Correspondence to: Yasunobu Okada, Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan. Fax:81-564-55-7735 E-mail:okada{at}nips.ac.jp.
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Abstract |
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In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl- channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around ±25 mV. The whole-cell current was selective for anions and sensitive to Gd3+. In on-cell patches, single-channel events appeared with a lag period of 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of
400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC50 of 12.3 mM and an electric distance (
) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC50 of 12.9 mM and
of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with PATP/PCl of 0.09. The single-channel activity was sensitive to Gd3+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.
Key Words: ATP-conducting channel, maxi chloride channel, osmotic cell swelling, volume regulation
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INTRODUCTION |
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Osmotic cell swelling is known to induce ATP release from a variety of cell types (
Although it was previously proposed that CFTR serves as a conductive pathway for ATP release (
In the present study, we searched for an alternative candidate anion channel that exhibits ATP conductivity and Gd3+ sensitivity in mouse mammary C127i cells. The volume- and voltage-dependent ATP-conductive large-conductance (VDACL) anion channel was found to exist in the plasma membrane and to share the same pharmacological profiles and CFTR-dependent modulation with swelling-induced ATP release.
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MATERIALS AND METHODS |
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Cells
A cell line of mouse mammary tissue origin, C127i, was obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium containing 10% FCS. A stable transfectant of C127i with the cDNA for human CFTR (C127/CFTR) was a gift from Dr. M.J. Welsh (University of Iowa), and was cultured in DMEM with 10% FCS and 200 µg ml-1 geneticin.
Solutions
The standard Ringer's solution contained the following (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 Na-HEPES, 6 HEPES, and 5 glucose, pH 7.4 (290 mosmol kg-1 H2O). The hypotonic solution was made by reducing the concentration of NaCl in Ringer's solution to 100 mM (210 mosmol kg-1 H2O). For selectivity measurements of whole-cell currents, 100 mM NaCl in Ringer's solution was replaced with 100 mM TEA-Cl or sodium glutamate. For single-channel selectivity measurements, 135 mM NaCl in Ringer's solution was replaced with 135 mM of TEA-Cl or Na salts of the anion that was being tested. For cation-to-anion selectivity measurements, NaCl concentration in Ringer's solution was lowered by isosmotic replacement with mannitol. For phosphate permeability measurements, 70 mM Na2HPO4 solution with pH 7.4 adjusted with HCl (calculated Cl- concentration: 11 mM) was used. For the experiments under Ca2+- and Mg2+-free conditions, CaCl2 and MgCl2 were removed from, and 1 mM EDTA was added to, Ringer's solution.
The pipette solution for whole-cell experiments contained the following (in mM): 125 CsCl, 2 CaCl2, 1 MgCl2, 5 HEPES, and 10 EGTA, pH 7.4 (adjusted with CsOH; 275 mosmol kg-1 H2O). In some whole-cell experiments, 15 mM ATP was added in the pipette solution. Pipette solution for cell-attached and inside-out experiments was standard Ringer's solution. When indicated, Cs-pipette and TEA-pipette solutions were used in which all monovalent cations were replaced with either Cs+ or TEA+. The pipette solution for outside-out experiments contained the following (in mM): 120 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4 (adjusted with NaOH), and 10 EGTA (pCa 7.7; 275 mosmol kg-1 H2O).
For experiments testing the effect of ATP on channel activity, 2.525 mM Na2ATP was added to the Ringer's bathing solution, and pH was adjusted to 7.4 with NaOH. For ATP-permeability measurements, 100 mM Na2ATP solution was used after pH was adjusted at 7.4 with NaOH. All ATP-containing solutions were kept on ice and warmed to room temperature immediately before the experiment.
GdCl3 was stored as a 30-mM stock solution in water and added directly to Ringer's solution immediately before the experiment. 5-Nitro-2-(phenylpropylamino)-benzoate (NPPB), glibenclamide, phloretin, 4-acetamido-4'-isothiocyanostilbene (SITS), niflumic acid (all from Sigma-Aldrich) were added to Ringer's solution immediately before use from stock solutions in DMSO to the final concentrations as indicated. The final DMSO concentration did not exceed 0.1% and DMSO did not have any effects, when added alone.
