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Originally published In Press as doi:10.1074/jbc.M312547200 on March 10, 2004

J. Biol. Chem., Vol. 279, Issue 20, 20643-20654, May 14, 2004
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Membrane-delimited Regulation of Novel Background K+ Channels by MgATP in Murine Immature B Cells*

Joo Hyun Nam, Ji-Eun Woo, Dae-Yong Uhm, and Sung Joon Kim{ddagger}

From the Department of Physiology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea

Received for publication, November 17, 2003 , and in revised form, March 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In WEHI-231, a representative immature B cell line, Ca2+ entry is paradoxically augmented by treatment with 2-aminoethoxydiphenyl borate (2-APB), a blocker of inositol 1,4,5-trisphosphate receptor and of nonselective cation channels (Nam, J. H., Yun, S. S., Kim, T. J., Uhm, D.-Y., and Kim, S. J. (2003) FEBS Lett. 535, 113–118). The initial goal of the present study was to elucidate the effects of 2-APB on membrane currents, which revealed the presence of novel K+ channels in WEHI-231 cells. Under whole-cell patch clamp conditions, 2-APB induced background K+ current (IK,bg) and hyperpolarization in WEHI-231 cells. Lowering of intracellular MgATP also induced the IK,bg. The IK,bg was blocked by micromolar concentrations of quinidine but not by tetraethylammonium. In a single channel study, two types of voltage-independent K+ channels were found with large (346 picosiemens) and medium conductance (112 picosiemens), named BKbg and MKbg, respectively. The excision of membrane patches (inside-out (i-o) patches) greatly increased the Po of BKbg. In i-o patches, cytoplasmic MgATP (IC50 = 0.18 mM) decreased the BKbg activity, although non-hydrolyzable adenosine 5'-({beta},{gamma}-imino)triphosphate had no effect. A pretreatment with Al3+ or wortmannin (50 µM) blocked the inhibitory effects of MgATP. A direct application of phosphoinositide 4,5-bisphosphate (10 µM) inhibited the BKbg activity. Meanwhile, the activity of MKbg was unaffected by MgATP. In cell-attached conditions, the BKbg activity was largely increased by 2-APB. In i-o patches, however, the MgATP-induced inhibition of BKbg was weakly reversed by the addition of 2-APB. In summary, WEHI-231 cells express the unique background K+ channels. The BKbgs are inhibited by membrane-delimited elevation of phosphoinositide 4,5-bisphosphate. The activation of BKbg would hyperpolarize the membrane, which augments the calcium influx in WEHI-231 cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Immune responses are initiated and regulated by the physical interactions of receptors and ligands on lymphocytes and antigen-presenting cells. In addition to the external factors, intrinsic changes of lymphocytes would participate in shaping the ongoing responsiveness of cells. An important intrinsic determinant of responsiveness could be the membrane potential (Vm) that is mainly determined by the potassium permeability of cell membrane and transmembrane gradient of [K+]. K+ channels, besides setting resting Vm, play critical roles in various cellular functions (1). Among them, the modulation of calcium signals by providing electrical driving force has been suggested as an essential role of K+ channels (1, 2). In human primary T cells, two kinds of K+ channels, the voltage-gated K+ channels Kv1.3 and the calcium-activated K+ channel IKCa1 (hSK4), are found to play such a role; Kv1.3 channels are essential for activation of quiescent cells, and signaling through protein kinase C pathway enhances expression of IKCa1 channels that are required for proliferation (3, 4). Besides Vm regulation, K+ channels also regulate the loss of intracellular K+, which is a prerequisite step in the normotonic- or the Fas-mediated apoptosis (5, 6). In contrast to the studies in T cells, the characteristics of K+ channels and their roles in B cells have been rarely investigated, and the types of reported K+ channels are restricted to the voltage-dependent (Kv) and Ca2+-activated (KCa) channels (7, 8).

A distinctive feature of the immune system is the balanced fine-tuning between growth and death by apoptosis. In bone marrow, the immature B cells with membrane-bound immunoglobulins reactive to autoantigens are arrested in the cell cycle and eliminated through the process of apoptosis, a crucial step preventing autoimmune diseases. In contrast, the mature B cells, once activated by specific antigens, undergo a second round of proliferation and selection in the secondary lymphoid organs to differentiate into memory B cells (9, 10). WEHI-231 cells are the representative murine B lymphoma cell line that reflects the characteristics of immature B cells, apoptosis by cross-linking B cell receptors (BCR)1 (11–13). The BCR ligation activates a series of protein-tyrosine kinases. Several of them (e.g. Syk and Btk) are involved in the activation of phospholipase C{gamma}, producing IP3 that subsequently mobilizes intracellular Ca2+ stores via the IP3 receptor (9, 10, 14). The resulting decrease in the Ca2+ content of intracellular stores triggers store-operated Ca2+ entry (SOCE), which is required for the tonic increase in cytosolic Ca2+ concentration ([Ca2+]c) (8, 15).

In our previous study, the increase in [Ca2+]c by SOCE was compared between cell lines Bal 17 (mature B cells) and WEHI-231 (16). In WEHI-231 cells, the increase in [Ca2+] is paradoxically augmented by an application of 2-aminoethoxydiphenylborate (2-APB) that has been reported as a blocker of SOCE channels in other cells (17). The unexpected effects might imply a 2-APB-activated Ca2+-permeable channels in WEHI-231, as recently identified in basophilic leukemia cells (18). Another possibility is that 2-APB indirectly enhances the Ca2+ influx via increasing the electrical driving force (i.e. membrane hyperpolarization). In the latter case, the K+ conductance of the WEHI-231 cell membrane would be increased by 2-APB. To address such hypotheses, it is essential to measure directly the membrane currents of WEHI-231 cells and examine the effects of 2-APB.

