Inhibition by cytoplasmic nucleotides of a new cation channel in freshly isolated human and rat type II pneumocytes
Norbert Mair,1
Manfred Frick,1
Cristina Bertocchi,1
Thomas Haller,1
Albert Amberger,2
Helmut Weiss,3
Raimund Margreiter,2,3
Werner Streif,4 and
Paul Dietl1
Departments of 1Physiology, 3General and Transplant Surgery, and 4Pediatrics, Medical University of Innsbruck, and 2Tyrolean Cancer Research Institute, A-6020 Innsbruck, Austria
Submitted 17 May 2004
; accepted in final form 12 August 2004
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ABSTRACT
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Here we report a 26- to 29-pS cation channel abundantly expressed in freshly isolated and primary cultured type II cells from rat or healthy human lungs. The channel was never spontaneously active in cell-attached patches but could be activated by cell permeabilization with
-escin. Excised patch-clamp experiments revealed activation by Ca2+ concentrations at the cytoplasmic side in the micromolar range. High concentrations of amiloride (>10 µM) at the extracellular side did not inhibit. The channel was equally permeable for K+ and Na+ but was essentially impermeable for Cl, Ca2+, and Mg2+. It was blocked by adenosine nucleotides (cytoplasmic side) with the following order of potency: AMP
ADP (EC50
10 µM) > ATP >> adenosine >> cyclic AMP. The blocking effect of ATP was reproduced by its nonhydrolyzable analogs AMPPNP or ATP-
-S. GTP did not inhibit. Cd2+ blocked the channel with an EC50
55.5 nM. We conclude that type II cells express a Ca2+-dependent, nucleotide-inhibited, nonselective, and Ca2+-impermeable cation channel (NSCCa/AMP) with tonically suppressed activity. RT-PCR confirmed expression of TRPM4b, a channel with functional characteristics almost identical with NSCCa/AMP. Potential physiological roles are discussed.
patch clamp; calcium; adenosine 5'-monophosphate; adenosine 5'-diphosphate; adenosine 5'-triphosphate; cadmium; lung; alveolus; nonselective cation channel
ION CHANNELS in alveolar type II cells have been intensively investigated. Most attention has been directed toward ion channels involved in transepithelial solute and water transport, one reason being the fascinating transition of the alveolus from a secretory to a reabsorptive organ during birth (reviewed in Ref. 32). Although the cellular mechanisms and physicochemical processes of this transition are still only partly understood, the general concept emerged that both secretion and reabsorption are active processes of the type II cell, the latter being dependent on the expression of functional
-subunits of the epithelial Na+ channel (ENaC) (11). Also in the adult alveolus, ion transport through type II cells is considered to play an essential role for alveolar fluid balance and edema clearance (reviewed in Ref. 25), although recent evidence suggests that type I cells may contribute to this task (14).
Substantial controversy has recently evolved about the role of Cl channels as well as the molecular identities, biophysical properties, and regulation of Na+ channels involved in transepithelial NaCl reabsorption in the adult alveolus (reviewed in detail in Refs. 20, 23, 24, 28, and 40). A particular matter of debate is a heterogeneous group of poorly selective cation channels, which have been described by several groups in both fetal distal epithelial (29) and adult type II cells (1, 5, 12, 13, 15, 36, 42). These channels have conductances between 10 and 30 pS and are differently sensitive to elevations of Ca2+ concentrations at the cytoplasmic side ([Ca2+]cyt). Some of them are considered to be associated with ENaC-mediated, transepithelial solute transport for several reasons. First, they are generally inhibited by high concentrations of amiloride, which also blocks ENaC and perinatal fluid reabsorption. Second, channel activity was reported to be stimulated by cyclic AMP or protein kinase A (1, 3, 36, 42), which is also thought to be the major signaling pathway of epinephrine-stimulated fluid reabsorption and perinatal fluid clearance. Third, and most specifically, antisense oligonucleotides targeting the
-subunit of ENaC decreased the density of the 20.6-pS, nonselective cation channel (12). As for all ENaC-related cation channels, ion selectivity may be determined by the coexpression pattern of the three subunits (7, 35, 37), giving rise to substantial functional diversity, depending on culture conditions and delay after isolation from lung tissue (13, 41). Another class of cation channels, activated by cyclic GMP, was suggested to account for an amiloride-insensitive fraction of alveolar fluid clearance (27).