The osmolality of all solutions was measured using a freezing-point depression osmometer (model OM802; Vogel) or a vapor pressure osmometer (model Vapro 5520; Wescor).
Electrophysiology
Patch electrodes were fabricated from borosilicate glass using a micropipette puller (model P-97; Sutter Instrument Co.) and had a tip resistance of 2 M
for whole-cell measurements and 28 M
for macro-patch and single-channel recordings when filled with pipette solution. Membrane currents were measured with an Axopatch 200A patch-clamp amplifier coupled to a DigiData 1200 interface (Axon Instruments) or an EPC-9 patch-clamp system (Heka-Electronics). The membrane potential was controlled by shifting the pipette potential (Vp). For whole-cell recordings, access resistance did not exceed 4 M
and was always compensated (to 7080%). Whole-cell capacitance was 17.5 ± 1.4 pF (n = 23) and ranged from 9 to 32 pF. If not indicated, currents were filtered at 1 kHz and sampled at 25 kHz. Instantaneous currents for whole-cell and macro-patch recordings were measured 510 ms after the onset of the test pulses. Data acquisition and analysis were done using Pulse+PulseFit (Heka-Electronics) or pCLAMP6 (Axon Instruments) and WinASCD software (provided by Dr. G. Droogmans, Katholieke Universiteit Leuven, Belgium). Whenever bath Cl- concentration was altered, a salt bridge containing 2 M KCl in 2% agarose was used to minimize bath electrode potential variations. Liquid junction potentials were measured by a separate patch electrode filled with 3 M KCl or calculated using pCLAMP8 algorithms, and both were found to be essentially the same (within 13 mV). The data were corrected for liquid junction potentials either on- or offline. All experiments were performed at room temperature (2025°C).
Luciferin-Luciferase ATP Assay
Bulk extracellular ATP concentration was measured by the luciferin-luciferase assay (ATP luminescence kit; model AF-2L1; DKK-TOA Co.) at room temperature with an ATP analyzer (model AF-100; DKK-TOA Co.), as described previously (15 min in hypotonic solution. Thus, the cells were incubated for 15 min at either 25 or 37°C, and an extracellular solution sample (100 µl) was collected for the luminometric ATP assay of swelling-induced ATP release. When required, drugs were added to the hypotonic solution to give the final concentrations as indicated.
Data Analysis
Single-channel amplitudes were measured by manually placing cursors at the open and closed channel levels. By dividing a mean macro-patch current by a single-channel amplitude, the mean number of channels open, Po · n, was calculated, where Po and n represent the open channel probability and the number of active channels, respectively. Doseresponse data for ATP block of single channel amplitudes were fitted to the equation:
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(1) |
where io is the single-channel amplitude in the absence of ATP, i is the single-channel amplitude in the presence of ATP at a given concentration [A], and Kd is the apparent dissociation constant.
The voltage dependence of blockade was examined using the Boltzmann equation:
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(2) |
where io is the single-channel current amplitude in the absence of ATP, i is the single-channel current amplitude in the presence of 25 mM ATP, is the fractional electrical distance from either extracellular or intracellular side, z is the valence of the blocker (z = -4 for ATP), Vo is a potential for half-maximal block, a is a limiting fractional current at high voltages, and F, R, and T have their usual thermodynamic meanings.
Permeability ratios for different anions (X) were calculated from Goldman-Hodgkin-Katz (GHK) equation:
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(3) |
where Erev is the reversal potential in the presence of a test anion at a concentration [X], [Cl]o is the Cl- concentration in the pipette (standard Ringer's) solution, and [Cl]i is the Cl- concentration in low Cl- bath solutions containing different test anions. PCl and PX are the permeabilities of Cl- and test anion, respectively.
