa. The 57 kDa protein was observed previously in Western blots from MCF-7 cells, a human mammary epithelial cancer cell line, and MG63 cells, a human osteoblast-like cell line (30, 32). Anti-TREK1 and anti-TREK2 antibodies labeled multiple proteins between ~47 and ~60 kDa. Earlier studies have distinguished five unique splice variants for TREK1 in human myometrial tissue (14, 50) and three splice variants for TREK2 associated with the NH₂-terminal domain of the channel (36). Whether the various proteins detected on Western blots in the present study represent splice variants of TREK1 or TREK2 is unknown; however, it is interesting to note that the TREK2b splice variant, which contains a PKC phosphorylation site that is not present in the other two variants, has a predicted molecular mass of ~51 KDa, consistent with one of the proteins detected for TREK2 (36). Other possibilities that could explain the existence of multiple bands include variations in glycosylation or perhaps other posttranslational modifications. Immunocytochemistry results using these same antibodies are presented in Fig. 8. Each of these K2P channel subtypes exhibited a labeling pattern similar to P2Y receptors, ENaC and CFTR at or near the apical membrane.

Fig. 7.

Messenger RNA and Western blots showing two-pore potassium (K2P) channel subtypes expressed in human mammary epithelial cells. A: relative mRNA expression of nine K2P channel subtypes (TWIK1, TWIK2, TREK1, TREK2, TASK 1–5) with GAPDH serving as the reference gene. Each value represents the mean ± SE (n = 5). B: representative Western blots showing expression of five K2P channel subtypes (TWIK1, TREK1, TREK2, TASK1, and TASK3) within biotinylated apical membranes obtained from three independent protein isolations.

Fig. 8.

Immunocytochemistry showing subcellular localization of five (TWIK1, TREK1, TREK2, TASK1, and TASK3) two-pore potassium (K2P) channel subtypes and either P2Y₂ or P2Y₄ receptors at or near the apical membrane. P2Y₂-R or P2Y₄-R (red); K2P subtypes (green); merged images (yellow), and nuclei (blue). The merged Z-stack images show P2Y-R and K2P channel expression in yellow.

DISCUSSION

In the present study, apical administration of the pan-selective K2P channel blockers bupivacaine and quinidine produced transient increases in I_sc that gradually returned to the initial baseline current, consistent with inhibition of K⁺ secretion. We speculate that the gradual decrease in I_sc resulted from apical membrane depolarization, which would be expected to reduce the driving force for benzamil-sensitive Na⁺ uptake across the apical membrane and net Na⁺ absorption. Experiments with amphotericin B-permeabilized monolayers made it possible to voltage clamp the apical membrane and uncouple ENaC-dependent Na⁺ absorption from K⁺ secretion. Under these conditions, treatment with apical K2P channel blockers produced a persistent increase in apical membrane current, indicating that constitutive K2P channel activity offsets a portion of the ENaC-dependent I_sc under basal conditions. Earlier studies using Calu-3 cells, a model human airway submucosal gland cell line, demonstrated the presence of multiple K2P channel subtypes (TWIK1, TREK1, TASK2) associated with the apical membrane (19). Inhibition of these channels with bupivacaine or quinidine significantly reduced the I_sc, indicating that the activity of K2P channels in the apical membrane support basal anion secretion by offsetting apical membrane depolarization associated with anion efflux. More recently, three K2P channel subtypes (TWIK1, TWIK2, and TASK2) were identified in the apical membrane of normal human bronchial epithelial cells and inhibition with bupivacaine or quinidine significantly reduced amiloride-sensitive Na⁺ absorption and forskolin-activated anion secretion (51). Basolaterally localized K2P channels are also known to contribute to Na⁺ absorption. In H441 cells, a human lung adenocarcinoma cell line, several K2P channel subtypes have been identified (TWIK1, TREK1, TASK2, TWIK2, KCNK7, TASK3, TREK2, THIK1, and TALK2) and basolateral treatment with bupivacaine or basolateral acidification produced inhibition of the ENaC-dependent I_sc (23).