Here we describe another, yet unknown, cation channel in the alveolar type II cell. This channel is most likely not involved in transepithelial solute transport, as it is amiloride insensitive and not activated by compounds known to stimulate epithelial transport. Consistently, channels with similar features have been described in various nontransporting and transporting cell types (10, 16, 30, 33, 34, 38). Adopting the classification of Halonen and Nedergaard (10), we refer to it as NSCCa/AMP, because Ca2+ is the only known activator and AMP (together with ADP) has the most potent blocking effect. Its high abundance in freshly isolated human and rat type II pneumocytes suggests an important function that remains to be determined.
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METHODS
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Rat type II cell preparation.
This was done according to the method of Dobbs et al. (4), with minor modifications (9). At the end of the cell preparation from adult male Sprague-Dawley rats (
200 g), type II cells were suspended in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 24 mmol/l NaHCO3 and seeded on glass coverslips at low density (
40 cells/mm2). Cells were given time to settle in 95% humidified air plus 5% CO2 (37°C) for 648 h before being used for patch clamp.
Human type II cell preparation.
Human type II cells were obtained from cadaveric donor lung lobes that were not considered suitable for lung transplantation due to adjacent organ injury. Lung preservation was carried out according to standardized protocols for human organ transplantation. In brief, lower pulmonary lobes were retrieved as part of multiorgan procurement (22). A median sternotomy was performed, and all of the abdominal dissections were finished before lung perfusion was started. After heparin administration, the lower lobe was completely inflated and resected by use of a staple-line instrument (Endo-GIA; Tyco Healthcare, Vienna, Austria). The respective pulmonary artery was then cannulated. Back-table perfusion started immediately thereafter as a cold high-volume/low-pressure perfusion (Perfadex; Vitrolife, Goeteborg, Sweden) at 4°C. Lung lobes were then triple-bagged in cold Perfadex solution and stored on ice. Type II cell preparation started after a cold ischemia time of up to 30 min. The remaining protocol was identical with cell preparation from rat, following lung removal, as described in Ref. 9. Before patch-clamp experiments, human cells subconfluently grown on glass coverslips were identified as type II cells by their typical appearance (multiple large vesicles), and cell viability was confirmed by active, pH-dependent uptake of LysoTracker Green DND-26 into lamellar bodies, as described in Ref. 9. In addition, cell membrane integrity was confirmed by the lack of permeation of the amphiphilic dye FM 1-43 from the bath solution into the cytosol (9).
This protocol has been designed and performed in accordance with the ethical standards in the Declaration of Helsinki and has been reviewed by the ethics committee of the University of Innsbruck.
Major findings obtained from rat type II cells were subsequently verified in human alveolar type II cells. Common features are listed in Table 1.
Single-channel patch-clamp experiments and fluorescence measurements.