Na+ to Cl- permeability was calculated according to the Equation 4:
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(4) |
where PNa is the sodium permeability, [Na]i and [Na]o are intracellular and extracellular Na+ concentrations, and Na and
Cl are the activity coefficients of Na+ and Cl- (
Permeability ratio for polyvalent anions (A: phosphate and ATP) was calculated from GHK equation:
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(5) |
where = F/RT; zA and zCl are valences of the anion and Cl-, respectively; [Cl]o and [Cl]i are the Cl- concentrations in the pipette and in the bath, respectively; [A]i is the polyvalent anion concentration in the bath; and Erev is the reversal potential. When no Cl- was present in the bath, and therefore [Cl]i = 0, the equation turns to the one used in
Data were analyzed in Origin 5.0 (MicroCal Software, Inc.). Pooled data are given as means ± SEM of observations (n). Statistical differences of the data were evaluated by paired or unpaired t test and considered at P < 0.05.
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RESULTS |
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Gd3+-sensitive Whole-Cell Anion Conductance Induced by Cell Swelling
Consistent with previous observations (
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In searching for possible ATP-conductive pathways, we modified the whole-cell patch-clamp experiments to eliminate VSOR Cl- currents in two ways: (1) by using a relatively selective blocker of VSOR channels, phloretin (
Steady-state current amplitude varied widely ranging from 240 pA up to 3,800 pA at +25 mV. Mean current density was 153 ± 54 pA/pF at +25 mV and -181 ± 56 pA/pF at -25 mV (n = 9). There was a delay of 320 min (mean 12.0 ± 1.2 min, n = 16) between hypotonic stimulation and current activation.
The phloretin-insensitive whole-cell current could be reversibly inhibited by 30 µM Gd3+ (Fig 2 A). Whole-cell currents were also sensitive to 100 µM NPPB and SITS, however, those agents were less effective than 30 µM Gd3+, as summarized in Fig 2 B. Niflumic acid failed to affect whole-cell currents at a concentration of 200 µM (data not shown, n = 5). Also, replacing Na+ ions with TEA+ had no appreciable effect on the I-V relationship (Fig 2 C) and voltage-dependent inactivation (data not shown). However, when 100 mM Cl- in the hypotonic bath solution was replaced with 100 mM glutamate, the reversal potential was shifted by 23 ± 2 mV (n = 5) (Fig 2 C), indicating anion selectivity of this swelling-induced phloretin-insensitive current.
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Swelling-induced Large-Conductance Single-channel Activity in Cell-attached Patches
Single-channel events were rarely observed in cell-attached patches on C127i and C127/CFTR cells bathed in isotonic Ringer's solution (Fig 3 A). When C127i cells were exposed to hypotonic solution with maintaining giga-sealed cell-attached patches, single-channel currents with a large conductance were observed (Fig 3 A, middle trace) after a lag period of 15.0 ± 3.0 min (n = 10). This channel had a single-channel amplitude of 10.2 ± 0.3 pA (n = 12) and -5.7 ± 0.2 pA (n = 13) at +25 mV and -25 mV of -Vp, respectively. The unitary i-V relationship was slightly outwardly rectifying (presumably due to a lower Cl- concentration within the cell) with a slope conductance of 402 ± 18 pS at positive voltages and 285 ± 15 pS at negative voltages, and the reversal potential was around -5 mV (Fig 3 B, open circles). After excision, the i-V relationship became linear (Fig 3 B, closed circles).
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Channels with the same amplitude (Fig 3 A, bottom trace) and i-V relation (Fig 3 B, open triangles) were also observed in the cell-attached patches on C127/CFTR cells after exposure to hypotonic solution with a lag time of 4.4 ± 1.1 min (n = 10), which is significantly shorter than that in C127i cells (P < 0.01).
Even though we did not use the VSOR Cl- channel blocker, phloretin, during single-channel recordings, we never detected VSOR Cl- channel activity, which has an intermediate unitary conductance of 5070 pS and profound outward rectification (
Excision-induced Activation of Voltage-dependent Large-Conductance Anion Channels
When silent patches from C127i cells bathed in isotonic Ringer's solution were excised, patch conductance rapidly increased up to 210 nS (Fig 4 A). As shown in Fig 4 B, the patch current inactivated at +25 mV in a stepwise manner revealing unitary events with an amplitude of 1012 pA.