Therefore, in this study, the initial goal was to elucidate the membrane conductance of WEHI-231 cells that is positively regulated by 2-APB. To our surprise, the patch clamp study indicates the presence of novel background K+ channels with very large unitary conductance (>340 pS) that are positively regulated by 2-APB. In addition, background K+ channels with medium size conductance (112 pS) are also present in WEHI-231 cells. Our experimental results also demonstrate that the large conductance K+ channels are negatively regulated by cytoplasmic ATP via phosphorylation of phosphoinositides in a membrane-delimited manner.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells—Mouse B lymphocytes with properties of immature B cells (WEHI-231) and mature B cells (Bal 17) were grown in 25 mM HEPES RPMI 1640 media (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 50 µM 2-mercaptoethanol (Sigma), and 1% penicillin/streptomycin (Invitrogen). All cells were incubated at 37 °C in 95% O2, 5% CO2.

Electrophysiology—Cultured cells were transferred into a bath mounted on the stage of an inverted microscope (IX-70, Olympus, Osaka, Japan). The bath (~0.15 ml) was superfused at 5 ml/min, and voltage clamp experiments were performed at room temperature (22–25 °C). Patch pipettes with a free-tip resistance of about 2.5 megohms were connected to the head stage of a patch-clamp amplifier (Axopatch-1D, Axon Instruments). Liquid junction potentials were corrected with an offset circuit before each experiment. Unless mentioned otherwise, a conventional whole-cell clamp was achieved by rupturing the patch membrane after making a giga-seal. In the perforated whole-cell patch clamp, a stock solution of nystatin in dimethyl sulfoxide (15 mg/ml) was added to the pipette solution, yielding a final concentration of 0.15 mg/ml. A steady-state perforation was usually achieved within 5 min after making a giga-seal. pCLAMP software version 7.0 and Digidata-1200A (both from Axon Instrument) were used for the acquisition of data and the application of command pulses. The resting membrane potential described in this study was measured under the zero-current clamp condition of the whole-cell patch clamp. Voltage and current data were low pass filtered (5 kHz) and stored using a digital tape recorder (DTR-1205, Biologic, Claix, France). Current traces were stored in a Pentium-grade computer and analyzed using pCLAMP software version 6.0 and Origin version 6.1 (Microcal Software Inc., Northampton, MA).

Single channel activities were recorded at 10 kHz in cell-attached (c-a) and inside-out (i-o) configurations using fire-polished glass pipettes (final resistance, 8–9 megohms). Recordings were performed at room temperature with Axopatch-200B (Axon Instruments). The voltage and current data were low pass filtered at 2 kHz and stored for later analysis using Fetchan and pSTAT version 6.0 software (Axon Instruments). Data were analyzed to obtain an amplitude histogram and open probability (Po). Data were represented as mean ± S.E. Student's t test was used to test for significance at the level of 0.05.

Experimental Solutions—The MgATP-free pipette solution for whole-cell patch clamp contained (in mM) 135 KCl, 6 NaCl, 10 HEPES, and 5 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, with a pH of 7.2 (titrated with KOH), and the final [K+] reached about 140 mEq/liter. To examine the effects of intracellular ATP on membrane currents, either MgATP or Na2ATP was added, as mentioned under "Results." The normal bath solution for the whole-cell patch clamp contained (in mM) 145 NaCl, 3.6 KCl, 1 MgCl2, 1.3 CaCl2, 5 glucose, and 10 HEPES, with a pH of 7.4 (titrated with NaOH). In some experiments testing the K+ selectivity of channels and the effects of pharmacological blockers, high KCl bath solution was used (in mM): 140 KCl, 5 NaCl, 1 MgCl2, 1.3 CaCl2, 5 glucose, and 10 HEPES, with a pH of 7.4 (titrated with KOH). The pipette solution for cell-attached (c-a) patch clamp contained (in mM) 140 KCl, 5 NaCl, and 5 HEPES with a pH of 7.4 (titrated with KOH). The pipette solution for i-o patch clamp and the bath solution for c-a and i-o patch clamp contained (in mM) 145 KCl, 1 EGTA, and 5 HEPES with a pH of 7.4 (titrated with KOH). To confirm the ion selectivity of recorded channels under the i-o recordings, the KCl of bath solution (cytoplasmic side) was totally replaced by NaCl, and the pH was titrated with NaOH.

Phosphoinositide 4,5-bisphosphate (PIP2) was initially delivered as a chloroform solution. The chloroform was evaporated with a stream of N2 to leave a filmy residue of PIP2. Recording solution was mixed with this residue and then sonicated for >30 min on ice in the dark. Wortmannin was first dissolved in dimethyl sulfoxide (Me2SO) to a concentration of 100 mM as a stock solution and was used at a concentration of 50 µM in the bath solution for i-o experiments. Considering the short half-life in the solution, the effect of wortmannin was tested within 2 h after dissolution. PIP2 was purchased from Avanti (Alabaster, AL). KR-252a was purchased from Biomol (Plymouth Meeting, PA). All the other chemicals and drugs used in this study were purchased from Sigma.