Cell-attached and inside-out (I-O) patch-clamp experiments were performed with borosilicate glass patch pipettes of 6- to 7-M
resistance and with an EPC9 (HEKA) setup as previously described (21). The I-O configuration was obtained by tearing the patch off the plasma membrane. Unless otherwise stated, the bath solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 D-glucose, and 10 HEPES, pH 7.4 (with NaOH). This control solution was also used as pipette solution unless stated otherwise. Single-channel currents were either recorded at various constant holding potentials (Vh) or during repetitive Vh ramps from 80 to +80 mV and vice versa (Fig. 1). Each ramp lasting for 6 s corresponded to a slope of ±0.053 mV/ms. The open probability (Po) is defined as the fraction of time during which the channels are in their open state within a patch. Po was calculated according to
 | (1) |
where tn is the open time of the channel in the nth level and n is the channel level. T is the total recording time. N is the maximum level of channel opening. Po was calculated without making any assumption about the total number of channels in a patch or the Po of a single channel. In experiments where Vh changed periodically (0/80/0/+80/0), Po values were separately determined for both the negative (P
) and the positive (P
) cycle of Vh (since single-channel currents are negligibly small near the reversing potential, Po was not determined for a Vh within the range of ±5.3 mV from the reversal potential). Single-channel currents were sampled at 5 kHz, filtered at 1 kHz with a low-pass Bessel filter, and stored on the hard disk of a PC using the Pulse + PulseFit software (version 8.53, HEKA).

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Fig. 1. A: holding potential (Vh) protocols used to study NSCCa/AMP (Ca2+-dependent, nucleotide-inhibited, nonselective, and Ca2+-impermeable cation channel). B: excision activation. B, left: current recording of a cell-attached membrane patch in an intact type II cell in response to Vh steps and ramps as shown in A. No current is detectable. B, right: same membrane as left after excision to the inside-out (I-O) configuration. The currents (I) in response to a Vh ramp (top tracing) and during a constant Vh of 60 mV (bottom tracing) reveal activation of 2 channels in the patch. Currents were leak corrected.
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For experiments done in the cell-attached mode, cells were preloaded with fura-2 AM (1.2 µM; Molecular Probes, Leiden, The Netherlands) for 30 min in the incubator. Excitation light of 340-, 380-, and 360-nm wavelengths was applied for 30 ms followed by 6 s of darkness. During illumination, the emitted fura-2 fluorescence was continuously detected by a photomultiplier tube throughout the experiments. The produced current was sampled at a rate of 5 kHz and averaged. The fluorescence signal at the isosbestic point (360 nm) was not Ca2+ dependent and was, therefore, taken to assess the integrity of the plasma membrane during the experiment. The ratio signal (340/380 nm) was used to follow changes in [Ca2+]cyt. Control of excitation light and data acquisition were performed with the fura extension of the Pulse + PulseFit software (version 8.53, HEKA).
RNA isolation and RT-PCR analysis.
Total RNA was isolated from freshly isolated rat type II cells using TriReagent (MRC, Cincinnati, OH). The reverse transcription reaction was performed in a solution of 1 µg RNA, 0.4 mM dNTPs, oligo(dT) primer, and Maloney murine leukemia virus reverse transcriptase (all from Promega, Mannheim, Germany) at 37°C for 60 min. For RT-PCR, 2 µl of template were used.
To design rat transient receptor potential channel, melastatin subfamily (TRPM)4-specific primers, we searched the National Center for Biotechnology Information database (available at www.ncbi.nlm.nih.gov/BLAST) with the mouse TRPM4 sequence (NM_175130
[GenBank]
) using the tblastN program. The Rattus norvegicus chromosome 1 WGS supercontiguous NW_047558 was predicted. Analysis of the NW_047558 sequence leads to the identification of 25 sequence segments that are equivalent to exon 1 to exon 25 of the mouse TRPM4 gene. The rat "exons" cover
30 kb of the NW_047558 sequence and exhibit
94% homology to the mouse sequence. Two sets of primer pairs were designed. Primer 1 covers exon 8 to exon 9 forward: 5'-CTCGGCTTACCTGGATGAGC-3', reverse: 5'-CTCCCAACAGAGTCCTCAG-3'. Primer 2 covers exon 19 to exon 22 forward: 5'-CAGCGACCTCTACTGGAAGG-3', reverse: 5'-GAGGCAGTAAGGCAGAGTGG-3'. Comparative RT-PCRs were done under the same conditions, and glyceraldehyde phosphodehydrogenase was amplified using published primers (31). The number of PCR cycles was 35.