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In excised patches, voltage and time dependency of inactivation of macro-patch currents (Fig 5 A) was similar to that of swelling-induced phloretin-insensitive whole-cell currents (Fig 1 B, b). The shape of the instantaneous current-voltage relationship (Fig 5 B, open circles) was more linear compared with whole-cell currents, but the steady-state current similarly displayed portions with "negative slope resistance" at potentials of greater than or equal to +20 and less than or equal to -30 mV (Fig 5 B, closed circles). The I-V curve obtained during ramp clamp also displayed similar inactivation (Fig 5 C). Steady-state Po values estimated from current responses in macro-patches (containing a number of channels) to step pulses (Fig 5 A) and ramp pulses (Fig 5 C) were both plotted against voltages in Fig 5 D (closed circles and solid line, respectively), together with steady-state Po estimated from the phloretin-insensitive component of the swelling-activated whole-cell currents (Fig 1 B, b). The bell-shaped voltage dependency of open-channel probability of the macro-patch current was strikingly similar to that of whole-cell currents (Fig 5 D, triangles). Based on these observations, we conclude that voltage-dependent large-conductance channels were responsible for the phloretin-insensitive volume-dependent whole-cell current. This conclusion was further confirmed in studies evaluating anion selectivity and pharmacology of this voltage-dependent large-conductance channel (see next paragraph).
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Channels activated in inside-out patches had a conductance of 398 ± 9 pS and a linear symmetrical i-V relationship (Fig 6 A, open circles). Neither the shape of the i-V relationship nor the reversal potential changed when Na+ in the bath (Fig 6 A, open squares) or in the pipette (Fig 6 A, open triangles) solution was replaced with TEA+. In contrast, replacement of chloride with other anion species in the bathing solution had a profound effect on the single-channel i-V relationship. Also, the reversal potential became more positive for iodide and bromide ions and more negative for fluoride, aspartate, and glutamate (Fig 6 B). The calculated permeability ratios showed a selectivity sequence of I > Br > Cl > F > phosphate > aspartate glutamate, as summarized in Table 1. When NaCl concentration was lowered by replacement with mannitol, shifts in reversal potential yielded PCl/PNa = 2126 (Fig 6 C). Thus, it appears that the large-conductance channels are not only largely selective to Cl-, but are also permeable to large organic anions as well.
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Open-channel Blocking of the Large-Conductance Channel Current by ATP from Both Outside and Inside
In the outside-out patches, single-channel amplitude of the large-conductance channel was significantly decreased by extracellular ATP (10 mM added to the bath) at positive potentials, whereas there was not much of an effect at negative potentials (Fig 7A and Fig B). The effects of different ATP concentrations on single-channel i-V relationships obtained in the outside-out mode are shown in Fig 7 C. The doseresponse curve for the outward current could be well fitted with an equation for a single ATP-binding site (Equation 1) with a Kd of 12.3 ± 1.1 mM (Fig 7 D). This site is located at an electrical distance of 0.47 ± 0.07 from the external entrance to the pore, as evidenced by the Boltzmann fit (Equation 2) of the voltage-dependent blockade (Fig 7 E).
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In the inside-out mode, in contrast to the outside-out mode, inward currents at negative potentials were depressed by intracellular ATP (10 mM added to the bath) more significantly than the outward currents (Fig 8A and Fig B). Dose-dependent blockade of ATP applied to the intracellular solution is shown in Fig 8 C. This block was very profound for inward currents and could be well described by binding of ATP to a single binding site with Kd = 12.9 ± 0.6 mM (Fig 8 D) located at the distance of = 0.40 ± 0.06 from the internal mouth of the pore (Fig 8 E).
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Taken together, we conclude that ATP causes a voltage-dependent open-channel blockade of these large-conductance anion channels. This inhibitory effect of ATP is likely to be very fast since no increase in channel noise could be detected, even when the recordings were performed at 5-kHz bandwidth (unpublished data; n = 8). Nearly identical values for the two dissociation constants and the halfway location of the binding site strongly suggest that ATP binds to a site located near the center of pore when it is applied from either the extracellular or intracellular side of the membrane patch. Therefore, the large-conductance channel can accommodate ATP anions deep inside the pore lumen.