Preparation of Cytosolic Fraction—WEHI-231 cells were washed twice with ice-cold phosphate-buffered saline without CaCl2 and MgCl2 (Invitrogen). Then cells were centrifuged again for 2 min at 1000 rpm. The pellet was resuspended in 15 ml of bath solution for i-o patch clamp study (see above). Cells were homogenized for 5 min in ice by using a pestle. Homogenized cells were then centrifuged at 12,000 rpm for 15 min at 4 °C (model 5415R, Eppendorf, Hamburg, Germany). The supernatant was placed in a test tube and diluted with the bath solution for i-o patch clamp. The experiment was done within 1 h after obtaining the cytosolic fraction. The protein concentration was determined by the Bradford assay (Sigma), and the final concentration of protein was 2.8 mg/ml.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Background K+ Conductance in WEHI-231 Cells—As the first step in examining the effects of 2-APB on the membrane currents and membrane potential of WEHI-231 cells, the nystatin-perforated whole-cell clamp was applied to preserve unknown cytosolic components other than monovalent ions. After achieving steady-state perforation, hyperpolarizing or depolarizing step pulses from the holding voltage of –60 mV were applied (100 ms, from –100 to +60 mV, 20-mV interval). On step depolarization, a delayed activation of outward current superimposed upon the instantaneous background component was observed (Fig. 1A). Brief current to voltage relations (I/V curve) were also obtained by depolarizing ramp pulses (from –90 to 60 mV, 0.1 V/s), which showed a voltage-dependent increase of slope conductance at depolarized membrane voltages above –20 mV (Fig. 1B). The application of 2-APB (50 µM) markedly increased the instantaneous component of membrane conductance and hyperpolarized the Vm from –43.5 ± 1.63 to –68.5 ± 2.35 mV (n = 12). From the I/V curves, it is evident that 2-APB increased the membrane conductance at negative as well as positive clamp voltages, and the curve reversed its direction at around –70 mV, close to the K+ equilibrium potential, suggesting an activation of background-type K+ conductance (IK,bg). For reasons yet unknown, the amplitude of IK,bg varied between WEHI-231 cells from different culture passages. In general, the amplitude of maximum IK,bg tended to decrease with the lapse of culture date. Therefore, throughout the present study, care was taken to compare the effects of experimental conditions with the responses of control cells on the same date of culture.



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  		FIG. 1.
Background-type K+ currents in WEHI-231 cells. A, membrane currents in control (left panel) and 2 min after 2-APB application (50 µM, right column) obtained by step pulses (see inset) under the nystatin-perforated whole-cell clamp conditions. B, I/V curves obtained by depolarizing ramp pulses from –90 to 60 mV in control and 2 min after 2-APB application (mean ± S.E., n = 6). C, summary of steady-state I/V curves after dialyzing the cells with KCl pipette solutions containing no MgATP, 3 mM Na2ATP, and with 3 mM MgATP (mean ± S.E., n = 5), respectively. Note that the initial I/V curve just after breaking-in patch membrane (control) is overlapped with the response to 3 mM MgATP. D, with 3 mM MgATP in pipette solution, 2-APB (50 µM) induced a small increase in outward current and the leftward shift of I/V curves (n = 3). E, after confirming the steady-state outward current under MgATP-free conditions, the extracellular concentration of KCl was increased by replacing with equimolar NaCl as indicated in the figure. F, no development of background-type K+ current in Bal-17 cells dialyzed with MgATP-free KCl solution (n = 5). The I/V curves obtained in the initial control (filled squares), 5 min after break-in (filled circles), and addition of 2-APB (50 µM, open triangles).

 
In the next experiments, the conventional whole-cell clamp was applied to control the intracellular environment more precisely (e.g. excluding the possibility of Ca2+ increase). A striking feature of WEHI-231 cells was that an outward current increased spontaneously after breaking in the patch membrane and dialyzing with MgATP-free KCl solution. On making a whole-cell configuration, the initial resting membrane potential was fluctuating around –30 mV, which hyperpolarized spontaneously to –75.0 ± 1.56 mV within 5–6 min after the break-in (n = 13). During the experiment, the membrane voltage was intermittently clamped at –60 mV, and ramp pulses (Fig. 1, C and D) were applied to monitor the changes in the membrane conductance. The slope of spontaneously developed membrane conductance was basically voltage-independent (Fig. 1C, n = 5), same with the IK,bg activated by 2-APB (Fig. 1, A and B). After full activation of IK,bg by the dialysis with the MgATP-free pipette solution, 2-APB had no further effect (n = 2, data not shown). The intracellular dialysis with di-sodium ATP (Na2ATP, 3 mM) without adding Mg2+ similarly increased the amplitude of IK,bg (Fig. 1C, n = 5). In contrast, no development of outward current was observed when 3 mM MgATP was included in the pipette solution (Fig. 1C, n = 4). These results suggest that the development of IK,bg is suppressed by cytoplasmic MgATP that can be used as a substrate for kinases in WEHI-231 cells. In Fig. 1D, we tested whether 2-APB could overcome the inhibition of IK,bg by MgATP. With 3 mM MgATP in the pipette, a bath application of 2-APB increased the outward current (n = 5). However, the increase was much smaller than the effects obtained in the nystatin-perforated conditions.

The voltage independence and K+ selectivity of the above background currents were confirmed more precisely. After the steady-state development of outward current under MgATP-free conditions, the extracellular K+ concentration ([K+]ext) was increased in a stepwise manner, and the I/V curve was obtained by ramp pulses from –90 to +60 mV (Fig. 1E). With symmetrical K+ concentrations in the pipette and the bath solution, the I/V curve became almost linear, confirming the voltage-independent background activity of K+ channels in WEHI-231 cells. Also, the reversal potential was right-shifted reflecting that a highly potassium-selective conductance was induced.