Materials.
Unless stated otherwise, drugs were purchased from SIGMA, Austria.
Statistics.
Data are reported as means ± SE. Experiments with no statistical difference between the control and experimental group are mentioned in the text but are not shown as numeric values or as graphs.
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RESULTS
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Channel expression and Ca2+ dependence.
No activity of this channel was observed under control conditions, i.e., 6 h up to 5 days after cell isolation, exposed to a control solution in the cell-attached patch-clamp configuration. This lack of channel activity was independent of Vh. Considering a membrane potential of the intact cell of approximately 30 mV (Frick and Mair, unpublished data), clamped potentials correspond to estimated effective potentials between 110 and +50 mV. In contrast, the channel was almost regularly observed after changing from the cell-attached to the I-O patch-clamp configuration under conditions of high [Ca2+]cyt (2 mM, excision activation occurred in
70% of all I-O patches, as exemplified in Fig. 1). This indicates that the channel is abundantly expressed in type II cells. As shown in Fig. 2, channel activation occurred when [Ca2+]cyt exceeded the nanomolar range. The concentration-response curve suggests that full channel activation was not even obtained when [Ca2+]cyt equaled the extracellular concentration of 2 mM.

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Fig. 2. A: on/off switch function of cytoplasmic Ca2+. Current tracing of an excised, I-O membrane patch that was alternatingly exposed to a Ca2+-free and a Ca2+-containing (2 mM) solution. The 2 channels are alternatingly switched off and on. B: Ca2+ concentration-response relationship. The open probability (Po) was determined in I-O patches exposed to various cytoplasmic Ca2+ concentrations. Dark gray filled circles represent Po values in the positive Vh range of repetitive ramps (see inset); light gray filled circles represent Po values in the negative Vh range (see inset). The maximum Ca2+ concentration was 2 mM, because this value is the upper limit that can be expected in vivo for Ca2+ concentrations at the cytoplasmic side ([Ca2+]cyt) to be reached under maximum cell permeabilization.
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Surrogate divalent ions as potential channel activators.
The candidates were Sr2+, Mn2+, Mg2+, Ba2+, Co2+, and Cd2+. In these experiments, 8 mM of each surrogate ion were applied to the cytoplasmic side of I-O patches, and Ca2+ was omitted (osmolarity was kept constant by adjusting NaCl). Figure 3 demonstrates that Sr2+ (n = 15) and Mn2+ (n = 16) could replace Ca2+ as channel activator to a certain extent, however, with a dramatically reduced Po. In contrast, Mg2+, Ba2+, Co2+, and Cd2+ were not able to replace Ca2+ (n = 15, 30, 21, and 11, respectively, data not shown). Readdition of 2 mM Ca2+ and omission of the surrogate ions reactivated the channel except for Cd2+, which appeared to block irreversibly (see below). Co2+ transiently increased channel activity before channel closure occurred (not shown).

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Fig. 3. The effects of cytoplasmic Sr2+ and Mn2+ in the absence and presence of 2 mM Ca2+. Original current tracings from 2 different I-O patches (top vs. bottom tracings) are shown (experimental sequence from left to right). Note that Sr2+ or Mn2+ can activate the channel to a small extent.
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Ion selectivity.
Ion replacement experiments (N-methyl-D-glucamine+ for Na+ or K+, gluconate for Cl) revealed a high selectivity for K+ and Na+ over Cl with approximately equal permeabilities for Na+ and K+ (Fig. 4). When N-methyl-D-glucamine Cl was replaced by equiosmolar amounts of CaCl2 (n = 18) or MgCl2 (n = 10) in the presence of 2 mM CaCl2, no changes of the current amplitude were observed. Hence, the channel has to be considered Ca2+ impermeable.

