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. 1999 Sep 15;519(Pt 3):645–655. doi: 10.1111/j.1469-7793.1999.0645n.x

A novel cGMP-regulated K+ channel in immortalized human kidney epithelial cells (IHKE-1)

J R Hirsch 1, G Weber 1, I Kleta 1, E Schlatter 1
PMCID: PMC2269539  PMID: 10457080

Abstract

  1. K+ channels from the apical membrane of immortalized human kidney epithelial (IHKE-1) cells were investigated in the cell-attached membrane configuration as well as in excised membranes using the patch clamp technique.

  2. In cell-attached membrane patches the open probability (Po) of the K+ channel was 0.42 ± 0.06 (mean ± s.e.m., n = 22) and its conductance was 94 ± 5 pS with 145 mm K+ in the pipette (n = 25). In excised membrane patches the Po of the channel was 0.55 ± 0.03 (n = 86) and its conductance was 65 ± 2 pS (n = 68) with 145 mm K+ on one side of the membrane and 3.6 mm K+ on the other. The I-V curve of the K+ channel was not rectifying.

  3. The channel was inhibited by several blockers of K+ channels such as 1 mM Ba2+ (cell-attached membrane: 78 ± 8 %, n = 9; excised: 80 ± 4 %, n = 26), 10 mm TEA+ (excised inside-out: 48 ± 5 %, n = 34; excised outside-out: 100 ± 0 %, n = 26), 0.1 mm verapamil (excised: 73 ± 9 %, n = 12), and 10 nm charybdotoxin (excised outside-out: 67 ± 9 %, n = 9).

  4. The K+ channel was activated by depolarization and rising cytosolic Ca2+. Half-maximal activity occurred at a cytosolic Ca2+ concentration of 200 nm. In the cell-attached membrane configuration the K+ channel was inhibited in a concentration-dependent manner by atrial natriuretic peptide (ANP). Powas blocked equally well by 10 nm ANP (52 ± 7 %, n = 10), brain natriuretic peptide (BNP; 37 ± 11 %, n = 6) and C-type natriuretic peptide (CNP; 44 ± 13 %, n = 8). 8-Bromoguanosine 3′,5′ cyclic monophosphate (8-Br-cGMP, 0.1 mm) also inhibited Poof this K+ channel, by 70 ± 10 % (n = 5).

  5. In excised membrane patches cGMP inhibited Po of this K+ channel in a concentration-dependent manner. The first significant effects were measured at a concentration of 1 μm (22 ± 7 %, n = 6), and greatest effects were obtained at 0.1 mm (34 ± 5 %, n = 15). cAMP (0.1 mm, n = 5) as well as GTP (0.1 mm, n = 5) had no significant effects on Po of this K+ channel. ATP (0.1 mm) had a weak inhibitory effect (17 ± 5 %, n = 14). Addition of Mg-ATP to cGMP did not increase the inhibitory effect (30 ± 4 %, n = 14). KT5823 (1 μm), a specific inhibitor of cGMP-dependent protein kinases, did not significantly alter the cGMP-induced reduction in Po of the K+ channel in three excised membrane patches.

  6. The results present the first electrophysiological characterization of a mammalian K+ channel that is directly regulated by cGMP.

K+ channels play an important role in renal electrolyte transport. They provide the negative membrane potential, are involved in cell volume regulation, recycle K+, which is transported into the different renal cells by the Na+-2Cl−-K+ transporter and Na+-K+-ATPases, and regulate K+ homeostasis via K+ secretion in the cortical collecting duct (Lang & Rehwald, 1992; Wang et al. 1992, 1997; Giebisch, 1995; Greger, 1996). Several different K+ channels have been reported for luminal and basolateral membranes of renal epithelia. In patch clamp experiments on the luminal membrane of the proximal tubule at least four different K+ channels were identified: a 33 pS inwardly rectifying K+ channel in the S3 segment of rabbit and mouse (Gögelein & Greger, 1984), a 60 pS linear K+ channel in the same segment of mouse (Gögelein, 1990), a 200 pS maxi-K channel and a 42 pS K+ channel in primary cultures of rabbit proximal tubules (Merot et al. 1989; Dubéet al. 1990). In the basolateral membrane of the S2 segment of rabbit a 50 pS inwardly rectifying K+ channel has been identified (Gögelein & Greger, 1987; Parent et al. 1988).