In contrast to the above results in WEHI-231 cells, no such IK,bg was observed in Bal 17 cells, a mature B cell line from mouse. The Bal 17 cells were voltage-clamped using the patch pipettes containing MgATP-free KCl solution, and various levels of depolarizing step pulses were applied. A prolonged dialysis with MgATP-free solution (>5 min) induced an increase of outward current only at highly depolarized clamp voltages (>40 mV) without shifting the reversal potential of the I/V curves (Fig. 1F). In this state, the application of 2-APB did not evoke IK,bg but reduced the currents at depolarized clamp voltages (Fig. 1F).

The IK,bg Is Resistant to TEA but Sensitive to Quinine/Quinidine—Tetraethylammonium (TEA) is an ion channel blocker with broad effects on various classes of K+ channels. In WEHI-231 cells, after dialyzing with MgATP-free pipette solution over several minutes to develop IK,bg, a bath application of 10 mM TEA weakly decreased the outward currents only at depolarized clamp voltages (>0 mV), and the reversal potential was not shifted (Fig. 2A). The resistance of IK,bg to TEA and its voltage independence were similar to the properties of the recently identified K+ channels with two tandem pore-loop domains (K2P channels) (19, 20). All the cloned K2P channels were resistant to TEA, whereas some subtypes showed variable sensitivity to quinine (quinidine) or to Ba2+ (19).



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  		FIG. 2.
Effects of pharmacological blockers on IK,bg. Drugs were applied after confirming the steady-state increase of IK,bg by dialyzing with MgATP-free KCl solution. A, effects of TEA (10 mM, filled circles). Asterisks indicate a statistically significant decrease in current amplitudes (n = 5, p < 0.05). B–F, effects of quinidine, quinine, and Ba2+ on the IK,bg in high KCl bath solution. Depolarizing ramp pulses from –90 to 60 mV were applied at various concentrations of blockers. The normalized amplitudes of remained currents (I/Icon) were measured at both positive (+60 mV) and negative (–60 mV) clamp voltages, and plotted against the test concentrations of blockers. The concentration-response relation was fitted by the function {I/Icon x 100(%) = 100/(1 + (tested concentration/IC50)n)}.

 
In the next experiment, therefore, the effects of quinine and quinidine on the IK,bg were examined. After confirming the full activation of IK,bg and membrane hyperpolarization under MgATP-free conditions, all extracellular Na+ was substituted to K+ (140 mM KCl), which revealed inward K+ currents at negative membrane voltages. The brief I/V curves were obtained with ramp-pulse protocols (from –90 to +60 mV, 0.1 V/s, 5-s interval), and various concentrations of blockers were applied to the bath (Fig. 2B). Both quinine and quinidine displayed inhibitory effects on IK,bg in a completely reversible manner. The effects of quinine and quinidine were voltage-dependent; both agents blocked IK,bg more effectively at positive voltages than at negative voltages. The concentration-response curves were obtained at –60 and at +60 mV, where the half-inhibitory concentrations (IC50) of quinine were 30.5 and 9.8 µM, respectively. Similarly, the IC50 values of quinidine were 51.7 and 6.2 µM at –60 and at +60 mV, respectively (Fig. 2, C and D). We also tested the effects of Ba2+, a nonselective K+ channel blocker, on IK,bg. The blocking effects of Ba2+ were relatively weak and more effective at negative membrane voltages, opposite to the effects of quinine or quinidine (Fig. 2, E and F).

As some types of K2P channels are sensitively regulated by extracellular pH and the stretch of cell membranes (19, 20), it was tested whether the extracellular acidification or osmotic stress affects IK,bg. An acidification of bath solution to pH 6.5 decreased the peak amplitude of outward currents by 21 ± 1.8, 45 ± 9.6, and 54 ± 9.6% of control at +60, 0, and –40 mV, respectively (data not shown). Thus the inhibitory effect by extracellular acidification was weakly voltage-dependent; a larger inhibition was observed at the negative clamp voltage. In another experiment, to exert an osmotic stress, 30 mM extracellular NaCl was replaced with 60 mM sucrose and regarded as an isotonic control. To apply hypotonic and hypertonic stimuli, 60 mM sucrose was omitted (–60 mosM) or newly added (+60 mosM, total 120 mM sucrose) for 3 min, respectively. Although not directly shown here, concomitant swelling or shrinkage of cells was clearly observable. However, neither the hypertonic shrinkage nor the hypotonic swelling had significant effect on IK,bg. Also, when the development of IK,bg was suppressed with 3 mM MgATP in the pipette solution, the hypotonic swelling could not induce IK,bg (data not shown).

Single Channel Recording of the Background K+ Channel—In the c-a condition with KCl (140 mM) pipette solution, single channel activities with large amplitudes of unitary currents were observed in WEHI-231 cells (Fig. 3A-a). The Po was initially very low but was greatly increased by the excision of membrane into MgATP-free KCl solution (Fig. 3A-b, see also Fig. 3C). When the K+ in the cytoplasmic side was totally replaced with Na+, the outward channel current at the positive clamp voltage was abolished (Fig. 3A-c), which confirmed the K+ selectivity of this channel. In the i-o conditions, the I/V relation under symmetrical KCl showed a weak inward rectification (Fig. 3B, closed circles). The slope conductance was 346 pS at negative voltages, and the Po was similarly high at both positive and negative membrane voltages (Fig. 3C). In the c-a recording with KCl pipette solution, presumably symmetrical K+ gradient across the patch membrane, the I-V curve was same with the one obtained under the i-o conditions (data not shown). In some cases of c-a recording, the KCl concentration in the pipette solution was reduced to 8.5 mM by an isomolar replacement with NaCl. With the low K+ pipette solution (i.e. low [K+]ext), the reversal potential of I-V curve was –70 mV, again indicating the K+-selective permeability of the 346 pS K+ channel (Fig. 3B, open circles, n = 4).