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Fig. 4. Examples of ion replacement experiments. Original current recordings (not leak corrected) of I-O patches during Vh ramps as shown above the current recordings. Solutions on both sides of the membrane patch are shown (right). A minimum of 2 mM Ca2+ at the cytoplasmic side was present in all experiments to maintain channel activity. Note that the current is generated by Na+ and K+. The solutions contained (in mM, in addition to 10 HEPES and 5 D-glucose, pH 7.4) for control: 140 NaCl, 5 KCl, 2 CaCl2, and 1 MgCl2; for 0 Na+ 0 K+: 145 N-methyl-D-glucamine, 2 CaCl2, and 1 MgCl2; for 93 Ca2+: 93 CaCl2 and 1 MgCl2; for 92 Mg2+: 2 CaCl2 and 92 MgCl2; for 145 Na+ 0 K+: 145 NaCl, 2 CaCl2, and 1 MgCl2; and for 0 Na+ 145 K+: 145 KCl, 2 CaCl2, and 1 MgCl2.
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Current-voltage behavior.
Po in the negative range of a voltage sweep (P
) was significantly lower than in the positive range (P
) (compare Figs. 1 and 4). The mean duration of a single channel opening was significantly longer at positive Vh. In contrast, the channel conductance was slightly smaller in the positive vs. negative Vh range (25.80 ± 0.05 pS and 28.86 ± 0.07 pS, respectively) (Figs. 1 and 4). The product Po x I (I represents current) was higher in the positive range (data not shown), suggesting that whole cell currents, if present, would exhibit an outward rectification.
Effects of potential inhibitors.
These experiments were performed in the presence of 2 mM [Ca2+]cyt. When amiloride (0.55 µM) was applied to the bath solution (I-O patch), Po was not significantly changed (n = 5). Likewise, channel activity was not significantly affected by the presence of 15 mM amiloride in the patch pipette (n = 14, data not shown).
Sr2+, Mn2+, Mg2+, and Ba2+ did not block the channel at concentrations of 8 mM in the presence of 2 mM [Ca2+]cyt (n = 6, 7, 6, and 7, respectively, data not shown). Co2+ even increased the ability of Ca2+ to activate the channel and arrested the channel in the open state (n = 8, data not shown). Only Cd2+ was a very potent channel blocker with an EC50
55.5 nM (Fig. 5).

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Fig. 5. Cd2+ concentration-response relationship. Po is expressed as % of control (i.e., in the presence of 2 mM bath Ca2+ in I-O experiments). As in Fig. 2, light gray filled circles represent values obtained during the negative Vh range of Vh ramps (see Fig. 2B inset); EC50 = 67 nM. Dark gray circles are values from the positive Vh range (see Fig. 2B inset); EC50 = 51 nM; average EC50 = 55.5 nM. Data were fitted with the general dose-response curve.
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Effects of agonists and intracellular messengers in cell-attached patch-clamp experiments.
We tested whether ATP (20 µM), a known Ca2+-dependent purinergic agonist in type II cells, or ()isoproterenol (10 µM), a cAMP-dependent stimulator of Na+ transport, could activate the channel on cell, i.e., within the plasma membrane of living type II cells. Agonists were applied in the bath solution. Neither ATP nor ()isoproterenol activated the channel in cell-attached patches (n = 18 and 32, respectively, data not shown), where the presence of this channel was verified at the end of the experiments by excision-activation. Hence, we can clearly state that even though the channel was present within the membrane patch of the pipette, it could not be activated on cell by these agonists. The cell-permeable cAMP analog 250 µM dibutyryl adenosine 3',5'-cyclic monophosphate and the Ca2+ ionophore ionomycin (110 µM) were also ineffective (n = 25 and 15, respectively, not shown), although we know from previous experiments (8) that these concentrations of ionomycin should yield a [Ca2+]cyt far above the threshold of channel activation, as shown in Fig. 2B. We therefore speculated that some cytosolic components keep the channel closed even at a high [Ca2+]cyt.