Not much is known about the regulation of these ion channels. The K+ channel of the basolateral membrane of proximal tubules as well as the maxi-K channel are activated by depolarization, while the former is also pH and ATP dependent. It has been speculated that they might be involved in the repolarization of the membrane potential after the occurrence of Na+-dependent transport (Greger & Gögelein, 1987; Lang & Rehwald, 1992). Furthermore, it was shown that the maxi-K channel of primary cultures of proximal tubules is involved in cell volume regulation (Dubéet al. 1990). The basolateral K+ channel is Ca2+ independent, but seems to be coupled to the function of the Na+-K+-ATPase and can be regulated by cytosolic ATP (Tsuchiya et al. 1992; Hurst et al. 1993). Most K+ channels are downregulated by intracellular acidification like the 50 pS K+ channel of the basolateral membrane of rabbit and mouse (Gögelein & Greger, 1984) and the 42 pS K+ channel of the luminal membrane from primary cultured proximal tubule cells (Merot et al. 1989).

The proximal tubule is the most important location in the kidney for the Na+-coupled uptake of substrates such as glucose, phosphate, sulphate and amino acids. Since the driving force for Na+-coupled substrate transport is provided by the chemical gradient for Na+ and the negative membrane potential (Lang et al. 1986), K+ channels are essential in this part of the nephron to guarantee the maintenance of this negative membrane potential.

In this study we provide evidence for a novel Ca2+-dependent, intermediate conductance, depolarization-activated K+ channel that is directly regulated via cGMP in human proximal tubule cells (IHKE-1).

METHODS

Cell culture

Immortalized IHKE-1 cells originating from human embryonic kidneys were cultured as described before (Tveito et al. 1989). In short, IHKE-1 cells were maintained in DMEM-F12 medium (1:1) with addition of 15 mM Hepes, pH 7.3, 1.6 nM epidermal growth factor (EGF), 100 nM hydrocortisone, 5 mg l−1 transferrin and insulin, 29 nM Na2SeO3, 44 mM NaHCO3, 20 mM L-glutamine, 1000 U l−1 penicillin-streptomycin and 1 % fetal calf serum (FCS) in an atmosphere of 8 % CO2-92 % air at 37°C. Subculturing was done using 0.05 % trypsin-0.02 % EDTA in Mg2+- and Ca2+-free phosphate buffer. Culture medium was exchanged twice a week. Cells were used from passages 162 to 188. Cells grew in a polarized manner on glass coverslips with the apical surface facing upwards and developed apical microvilli (Hirsch et al. 1999). Cells from confluent monolayers did not respond to 10−4 M ouabain while those from subconfluent monolayers depolarized (0.7 ± 1.3 and 2.8 ± 0.4 mV, respectively, n = 3 each). There are several pieces of evidence that these cells are derived from proximal tubules, e.g. they have proximal tubule-specific enzyme markers (maltase, alkaline phosphatase and leucine aminopeptidase) in the apical membrane and, furthermore, they have several Na+-dependent and -independent amino acid as well as organic cation transport systems (Jessen et al. 1994; Hirsch et al. 1998; Hohage et al. 1998).

All media, buffers and growth factors were purchased in the highest available purity from Gibco, Biochrom/Seromed (Berlin, Germany), Calbiochem, Sigma and Merck (Darmstadt, Germany).