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  		FIG. 3.
Large conductance background K+ channels (BKbg) in WEHI-231 cells. A, after confirming the channel activity at the negative (–60 mV, left panels) and positive (+60 mV, right panels) clamp voltages under the c-a condition (a), the patch membrane was excised into the KCl bath solution (b), which increased the Po dramatically. On replacing the K+ in the bath solution (cytoplasmic side) with equimolar Na+ (c), the outward current was completely abolished (right panel), whereas the inward current was not affected (left panel). The arrows with c and o indicate closed and open state of channel, respectively. B, the I/V curves of BKbg under the c-a conditions with low [K+]ext (8.5 mM) in the pipette solution (open circles, n = 4), and under the i-o conditions with symmetrical concentrations of K+ (closed circles, n = 8). Each point is the mean ± S.E. A linear fitting at negative voltages yields 346 pS of slope conductance. C, means of Po of BKbg at positive (60 mV) and negative (–60 mV) membrane voltages.

 
From these results, we designated the background K+ channel with a maximum slope conductance of 346 pS as BKbg. The BKbg were observed in 95 cases out of total 480 trials of i-o patches. Although the i-o recording of BKbg was done in Ca2+-free conditions with 1 mM EGTA, the size of unitary conductance suggested that the BKbg might be related with maxi-K, the large conductance Ca2+-activated K+ channels. However, an inclusion of iberiotoxin (500 nM), a well known blocker of maxi-K, in the pipette solution of c-a patches did not block the BKbg (n = 4, data not shown). Also, the involvement of recently found Na+-activated K+ channels with unitary conductance of 141 pS, Slick channel (21), was excluded because an increase of [Na+] to 50 mM in cytoplasmic bath solution had no effect on BKbg activity (n = 5, data not shown).

In addition to BKbg, K+ channels with smaller conductance, designated MKbg (medium conductance background K+ channels), were observed at both positive and negative clamp voltages (Fig. 4). In the i-o conditions, the I-V curve of MKbg also showed a weak inward rectification with maximum unitary conductance of 112 pS at negative membrane voltages (Fig. 4A). The replacement of cytoplasmic K+ with Na+ completely abolished the channel activity, proving the K+ selectivity of MKbg (Fig. 4B). Although the unitary conductance was larger at negative voltages, the Po of MKbg was ~2-fold higher at +60 mV than at –60 mV (Fig. 4C). The MKbgs were observed in 46 cases out of a total 480 trials of i-o patches. Because the unitary conductance of MKbg was similar to that of TREK-2 channels (22), it was tested whether the acidification of intracellular pH or membrane stretch facilitates the activity of MKbg. However, the intracellular acidification (pH 6.0) inhibited MKbg (n = 4, Fig. 4D), and the membrane stretch by a negative pressure (–40 cm of H2O in the pipette) had no effect (n = 3, Fig. 4E).



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  		FIG. 4.
Medium conductance background K+ channels (MKbg) in WEHI-231 cells. I/V curves (A) and representative current traces (B) of MKbg recorded under the i-o conditions with symmetrical K+ concentrations (145 mM). Each point of A is the mean ± S.E. The K+ selectivity of recorded channel was confirmed from the elimination of channel activity at 60 mV by replacing the cytoplasmic K+ with Na+ (B, lowermost trace). C, the Po of MKbg at positive clamp voltage (+60 mV) was higher than at negative voltage (–60 mV, p < 0.05, n = 4). D, representative traces of MKbg at –60 mV demonstrating the inhibitory effects of decreasing the cytoplasmic pH. Time breaks indicate 20 s. E, no effect of membrane stretch on MKbg activity. Negative pressure (–50 cm of H2O) was applied to the patch pipette through a U-tube filled with water. The holding voltage was –60 mV.

 
Inhibition of BKbg by Intracellular MgATP—As mentioned above, the excision of membrane patches yielded a large increase of the Po of BKbg, suggesting the washout of "inhibitory" cytosolic components. Because the whole-cell current (IK,bg) was sensitive to MgATP in the cytoplasmic solution, we tested the effects of MgATP on the K+ channels under the i-o conditions. The Po of BKbg decreased dramatically with the application of MgATP (1 mM) to the cytoplasmic side, which was completely reversed by washout (Fig. 5A). In contrast, the di-sodium form of ATP (Na2ATP) had no effect on BKbg activity (n = 3, data not shown). BKbg activity was not affected by AMP-PNP (1 mM), a non-hydrolyzable analogue of ATP (Fig. 5B). These results suggest a phosphorylation-dependent regulatory mechanism for the inhibition of BKbg. Moreover, the application of ATP{gamma}S (1 mM), which is commonly used for permanent phosphorylation of substrates, exerted a non-washable inhibition of BKbg (Fig. 5C). Because the commercially available AMP-PNP and ATP{gamma}S are provided as lithium salts, 0.5 mM MgCl2 was added along with the application of AMP-PNP or ATP{gamma}S in the above experiments. The application of Li2ATP{gamma}S (1 mM) had no effect on BKbg activity (n = 2, data not shown). The current trace of Fig. 5C demonstrates a representative case where both BKbg and MKbg were present in the same patch of membrane. In contrast to the non-reversible inhibition of BKbg, it was evident that the activity of MKbg was persistent in the presence of ATP{gamma}S. The resistance of MKbg to MgATP was confirmed in eight patches (see also Fig. 6A). The concentration dependence of the inhibitory effects on BKbg was obtained from the decrease of the Po by various concentrations of MgATP, where the IC50 was 0.18 mM (Fig. 5D).