Apical membrane current was likewise increased in the presence of the K_Ca3.1 channel blocker TRAM-34, suggesting that these channels also participate in K⁺ secretion under basal conditions. Immunocytochemistry experiments demonstrated that K_Ca3.1 immunoreactivity was associated with the apical region of the epithelium, similar to both α and γ subunits of ENaC. Furthermore, results from immunocytochemistry experiments demonstrated that α₁ and α₂ subunits of the Na⁺-K⁺ ATPase are expressed at or near the basolateral membrane as well as in the cytoplasm. Previous studies have demonstrated that α/β subunit heterodimers display distinct Na⁺ affinities, where α₁/β₁ and α₁/β₂ dimers exhibit greater affinity than their corresponding α₂/β dimer complements in heterologous expression systems as well as in native skeletal muscle fibers (5, 15, 16, 27, 34). We speculate that the α₂ subunits are distributed within membrane vesicles associated with the basolateral membrane and that under conditions where transepithelial Na⁺ absorption is stimulated [e.g., during basolateral exposure to purinergic agonists (31)], recruitment of α₂ subunits into the membrane may occur as a mechanism to increase Na⁺ efflux across the basolateral membrane. The lower Na⁺ affinity of the α₂/β heterodimers could potentially serve to extend the range of intracellular Na⁺ concentrations where Na⁺-K⁺ ATPase activity remains below its maximum transport rate.

Our previous studies of purinergic regulation of Na⁺ and K⁺ transport across human mammary epithelial cell monolayers demonstrated that apical administration of UTP or a nonhydrolyzable analog of ATP (ATP-γ-S) evoked a transient decrease in I_sc that was blocked by inhibitors of K_Ca3.1 channels, consistent with activation of K⁺ secretion (38). Immunocytochemistry data reported in the present study revealed apical localization of multiple P2Y receptor subtypes capable of activation by UTP and ATP-γ-S. Moreover, experiments using amphotericin B-permeabilized monolayers indicated that treatment with apical UTP induced a biphasic effect on the apical membrane current beginning with an initial decrease, reflecting enhanced K⁺ efflux followed by a slow and sustained increase, consistent with inhibition of basal K⁺ secretion. The increase in K⁺ efflux was blocked by pretreatment with an intracellular Ca²⁺ chelating agent (BAPTA), suggesting inhibition of Ca²⁺-activated K⁺ channels whereas the secondary sustained inhibition of K⁺ efflux was reduced in the presence of BAPTA. Activation of PKC activity with an exogenous phorbol ester compound was capable of reproducing most of the UTP-dependent inhibition of K⁺ efflux whereas pretreatment with a pan-selective inhibitor of PKC produced nearly complete block of the inhibitory effect of UTP, but not the initial increase in Ca²⁺-dependent K⁺ efflux. These findings suggest that the inhibitory effects of UTP on K⁺ secretion are primarily PKC dependent. Furthermore, we observed that pretreatment with a selective TASK1/TASK3 channel inhibitor (PK-THPP) reduced most of the response, suggesting that TASK channel inhibition by PKC may be responsible for a substantial portion of the inhibitory effects of UTP on K⁺ secretion. These results are consistent with previous studies of recombinant TASK-3 channels expressed in HEK293 cells demonstrating that activation of the classical (Ca²⁺-dependent) PKCα isoform leads to inhibition of channel activity (48). PKCα was shown to directly phosphorylate T341 within the COOH-terminus of the protein and reduce channel current. Similarly, activation of PKCε (a novel, Ca²⁺-independent PKC), following stimulation with platelet-activating factor in cardiac ventricular myocytes, produced inhibition of TASK1 channels by phosphorylating T381, also located within the COOH-terminal domain of the channel (21, 22). The results of the present study demonstrate that multiple K2P channel subtypes are expressed in human mammary epithelial cells and appear to contribute to basal K⁺ secretion. At least four of these subtypes (TREK1, TREK2, TASK1 and TASK3) have been shown to be regulated by different PKC isoforms (21, 29, 47, 52). Additionally, experiments involving BAPTA-AM pretreatment followed by UTP stimulation suggested that Ca²⁺-dependent and Ca²⁺-independent PKCs were required to produce maximum inhibition of the apical K2P current.