To test this hypothesis, we aimed at permeabilizing the plasma membrane to allow leakage and depletion of cytoplasmic compounds. We used
-escin, a saponin derivative extracted from horse chestnuts, which was reported to form holes permeable to high-molecular-weight substances (17).
-Escin (540 µM) was added to the bath. In these experiments, the cells were preloaded with fura-2 (see METHODS), and the single cell fura-2 ratio was simultaneously recorded with the channel activity. An example of such an experiment is shown in Fig. 6. In all experiments (n = 27), [Ca2+]cyt rose immediately after addition of
-escin, which can be explained as the initiation of permeabilization. Fura-2 leakage from the cell occurred one to several minutes later (Fig. 6) and was dependent on the
-escin concentration. Notably, the beginning of fura-2 leakage coincided with channel activation (Fig. 6). Konishi and Watanabe (17) reported that with a low concentration of
-escin (up to 5 µM), most of the cellular ATP is lost within 3040 min. Their results demonstrated that
-escin concentrations of 5 µM render the cell membrane permeable to relatively small molecules (ATP) without a substantial loss of large cytosolic proteins. The molecular size of ATP is
0.5 kDa and that of fura-2 is
0.65 kDa. We assumed, therefore, that it was the loss of nucleotides that activated the channel (in addition to the high [Ca2+]cyt) and tested the hypothesis below.
Effects of nucleotides at the cytoplasmic side of the channel.
ATP, ADP, AMP, and adenosine were added to the control bath solution in I-O patch-clamp experiments. The calcium and magnesium ions bound by nucleotides were corrected according to Chang et al. (2) to keep the free [Ca2+]cyt and [Mg2+]cyt constant in all experiments. The results are shown in Fig. 7. The nucleotides blocked the channel in the following order of potency: AMP
ADP > ATP > adenosine (see Fig. 7 legend for EC50 values). A comparison of the inhibitory potencies of these and other nucleotides is shown in Fig. 8. cAMP also acted as inhibitor, although not as effectively as ATP or adenosine. GTP had no effect (n = 14, Fig. 8).

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Fig. 7. Nucleotide concentration-response relationship. Nucleotides were added to the bath solution containing 2 mM Ca2+ in I-O patch-clamp experiments. (For further details, see Fig. 5.) The EC50 values for these substances in the negative and positive Vh range, respectively, are ATP: 64.8 and 103 µM, ADP: 6.7 and 10.7 µM, AMP: 6.3 and 10.1 µM, and adenosine: 211.5 and 235.2 µM. The ranking order in terms of inhibition is AMP = ADP > ATP > adenosine.
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Fig. 8. Comparison of the effects of cytoplasmic adenosine nucleotides, guanosine nucleotides, and adenosine, each given at a concentration of 0.5 mM, on the Po in I-O patches. Dark and light gray values are as described in Fig. 5. AMPPNP, adenosine 5'-(b,g-imido)triphosphate tetralithium salt hydrate.
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Hydrolysis is not required for channel inhibition because the nonhydrolyzable ATP analog ATP-
-S (0.05 mM) reduced Po even more than the same amount of ATP (Po = 40.2 ± 8.3 and 61.1 ± 4.9% of control, respectively, n = 16 and 19). Adenosine 5'-(
,
-imido)triphosphate tetralithium salt hydrate (0.5 mM), another nonhydrolyzable ATP analog, was equally effective (P
= 8.76 ± 2.8%; P
= 21.02 ± 5.28% of control, n = 17, Fig. 8), indicating that ATP hydrolysis is not required for channel inhibition.
A potential role of the cytoskeleton for channel activation was also examined. Preincubation with 1 µM latrunculin B, which inhibits actin polymerization and disrupts microfilament organization, did not result in channel activation in cell-attached patches (n = 15). Nocodazol, a tubulin depolymerization promoter, also had no effect.
Expression of rat TRPM4b.