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  		FIG. 5.
Inhibition of BKbg activity by cytoplasmic application of MgATP. The recordings were commonly obtained under the i-o patch clamp conditions at –60 mV with symmetrical KCl (145 mM) solutions. A, application of MgATP (1 mM) to the cytoplasmic side of membrane greatly reduced the activity of BKbg, which was completely reversed by the washout with control solution. Note the time break of 90 s. Histogram of Po obtained from the experiment shown above (lower panel). The numbers in parentheses indicate the moment where displayed current traces were recorded, respectively. B, bath application of AMP-PNP (1 mM) had no effect on the activity of BKbg that was sensitively decreased by MgATP (1 mM). C, a representative case where BKbg and MK activities were concomitantly recorded (note two open states with different amplitudes). A bath application of ATP{gamma}S (1 mM) abolished the BKbg activity, whereas the MKbg was not affected. Note that the inhibition of BKbg by ATP{gamma}S is not reversed by washout. D, concentration-dependent effects of MgATP on BKbg activity. The Po measured of BKbg at each concentration of MgATP was normalized to the control Po (Po,con), and the means ± S.E. were plotted, which was fitted by the function {Po/Po,con x 100 (%) = 100/(1 + (tested concentration/IC50)n)}.

 



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  		FIG. 6.
Mechanism of the inhibitory effects of MgATP on BKbg. The recordings were commonly obtained under the i-o patch clamp conditions at –60 mV with symmetrical KCl (145 mM) solutions. 1 mM MgATP was applied. A, in the presence of K252a (400 nM), MgATP suppressed BKbg activity, whereas the MKbg was not affected. B and C, the application of Al3+ (50 µM) or wortmannin (50 µM) blocked the inhibitory effects of MgATP on BKbg. In some cases, Al3+ alone increased the activity of BKbg as demonstrated in B. D, the application of PIP2 (10 µM) largely abolished the activity of BKbg.

 
Next, the inhibitory mechanism of MgATP was investigated. K-252a (400 nM), a nonspecific inhibitor of cAMP-dependent protein kinase, protein kinase C, protein kinase G, and calmodulin-dependent kinase at this concentration (23) did not block the effect of MgATP (Fig. 6A). Chelerythrin (5 µM), a protein kinase C inhibitor, also did not block the inhibition by MgATP (n = 3, data not shown). The relatively high IC50 value of ATP and the insensitiveness to protein kinase inhibitor suggested that a lipid phosphorylation might mediate the inhibition of BKbg by MgATP. It was reported recently (24–26) that cytoplasmic application of ATP regulates various ion channels and transporters by generating PIP2 in excised patch clamp conditions. Aluminum ion (Al3+) is known to form a highly stable complex with PIP2 and blocks the PIP2-dependent regulation of channels and transporters (25). In the i-o recording of BKbg, a pretreatment with Al3+ (50 µM) completely blocked the inhibitory effects of MgATP (n = 8, Fig. 6B). Also, the pretreatment with wortmannin (50 µM), a PI-3 and PI-4 kinase inhibitor at this concentration, blocked the inhibitory effects of MgATP (n = 6, Fig. 6C). Finally, a direct application of PIP2 (10 µM) to the cytoplasmic side (bath solution) suppressed the BKbg activity (n = 5, Fig. 6D). These results commonly suggest that the cytoplasmic ATP-dependent inhibition of BKbg is tightly related with PIP2 in the membrane.

Effects of 2-APB on BKbg—The sensitivity of BKbg to MgATP strongly suggested that most of the whole-cell current, namely IK,bg, was because of the activity of BKbg. Therefore, we tested whether 2-APB could also stimulate BKbg. In the c-a recordings of BKbg, a bath application of 2-APB (50 µM) induced a huge increase of Po, which was reversed by washout of 2-APB (Fig. 7A). Similar responses to 2-APB were observed in more than 20 c-a recordings of BKbg.



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  		FIG. 7.
Effects of 2-APB on BKbg. Representative cases of c-a recording (A) and i-o recordings (B and C) are shown. Holding voltage was –60 mV. A, bath application of 2-APB (50 µM, b and c) slowly increased the Po of BKbg, which was reversed by washout of 2-APB. The lowermost panel shows a histogram of Po plotted against the recording time. The lowercase letters indicate the moment where the above current traces were recorded. B, after confirming the inhibition of BKbg activity by MgATP (1 mM, a), 2-APB (50 µM) was added to the cytoplasmic side, which induced a transient partial recovery of BKbg activity (b). Lower panels show histograms of Po obtained from the experiment shown above. Numbers in parentheses indicate the time where the above current traces were recorded. In the lowermost panel, a part of the above histogram was expanded to demonstrate the washout effect of 2-APB in the presence of MgATP (see also c of B). C, the addition of cytosolic fraction (2.8 mg of protein/ml) did not facilitate the effect of 2-APB (50 µM) on BKbg.