Patch clamp studies

Coverslips with cultured cells which had been grown to confluency were fixed at the bottom of a perfusion chamber mounted on an inverted microscope (IM 35, Zeiss, Oberkochen, Germany). The perfusion chamber was continuously perfused at a rate of 10-30 ml min−1 with a standard solution containing (mM): NaCl, 145; KH2PO4, 0.4; K2HPO4, 1.6; D-glucose, 5; MgCl2, 1; and calcium gluconate, 1.3; pH was adjusted to 7.4. All agonists were dissolved in this standard solution immediately before use. All experiments were performed at 37°C. For the recording of channel currents across membranes in the cell-attached or excised configuration, pipettes were filled with a solution containing (mM): KCl, 145; Na2HPO4, 1.6; NaH2PO4, 0.4; calcium gluconate, 1.3; magnesium gluconate, 1; and D-glucose, 5; pH was adjusted to 7.4. For cell-attached nystatin (CAN) experiments 20 mg l−1 nystatin (Sigma) was added to the pipette solution. Under these conditions single channel currents as well as membrane voltage can be measured depending on the amplifier setting (Vc, voltage clamp mode to resolve single channels; Cc, current clamp mode to visualize the membrane voltage) as has been described before (Greger & Kunzelmann, 1991). The input resistance of the patch pipettes was 8-15 MΩ. Single channel activity was recorded continuously with a patch clamp amplifier (U. Fröbe, Physiologisches Institut, Universität Freiburg, Germany) at a cut-off frequency of 10 kHz and stored directly after A/D conversion on a Pentium computer system. Single channel currents and open probabilities were analysed after low-pass filtering between 1 kHz and 200 Hz (eight-pole Bessel filter, type 902; Krohn-Hite, Avon, MA, USA) using patch clamp analysis software (U. Fröbe, Physiologisches Institut). The noise band allowed the detection of single channel activity with current amplitudes above 0.5 pA.

Biochemicals

All standard chemicals and nucleotides were obtained in the highest available purity from Merck, Calbiochem or Sigma. KT5823 was obtained from Calbiochem. The natriuretic peptides ANP, BNP and CNP were synthesized in the Niedersächsisches Institut für Peptidforschung (Hannover, Germany; we gratefully acknowledge the help of Dr K. Adermann).

Statistics

Data are presented as original recordings from individual experiments or as mean values ±s.e.m. with n referring to the number of observations. Patch clamp experiments were performed in a paired fashion with pre- and postcontrol periods for each experimental manoeuvre. Pre- and postcontrol values were averaged and compared with the corresponding experimental value. Thus, Student's two-tailed paired t test was used to test for statistical significance of the effects. P < 0.05 was set as the significance level.

RESULTS

Recently, we described a K+ conductance in immortalized human kidney epithelial cells (IHKE-1) that was downregulated by natriuretic peptides and 8-Br-cGMP (Hirsch et al. 1999). In order to identify this K+ conductance at the single channel level we investigated the apical membrane of IHKE-1 cells with the patch clamp technique. In 100 experiments with IHKE-1 cells we identified a 10 pS and a 22 pS K+ channel, a 163 pS maxi-K channel (all excised patches) and a fourth channel which caught our interest since it was highly active on the cell and different from all K+ channels described so far for the proximal tubule. The characterization of the biophysical and pharmacological properties of this channel as well as its regulation by intracellular factors in cell-attached experiments and excised membrane patches is described below.

In 25 cell-attached experiments this novel intermediate conductance K+ channel was recorded 25 times and therefore was the most prominent one. The small 22 pS channel was seen in 11 of these cell-attached recordings while the maxi-K channel was never active on the cell. In excised membrane patches we recorded the intermediate conductance K+ channel 87 times, the 22 pS channel 10 times and the maxi-K channel 53 times. Besides these three K+ channels in the cell-attached configuration the very small, 10 pS K+ conductance that was no longer detected in the presence of Ba2+ was recorded occasionally. Since it was often hidden among the other types of K+ channels we were not able to further characterize this K+ channel. This channel is also the reason for most of the background noise in some of the cell-attached and excised membrane recordings.

Biophysical properties of the intermediate conductance K+ channel

The mean open probability (Po) of the intermediate conductance K+ channel in cell-attached experiments was 0.42 ± 0.06 (n = 22), and its conductance was non-rectifying and reached 94 ± 5 pS with 145 mM K+ in the pipette (n = 25). In excised membrane patches Po of this K+ channel was 0.55 ± 0.03 (n = 86) and its conductance was 65 ± 2 pS (n = 68) with 145 mM K+ on one side of the membrane and 3.6 mM K+ on the other. Its permeability after excision was 320 ± 12 cm2 s−1 (n = 68). The K+ channel had an extrapolated reversal potential, Vrev, of 99 mV which leaves practically no significant permeability for Na+. Besides K+, this K+ channel was also capable of transporting NH4+ with a permeability ratio of NH4+/K+ of 0.52 (n = 3). Original current traces and current-voltage relationships (I-V curves) of the intermediate conductance K+ channel in the cell-attached and excised membrane configuration are shown in Fig. 1.