ATP Permeation through Large-Conductance Anion Channels
When the Ringer's bath solution was replaced with 100 mM ATP4-, current amplitude of an inside-out patch containing a number of large-conductance channels was inhibited, the I-V relationship became outwardly rectifying, and the inactivation gating became less prominent (Fig 9 A). Small inward currents could be detected both under ramp clamp (Fig 9A and Fig B) and by applying step pulses (Fig 9, BD). The small inward currents were found to be blocked by 100 µM NPPB or 200 µM SITS, but not Gd3+ (30 µM), added to inside-out patches from the intracellular side, whenever the outward currents were blocked (unpublished data; n = 3 each). If the inward current was due to sodium flux from the pipette, then the reversal potential of -20 mV would suggest PCl/PNa = 2.2, which is 10 times less than it was actually measured to be in our experiments (see data in Fig 6 C). When Na+ in the pipette was replaced with Cs+ or TEA+ in the presence of 100 mM ATP4- in the bath, the i-V relationship and the reversal potential were not affected (Fig 9 B and Table 2). Therefore, it is unlikely that the inward current is due to cationic flux from the pipette. Since no other anion except ATP4- was present in the bath, the inward current component should be due to the conductance to ATP4-. However, it is also possible that this inward current could be carried by inorganic phosphates produced by ATP degradation, since the channel exhibited a considerable permeability to phosphate (Table 1). To produce a reversal potential of -20 mV,
40 mM of HPO42- would be needed in the absence of significant ATP4- permeability of this channel. However, luciferin-luciferase measurements of the 100-mM ATP solutions, used in these experiments, gave an ATP concentration of 103 ± 3 mM (n = 5), which is not statistically different from 100 mM, suggesting that no significant ATP degradation occurred in the solutions used in our experiments. There is a possibility that the effects of 100 mM ATP were simply due to divalent ion chelation by ATP. However, single-channel conductance and channel gating were not essentially affected by applying Mg2+- and Ca2+-free Ringer's solution containing 1 mM EDTA (unpublished data; n = 9). Thus, it is concluded that the large-conductance anion channel is ATP-permeable with a PATP/PCl ratio of 0.080.1 (Table 2). This value is comparable to the permeability of the ATP-conductive pathway in CFTR-expressing cells (PATP/PCl = 0.2 [
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Pharmacological Profile of Large-Conductance Single-channel Activity
Fig 10 illustrates a pharmacological profile of the large-conductance anion channel at the single-channel level. This channel in outside-out patches was readily blocked by bath application of 30 µM Gd3+ (Fig 10A and Fig B, a, respectively), whereas, in the inside-out mode, 30500 µM Gd3+ had no effect on the channel activity from the intracellular side (Fig 10A and Fig 10 B, b, respectively). NPPB, produced a fast open-channel block: in the presence of 100 µM NPPB in the bathing solution, the large-conductance channels recorded in outside-out (a) and inside-out (b) patches had smaller amplitudes with increased noise (Fig 10 A). Another conventional Cl- channel blocker, SITS, produced a profound flickery open-channel block that is shown in traces from both outside-out (Fig 10 A, a) and inside-out (Fig 10 A, b) patches. The large-conductance anion single-channel in C127i cells was insensitive to 200 µM niflumic acid, 200 µM glibenclamide, and 300 µM phloretin from both extracellular (Fig 10 B, a) and intracellular (Fig 10 B, b) sides. Thus, the pharmacological properties of the single-channel currents from the voltage-dependent, large-conductance anion channel are virtually identical to those of the phloretin-insensitive volume-dependent whole-cell currents (Fig 1 B and 2 B).
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Pharmacological Profile of Swelling-induced ATP Release
Fig 11 A summarizes the pharmacological profiles of the swelling-induced ATP release from C127i cells under standard experimental conditions (37°C and 56% hypotonic stress) used in our previous paper (
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We then measured ATP release from C127i cells under the conditions similar to those for patch-clamp experiments (at 25°C and 71% osmolality). Fig 11 B summarizes the pharmacological profiles of the swelling-induced ATP release observed under these conditions. Extracellular Gd3+ again exhibited a strong inhibiting effect on the ATP release, whereas extracellular SITS and NPPB were less effective, and phloretin, glibenclamide, and niflumic acid had again no significant effect on the release of ATP. The pharmacological profile of swelling-induced ATP release is essentially the same as those found for whole-cell (Fig 1 B and 2 B) and single-channel currents (Fig 10).