 
In the i-o recordings without MgATP, the effect of 2-APB was hard to determine because the Po of BKbg was already very high (n = 3, data not shown). Therefore, it was tested whether an application of 2-APB could overcome the inhibitory effects of MgATP on BKbg. In the presence of MgATP, the application of 2-APB (50 µM) to the cytoplasmic side induced a transient increase of Po followed by a slight tonic increase (Fig. 7B, n = 5). Because the positive effect of 2-APB on BKbg was largely abolished, a cytosolic molecule mediating the effect of 2-APB might have been washed off in the i-o conditions. To test this hypothesis, we prepared a cytosolic fraction of WEHI-231 cells and applied it together with 2-APB. However, the co-application of cytosolic fraction (2.8 mg protein/ml) and 2-APB could not overcome the inhibitory effect of MgATP (Fig. 7C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study for the first time demonstrates background-type K+ channels (BKbg and MKbg) in lymphocytes. Besides their voltage-independent activity, BKbg displayed intriguing properties including the following: 1) conspicuous large unitary conductance (346 pS); 2) inhibition by cytoplasmic MgATP most likely mediated by phosphoinositide phosphorylation; and 3) facilitation by 2-APB. The responses of BKbg to MgATP and 2-APB strongly suggest that the whole-cell K+ current of WEHI-231, namely IK,bg, was largely due to the activity of BKbg. Because membrane hyperpolarization could provide a driving force for Ca2+, the activation of BKbg by 2-APB could explain our previous finding that 2-APB facilitates Ca2+ influx in WEHI-231 cells (16).

Background-type K+ Channels in WEHI-231 Cells—To our knowledge, the unitary conductance of the BKbg is larger than any other classes of K+ channels including the large conductance Ca2+-activated K+ channels (maxi-K channels, ~200–250 pS) (1). The involvement of maxi-K channels in the present study is highly unlikely because the cytoplasmic Ca2+ activity was clamped close to zero throughout the experiment, and iberiotoxin or TEA did not block the current. Moreover, the Po of BKbg is voltage-independent, whereas the maxi-K channels are well known for their voltage-dependent increase of channel activity (1).

Leak or background-type K+ channels are defined by the lack of voltage dependence in channel activity. In this study, the Po of BKbg is clearly voltage-independent. Although the Po of MKbg is about 2-fold higher at +60 mV than at –60 mV, such a difference is much weaker than classical voltage-gated K+ channels or Ca2+ channels. The BKbg and MKbg in WEHI-231 cells commonly display weak inward rectification in terms of their unitary current to voltage relations. As a whole, such properties of single channel currents would be reflected as a linear or sublinear whole-cell current to voltage relation in symmetrical K+ gradient, as demonstrated in this study (Fig. 1E and Fig. 2).

The voltage-independent activity and resistance to TEA of IK,bg are similar to the traits of those K+ channels with two pore domains in tandem, called K2P channels (19, 20). However, the very large conductance of BKbg and the inhibitory actions of intracellular MgATP are unprecedented in the K2P channels. Although IK,bg decreased moderately by acidification of extracellular fluid (pH 6.4), the contribution of TASK channels is unlikely considering a large difference in the single-channel conductance (19). The conductance of MgATP-insensitive MKbg (112 pS) is quite comparable with that of TREK-2 channels (22). However, the inhibition of MKbg by intracellular acidification and no response to membrane stretch are inconsistent with the properties of TREK-2 (22). These findings suggest that the background K+ channels in WEHI-231 cells, especially BKbg, represent novel classes of K+ channels, the molecular nature of which remains to be identified.

Regulation of BKbg Activity by Intracellular MgATP—Both whole-cell dialysis with MgATP-free solution and direct application of MgATP to i-o patches document the strong inhibitory action on BKbg. At a glance, the inhibition by intracellular ATP reminded us of the behavior of ATP-sensitive K+ channels (KATP) and Slick, a Na+-activated K+ channel inhibited by ATP (1, 21). However, glibenclamide (10 µM), a selective inhibitor of KATP, had no effect on IK,bg (n = 3, data not shown). Also, neither the application of KATP openers (e.g. levcromakalim, 5 µM) nor an increase in cytoplasmic Na+ (50 mM) affected BKbg activity (n = 4 and 5, respectively, data not shown). Because only hydrolyzable MgATP, and neither Na2ATP nor AMP-PNP, exerts inhibitory effects, the regulatory mechanism is strongly assumed to be a phosphorylation-mediated pathway rather than a direct binding of ATP (26).

Because the inhibitory effect of MgATP was reversible in i-o patches, the relevant kinase and phosphatase seem to be tightly associated with BKbg in the same patch of membrane. Such a "membrane-delimited" regulation of ion channels has been reported in Ca2+-activated K+ channels of the brain and in smooth muscle cells (27, 28). Another recently found means of membrane-delimited regulations of ion channels are lipid kinases associated with phosphoinositides (24–26, 29). In this study, wortmannin or Al3+ effectively blocked the inhibitory action of MgATP on BKbg, whereas inhibitors of protein kinases were ineffective. Also, the IC50 value of MgATP was relatively high (0.18 mM, Fig. 5D) compared with the typical concentrations (10–5–10–6) used for protein kinases. Finally, a direct application of PIP2 abolished BKbg activity in i-o recordings. All these results suggest that the level of phosphoinositide phosphates, especially PIP2, plays critical roles in the regulation of BKbg. The inhibition of BKbg by PIP2 is a unique phenomenon because all the other PIP2-sensitive K+ channels are positively regulated by raising the level of PIP2 (24, 26). Most likely the action mechanism of PIP2 on BKbg would be quite different from the case with other K+ channels, which needs to be investigated further. Also, one should be careful in the interpretation of the above results because the strongly charged PIP2 might have nonspecific effects that mimic the action of MgATP. In fact, a recent report in the G protein coupled K+ channel, a well known example of the interaction between PIP2 and K+ channel, demonstrates that the effects of MgATP on G protein coupled K+ channel activity cannot be simply equated with PIP2 (30).