The efficiency of nucleotide inhibition and other features of NSCCa/AMP resemble a recently described splice variant of the TRPM4 channel TRPM4b, where TRPM stands for transient receptor potential channel, melastatin subfamily. We analyzed TRPM4b by RT-PCR in two independent preparations of rat type II cells. Two primer pairs were used: primer 1 amplified a 365-bp product from exon 8 to exon 9 of the rat TRPM4 gene, and primer 2 amplified a 443-bp product from exon 19 to exon 22, respectively. Importantly, the amplified products cannot stem from genomic DNA since the primers cross exon/intron boundaries. As shown in Fig. 9, both PCR products could be amplified from type II cells, demonstrating that the TRPM4b gene is expressed.

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Fig. 9. Expression of transient receptor potential channel, melastatin subfamily (TRPM)4. RT-PCR of 2 independent preparations of type II cells (a and b). Two PCR products specific for rat TRPM4 were detected: primer 1 amplified a 365-bp fragment, and primer 2 amplified a 443-bp fragment. For controls, RT-PCR was performed without reverse transcriptase.
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DISCUSSION
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The major differences between NSCCa/AMP and other published cation channels in type II cells are its inactivity under normal physiological conditions, its amiloride insensitivity, and the lack of ()isoproterenol or cAMP-dependent activation, but, instead, nucleotide-dependent inhibition. With regard to both features, NSCCa/AMP is certainly different from a 20- to 25-pS channel purified from type II cells and reconstituted in lipid bilayers, because in this channel, ATP at the cytoplasmic side did not have an inhibitory effect (36). Furthermore, since NSCCa/AMP is clearly not activated by the cAMP pathway, it is different from cation channels involved in cAMP-dependent, transepithelial Na+ reabsorption.
Despite its Ca2+ dependence, NSCCa/AMP was never activated on cell by extracellular ATP, although this agonist evokes an elevation of [Ca2+]cyt in type II cells that is usually >500 nM (8) and hence in the range where channel openings should be observed. All evidence presented here suggests that this is due to a strong inhibition by cytoplasmic adenine nucleotides, overriding potential activation by Ca2+. This is another argument that the channel is different from nonselective cation channels associated with transport because these channels are spontaneously active on cell (42). Importantly, the abundance and features of NSCCa/AMP did not change within a time frame between a few hours and many days after cell preparation, which is also different from channels involved in transport (13). Taking together all these differences, it appears justified to conclude that NSCCa/AMP has an entirely different function than transepithelial transport and is "silent" under conditions when transport channels may be fully or partially active.
NSCCa/AMP channels of identical or similar characteristics have been described in various cell types, such as brown adipocytes (10, 16), pancreatic
-cells (34, 38), kidney epithelium (18, 30), and eye (corneal) endothelium (33). Until now, neither clear structural nor functional information has been available about them. Recently, however, a subtype of the TRP channel family TRPM4b (in contrast to the short splice variant TRPM4a) with striking similarities to NSCCa/AMP was expressed in HEK cells (19, 26). This channel is also cation selective, Ca2+ impermeable, Ca2+ dependent, and inhibited by nucleotides in the micromolar range (19, 26). A detailed comparison between NSCCa/AMP and TRPM4b is presented in Table 2. Hence, TRPM4b is a very strong candidate for NSCCa/AMP. Consistently, our RT-PCR experiments indicate that this channel is indeed expressed in type II cells. The appearance of multiple channels within one excised membrane patch is also consistent with the reported ability of TRPM4b to homoassociate and form homomultimeric channels (19).
Potential roles of NSCCa/AMP.
Expression of NSCCa/AMP in various cell types suggests involvement in a function that is common to cells of different origin and specialization. As long as the physiological (pathophysiological?) mode of activation of this channel remains obscure, we can only speculate about its role. Importantly, the cell permeabilization experiments with
-escin definitely rule out that excision activation (I-O patch) was a result of a physical disruption between the channel and cellular components (such as channel-anchoring elements).