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DISCUSSION |
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In response to many different stimuli, ATP has been demonstrated to be released from neuronal and nonneuronal cells. One reason that ATP may be released into extracellular fluid is to serve as a signaling molecule involved in cellcell communication in a wide variety of cell types (
Since most ATP molecules exist in anionic forms at physiological pH, there is a possibility that some anion channels can conduct ATP. In fact, recent patch-clamp studies have provided data showing ATP-conducting currents in association with the expression of CFTR (
Previously, in mouse mammary C127i cells, swelling-induced ATP release was shown to be independent of CFTR and VSOR Cl- channels (
Here, we identified and characterized the volume-dependent VDACL anion channels in C127i cells. We conclude that this channel serves as a pathway for swelling-induced ATP release in this cell line for the following reasons: (1) this channel is activated by osmotic cell swelling in both cell-attached and whole-cell modes; (2) the channel pore has an ATP-binding site that is about halfway from both extracellular and intracellular sides, thereby resulting in ATP accumulation within the pore; (3) the current that was carried by ATP4- was actually detected in inside-out patches; (4) this channel shares the same pharmacological profile as swelling-induced ATP release, i.e., sensitivity to Gd3+, SITS, and NPPB as well as insensitivity to phloretin, niflumic acid, and glibenclamide; and (5) swelling-induced activation of this channel was facilitated in CFTR-expressing cells in agreement with the upregulation of swelling-induced ATP release by CFTR (105 ATP4- s-1 at -30 mV. This is comparable to the measured rate of ATP release, which was 2 x 104 s-1 cell-1 in response to cell swelling (
Biophysical and physiological characteristics of VDACL anion channels in C127i cells are quite different, in a number of ways, compared with VSOR Cl- channels in C127/CFTR cells (400 pS) was much greater than that of VSOR channels (5070 pS). Second, single-channel current-voltage relationship was linear for VDACL channels, but was outwardly rectifying for VSOR channels. Third, voltage-dependent inactivation was observed in VDACL currents not only at large positive potentials, but also at large negative potentials, thus exhibiting the bell-shaped voltage dependency of open-channel probability, whereas it was observed in VSOR currents only at large positive potentials. Fourth, VDACL, but not VSOR, channels were activated in cell-attached patches when cells were exposed to a hypotonic solution after giga-sealed formation. Fifth, VDACL, but not VSOR, channels could be activated by patch excision without reducing bath osmolarity. Sixth, the presence of intracellular ATP was not required for VDACL channel activity, but was a prerequisite to VSOR channel activity. The pharmacological profile for inhibition of VDACL channel activity was also distinct from that of VSOR channel. VDACL channels were sensitive to Gd3+, which was ineffective for VSOR channels, and insensitive to known blockers of VSOR channels, such as phloretin, niflumic acid, and glibenclamide.
The large unitary conductance and bell-shaped voltage dependency of VDACL channels in C127i cells are similar to those of the voltage-dependent anion channel (VDAC) expressed in the outer mitochondrial membrane (
Previous studies have suggested various signaling pathways involved in activation of the VDACL anion channels in other cell types: G proteins (
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Footnotes |
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* Abbreviations used in this paper: VDAC, voltage-dependent anion channel; VDACL, voltage-dependent ATP-conductive large-conductance; VSOR, volume-sensitive outwardly rectifying.
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Acknowledgements |
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The authors are grateful to P.D. Bell for reading the manuscript, to S. Tanaka and K. Shigemoto for technical assistance, and to T. Okayasu for secretarial assistance.
This work was supported by Grant-in-Aid for Scientific Research (to R.Z. Sabirov) and by that for Priority Areas of "ABC Proteins" (to Y. Okada) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by grants from Houansha Foundation and Salt Science Foundation (to Y. Okada).
Submitted: 2 March 2001
Revised: 16 July 2001
Accepted: 18 July 2001
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