Effects of 2-APB on IK,bg and BKbg—The facilitation of IK,bg by 2-APB was an unexpected phenomenon because 2-APB has been widely used as a blocker of various cationic channels (17). After the initial suggestion as a membrane-permeable blocker of inositol 1,4,5-trisphosphate receptors (31), the blocking effects of 2-APB on the various membrane channel proteins, including Ca2+ release-activated calcium channel, gap junction, and sarco/endoplasmic reticulum Ca2+-ATPase, have been reported (17, 31–33). On the other hand, depending on the concentration used, 2-APB facilitates the cytosolic regulator of adenylyl cyclase or other Ca2+-permeable cation channels (18, 34, 35). However, the positive effect of 2-APB on BKbg is still a novel finding, and the subsequent hyperpolarization explains our previous finding that 2-APB augments the Ca2+ influx in WEHI-231 cells (16).

For reasons not clear yet, the positive effects of 2-APB on BKbg are remarkable only in the c-a or nystatin-perforated conditions (Fig. 1 and Fig. 7). Such dependence on the intact intracellular environment initially suggested an involvement of washable cytosolic molecules. However, the effects of 2-APB were not recovered by co-treatment with the cytosolic fraction (Fig. 7C). One possibility is that the unidentified molecules necessary for the action of 2-APB are fragile and easily lose their function after fractionation. Another possible explanation is that 2-APB somehow decreases the level of cytoplasmic MgATP and subsequently relieves the tonic inhibition of BKbg. Because 2-APB can block various ion channels permeable to divalent cations (17), an inhibition of Mg2+ influx by 2-APB might decrease the level of phosphorylatable ATP, namely MgATP in the cytosol. The exact target sites and action mechanism of 2-APB on BKbg needs further investigation.

Role of K+ Channels in the Signal Transduction of Immune Cells—Previous studies in T cells emphasized the role of voltage-gated K+ channel (Kv1.3) and the calcium-activated K+ channel (IKCa1, SK4) regulating the membrane potential and the Ca2+ signaling (2, 3, 8, 36). In T cells, the expression of IKCa1 is dramatically up-regulated by T cell receptor activation (3), explaining the stronger Ca2+ responses to secondary T cell receptor stimulation in activated T cells (37). In another study, however, repetitive stimulation of naive T cells induces terminally differentiated effector memory T cells with the characteristic expression pattern of high Kv1.3 and low IKCa1 (38). Although studies on B cells are rare, intriguing similarities like the up-regulation of IKCa1 channels by BCR stimulation have been reported (7, 8). A recent study in DT-40 B cells has demonstrated that BCR cross-linking exerts inhibitory effects on the thapsigargin-induced SOCE, part of which is mediated by a membrane depolarization (39). In a similar context, the regulation of IK,bg of WEHI-231 cells might play a role in the regulation of [Ca2+]c.

At the normal intracellular concentration of ATP in the intact B cells, it seems likely that the BKbg would be under the tonic inhibitory control. In fact, the Po of BKbg under the c-a conditions was very low compared with their maximum capacity revealed after the excision of patch membrane (Fig. 3C). Considering their large unitary conductance, however, only a partial recruitment of BKbg might be sufficient to change the membrane voltage of B cells. As the BKbg activity was inhibited by PIP2, a plausible mechanism of BKbg activation is a decrease of PIP2. The BCR cross-linking and subsequent activation of phospholipase C{gamma} (9, 10, 14) would degrade PIP2 to produce inositol 1,4,5-trisphosphate, which might facilitate BKbg activity in WEHI-231 cells. However, it is also documented that the net synthesis of PIP2 is increased in B cells after BCR stimulation, which is signaled via Btk, a Tec family cytoplasmic tyrosine kinase (40). The physiological role and direction of BKbg regulation in the immature B cells will be an interesting theme to be pursued and should be interpreted depending upon the signaling pathways recruited.

In contrast to WEHI-231 cells, the IK,bg was not found in mature B cells (Fig. 1F). Considering the apoptotic tendency of WEHI-231 cells, it would be an intriguing question whether an aberrant activation of IK,bg might play a role in the cell death of immature B cells (9, 10). Persistent loss of potassium ions and the decrease of cell volume have been suggested as critical steps during cell death (1, 5, 6). In addition, roles of background-type K2P channels have been suggested in the apoptosis of embryonic cells and cerebellar granule neuron (41, 42). Thus, it remains to be investigated whether the background K+ channels play a similar role in the apoptotic response of immature B cells to BCR stimulation.

In summary, we report a background-type K+ current (IK,bg) and corresponding channels with unique properties (BKbg, MKbg) in WEHI-231, murine immature B lymphocytes. The activity of BKbg is sensitively regulated by cytosolic MgATP, which is most likely mediated by the level of PIP2. Future investigations will be focused on the modulation of background K+ channels by immunological signals and their roles in the immature B cells.


   FOOTNOTES
 
* This work was supported by Samsung Grants SBRI B-A2–302 (to D.-Y. U.) and B-A2–102 (to S. J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Physiology, Sungkyunkwan University School of Medicine, Changan-Gu, Cheoncheon-Dong 300, Suwon 440-746, Korea. Tel.: 82-31-299-6104; Fax: 82-31-299-6129; E-mail: sjoonkim{at}med.skku.ac.kr.

1 The abbreviations used are: BCR, B cell antigen receptor; IK,bg, background K+ current; Kbg, background K+ channel; i-o, inside-out; c-a, cell-attached; 2-APB, 2-aminoethoxydiphenyl borate; [Ca2+]c, cytosolic Ca2+ concentration; SOCE, store-operated Ca2+ entry; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphoinositide 4,5 bisphosphate; pS, picosiemens; TEA, tetraethylammonium; AMP-PNP, adenosine 5'-({beta},{gamma}-imino)triphosphate; adenosine 5'-O-(thiotriphosphate). Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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