In HEK-293 cells overexpressing TRPM4b, Launay et al. (19) reported transient TRPM4b channel activation on cell in an experimental setting where Ca2+ entry was greatly supported by a high, negative holding potential and simultaneous activation of store-operated Ca2+ influx. Unfortunately, these results are difficult to interpret, because their patch pipette-filling solution did not contain ATP, and actual cytoplasmic nucleotide concentrations are impossible to estimate. They suggested that localized (subplasmalemmal) rather than global Ca2+ concentrations would be able to activate TRPM4b, inducing oscillatory depolarizations of the membrane potential, thereby reducing the electrical driving force for Ca2+ entry and "fine-tuning" capacitative Ca2+ entry. This model implies a very close vicinity between a Ca2+-permeable channel and TRPM4b, where TRPM4b exerts a voltage-mediated negative feedback control on Ca2+ entry. If this hypothesis holds true for type II cells, TRPM4b might play an important protective role in the alveolus by preventing cellular Ca2+ overload during various types of stimulation. Because Ca2+-permeable cation channels are also considered to be involved in apoptosis (39), NSCCa/AMP could be, in analogy to its potential role as "anticapacitative Ca2+ entry channel," an "antiapoptosis channel." It should be noted that the inability to activate NSCCa/AMP on cell by agonists in our study may be due to a relatively low membrane potential of isolated type II cells (
30 mV, see above). This constitutes an unfavorable condition (small driving force) for agonist-induced (capacitative) Ca2+ entry, and hence, subplasmalemmal Ca2+ concentrations needed to activate NSCCa/AMP in cell-attached patches may be even more difficult to generate.
On the basis of the same considerations, Nilius et al. (26) suggested that TRPM4b might play a protective role during metabolic injury by slowing down Ca2+ entry and Ca2+-dependent metabolic processes. This prima vista appealing hypothesis suffers from the lack of a conceivable metabolic situation in which all three nucleotides (ATP, ADP, and AMP) should be depleted.
The main function of type II cells is the production and secretion of surfactant via exocytosis of lamellar bodies. Because exocytosis is an ubiqitous process, the widespread expression of NSCCa/AMP would be consistent with a role therein. It is unclear, however, how a Ca2+-impermeable cation channel could contribute to membrane fusion or fusion pore expansion. Consistent with its presumed Ca2+ entry-inhibiting function (see above), it would impede rather than stimulate surfactant secretion in type II cells, in particular when caused by cell strain, which is critically dependent on Ca2+ entry (6).
Korbmacher et al. (18) speculated that a similar cation channel in kidney collecting duct cells may be involved in volume regulation. We tested a potential mechanosensitivity by applying suction to the patch pipette. Channel activation was not detected under this condition (data not shown).
It appears that nucleotides are used by nature as constant, "reliable" inhibitors of this channel, preventing a fatal breakdown of the membrane potential under "normal" conditions. The search for a physiological situation where such inhibition is overridden in the type II cell (see above) will be necessary to gain more insight into its role in the lung. Beyond Ca2+ microdomains, hypothetical nucleotide-free cytoplasmic zones and potential cellular components in addition to Ca2+, which could overcome nucleotide inhibition, should be considered.
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GRANTS
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This work was supported by the Fonds zur Förderung der Wissenschaftlichen Forschung (FWF) Grants P15742 and P15743
[GenBank]
, Austrian National Bank Grant 9640, Fonds zur Förderung der Forschung an den Universitätskliniken Innsbruck Grant 16, and the Austrian Ministry of Science GZ 140.139/1-VI/Ia/2002.
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ACKNOWLEDGMENTS
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We thank Gabriele Buemberger for proofreading the manuscript. Technical assistance by Gerlinde Siber and Irina Öttl is gratefully acknowledged.
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FOOTNOTES
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Address for reprint requests and other correspondence: P. Dietl, Dept. of Physiology, Medical Univ. of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria (E-mail: paul.dietl{at}uibk.ac.at)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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