Figure 1. Original current traces at the membrane voltage and I-V relationships for the intermediate conductance K+ channel of IHKE-1 cells in the cell-attached (left) and excised (right) membrane configuration.

Figure 1

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The I-V relationship on the left with symmetrical K+ concentrations can be fitted linearly, the one on the right under asymmetrical K+ concentrations with the Goldman-Hodgkin-Katz equation. The K+ channel had an extrapolated reversal potential, Vrev, of 99 mV which indicates practically no significant permeability for Na+. The conductance of the K+ channel in excised membrane patches was estimated at a clamp voltage of 0 mV.

Inhibitors and regulators of the intermediate conductance K+ channel under cell-attached conditions

In order to identify the channel under cell-attached conditions as a K+ channel, in addition to the determination of its reversal potential, its inhibition by 1 mM Ba2+ was tested. Ba2+ enters the cell via Ca2+ channels and blocks K+ channels from the cytosolic side (Bleich et al. 1990). Compared with control, Po was reduced in the presence of Ba2+ by 78 ± 8 % (n = 9). To actually show that this K+ channel was the one responsible for the K+ conductance regulated by the natriuretic peptides in these cells (Hirsch et al. 1999), ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and CNP (C-type natriuretic peptide) were tested in the cell-attached membrane configuration. ANP at 10−12 M reduced Po by 7 ± 3 % (n = 3), 10−10 M ANP reduced it by 34 ± 10 % (n = 7), 10−9 M ANP by 41 ± 8 % (n = 6), and 10−8 M ANP by 52 ± 7 % (n = 10). An original current trace of the ANP effect is shown in Fig. 2A. Comparable with the effect of ANP, CNP (10−8 M) reduced Po of this K+ channel by 51 ± 13 % (n = 7). An original current trace of the CNP effect is displayed in Fig. 2B. The membrane-permeable analogue of the respective second messenger 8-Br-cGMP (0.1 mM) blocked Po of this ion channel by 70 ± 10 % (n = 5) and an original current trace displaying the inhibitory effect of 8-Br-cGMP is shown in Fig. 2C. An example of the effect of BNP (10−8 M) which also inhibited Po by 39 ± 13 % (n = 5) using the CAN method is displayed in Fig. 2D. In four experiments the CAN method was chosen to detect a parallel decrease in membrane voltage (Vm) and single channel current. A summary of all natriuretic peptide effects on Po of this K+ channel is shown in Fig. 3.

Figure 2. Effects of natriuretic peptides and 8-Br-cGMP on Po of the intermediate conductance K+ channel in cell-attached membrane patches of IHKE-1 cells.

Figure 2

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A, effect of ANP (10 nM) on Po of this K+ channel. B, effect of CNP (10 nM) on the K+ channel Po. C, effect of 8-Br-cGMP (0.1 mM) on Po of this intermediate conductance K+ channel. C → indicates the closed state of the channels. D, effect of BNP (10 nM) on Po of this K+ channel in a cell-attached membrane patch of IHKE-1 cells while the pipette solution contained nystatin (20 mg l−1) to allow measurement of single channel currents parallel to membrane voltage (CAN method). BNP depolarized Vm by 12 mV (current clamp mode indicated by spikes) and reduced Po of the K+ channel (voltage clamp mode). C → indicates the closed state of the channels, which was determined in the presence of Ba2+ following the BNP manoeuvre.

Figure 3. Summary of the effects of natriuretic peptides on Po of the intermediate conductance K+ channel in cell-attached membrane patches of IHKE-1 cells.

Figure 3

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ANP reduced Po of the channel in a concentration-dependent manner with strongest effects at 10−8 M. At this concentration BNP and CNP were equally potent. * Statistically significant effect. Po was analysed over a period of at least 60 s.