P2Y receptors have been previously shown to bind Gq family G-proteins that activate phospholipase C (PLC), catalyzing the production of IP₃ and subsequent mobilization of Ca²⁺ from internal stores. Earlier studies in glioma cells (NG-108–15) showed that P2Y₂ receptors distribute within lipid raft domains within the membrane and that treatment with MβCD disrupted the lipid raft structure as well as P2Y₂ receptor coupling to downstream effector pathways involved in UTP-dependent regulation of cell migration (3). A similar effect of MβCD on P2Y₂, P2Y₄ and P2Y₆ receptor coupling has been reported in PC12 cells and human neuroblastoma cells (SH-SY5Y) supporting the concept that P2Y receptor signaling can be dependent on localization within membrane microdomains (17, 18, 46). Results of the present investigation demonstrated disruption of the inhibitory effect of UTP on K⁺ secretion following pretreatment of the cells with MβCD. However, no significant change in the initial decrease in apical membrane current was observed, indicating that activation of Ca²⁺-dependent K⁺ efflux was unaffected by MβCD. This observation suggests that UTP was capable of increasing intracellular [Ca²⁺], but unable to induce PKC-dependent inhibition of K2P channels in the apical membrane. One possible explanation for this effect could be that lipid raft disruption dissociates a P2Y/PLC/K2P signaling complex necessary for regulating channel activity, but does not disrupt P2Y receptor coupling to PLC within membrane domains that are not associated with lipid rafts. Additional experiments will be required to determine if such signaling complexes exist and if they localize within lipid rafts within the apical membrane.

Epithelial cells express multiple K2P channel subtypes that are located in both apical and basolateral membranes (10, 19, 23, 24, 33, 39, 43, 44, 51). In the present study we performed qRT-PCR experiments and identified nine K2P channels including TWIK1, TWIK2, TREK1, TREK2 and five distinct TASK channels. Of these, five channels were detected on Western blots of proteins obtained from biotinylated apical membranes. Immunocytochemistry experiments performed with these antibodies showed that TASK1, TASK3, TREK1, TREK2, and TWIK1 channels are associated with the apical region of the epithelium. Although we did not specifically examine the localization of all nine K2P channels detected by qRT-PCR, we acknowledge that other subtypes may also be present in the apical membrane.

The major conclusions from this investigation are illustrated in the model presented in Fig. 9. Our results indicate that multiple K2P channels are associated with the apical membrane of human mammary epithelial cells and that they participate in basal K⁺ secretion occurring in parallel with ENaC-dependent Na⁺ absorption. We speculate that this underlying K⁺ secretion contributes to the elevated K⁺ concentration observed in human milk. Moreover, at least some of these K2P channel subtypes appear to exist within lipid raft domains in association with P2Y receptors. Apical stimulation with UTP initially increased K⁺ secretion by activation of TRAM-34 sensitive, Ca²⁺-dependent K⁺ channels. This initial increase in secretion was followed by a time-dependent decrease in apical K⁺ efflux resulting from K2P channel inhibition. We hypothesize that a combination of previously identified, PKC-regulated K2P channels including TREK1, TREK2, TASK1, and TASK3 are most likely responsible for the inhibitory effects of UTP on K⁺ secretion.

Fig. 9.

A model showing apical two-pore potassium (K2P) channels and their regulation following apical P2Y receptor stimulation. Details are presented in the discussion.

GRANTS

This study was partly supported by a Royal Golden Jubilee PhD program Fellowship from The Thailand Research Fund (PHD/0204/2551) to Y. Srisomboon and C. Deachapunya. The study was also supported by NIH National Institute of Allergy and Infectious Diseases Grant R01 AI128729-01 to S. M. O’Grady.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Y.S., N.A.Z., C.D., and S.M.O. conceived and designed research; Y.S., N.A.Z., and P.J.M. performed experiments; Y.S., N.A.Z., P.J.M., C.D., and S.M.O. analyzed data; Y.S., N.A.Z., C.D., and S.M.O. interpreted results of experiments; Y.S. and N.A.Z. prepared figures; Y.S. drafted manuscript; Y.S., N.A.Z., P.J.M., C.D., and S.M.O. edited and revised manuscript; Y.S., N.A.Z., P.J.M., C.D., and S.M.O. approved final version of manuscript.