Inhibitors and regulators of the intermediate conductance K+ channel in excised membrane patches

To further investigate the channel properties and its regulation, experiments with excised membrane patches were performed. In this configuration Po of the K+ channel was inhibited by various K+ channel blockers. In the inside-out configuration Ba2+ (1 mM) reduced Po by 80 ± 4 % (n = 26). Verapamil (0.1 mM), a known Ca2+ channel blocker that also inhibits K+ channels (Schlatter et al. 1993), inhibited Po of this K+ channel by 73 ± 9 % (n = 12). TEA+ (10 mM) reduced the current amplitude of the channel by 24 ± 3 % and surprisingly also Po by 48 ± 5 % (n = 34) in the inside-out configuration. In the outside-out membrane configuration TEA+ blocked Po of the channel completely (n = 26). Figure 4 shows typical current traces of the TEA+ effects on Po of the K+ channel in outside-out and inside-out membrane configurations. The unique effect of TEA+ on Po in the inside-out configuration was also used to distinguish this K+ channel from others also found in excised membrane patches, mostly the maxi-K channel, which responded to TEA+ in the inside-out membrane configuration with no reduction in Po. Furthermore, like the maxi-K channel, the intermediate conductance K+ channel was blocked by charybdotoxin (CTX) in the outside-out configuration. CTX at 10−10 M reduced Po by 9 ± 9 % (n = 3), 10−9 M CTX reduced it by 28 ± 2 % (n = 4) and 10−8 M CTX by 67 ± 9 % (n = 9). The sensitivity of this channel to CTX had an IC50 of 5 nM, similar to the sensitivity of the maxi-K channel (IC50 of 10 nM) described in rat cortical collecting duct (Schlatter et al. 1993).

Figure 4. Effects of TEA+ (10 mM) on Po of the intermediate conductance K+ channel in excised outside-out and inside-out membrane patches of IHKE-1 cells.

Figure 4

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In an outside-out patch (upper panel) Po of the K+ channel was completely blocked by TEA+. In an inside-out membrane patch (lower panel) TEA+ displayed a ‘Ba2+-like’ inhibition pattern of the K+ channel which was also used to distinguish this K+ channel from other K+ channels also found in these cells. C → indicates the closed state (zero current level) of K+ channels. The Po of each trace was analysed over a period of at least 60 s. The clamp voltage in the excised membrane patch configurations was 0 mV.

The sensitivity of maxi-K or intermediate conductance K+ channels for CTX is in most cases typical for Ca2+-dependent K+ channels. Therefore, we also examined the Ca2+ dependence of this channel in inside-out membranes. A 50 % activation of the K+ channel was seen at a cytosolic Ca2+ concentration of 200 nM. At a more physiological Ca2+ concentration of 100 nM, Po of the channel was 36 ± 13 % (n = 8) compared with the normalized control containing a Ca2+ concentration of 1 mM. This result fits with the observed Po of 0.44 ± 0.07 (n = 23) in the cell-attached membrane configuration. A concentration-response curve for the sensitivity of this channel for Ca2+ is given in Fig. 5. This K+ channel also showed a voltage dependence. With depolarizing clamp voltages, Po of the K+ channel increased (Fig. 6).

Figure 5. Relationship between Po and cytosolic Ca2+ concentration in inside-out membrane patches with the intermediate conductance K+ channel from the apical membrane of IHKE-1 cells.

Figure 5

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The clamp voltage in the excised membrane patch configuration was 0 mV. Control activity in the presence of a Ca2+ concentration of 10−3 M was normalized to a Po of 100 %. Data are presented as mean values ±s.e.m. The numbers in parentheses refer to the number of experiments at any given Ca2+ concentration. Half-maximal activity of the K+ channel was reached at a Ca2+ concentration of 200 nM, making this channel active under physiological conditions.

Figure 6. Voltage dependence of the intermediate conductance K+ channel of IHKE-1 cells.

Figure 6

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The upper panel shows original current traces of the K+ channel at different clamp voltages (Vm) in an inside-out membrane patch. Po was analysed over a period of at least 60 s. The lower panel displays the relationship between Po and Vc of the intermediate conductance K+ channel in excised inside-out membrane patches. Experiments were performed with 145 mM KCl either in the pipette or in the bath. Data are given as mean values ±s.e.m. The numbers in parentheses refer to the number of experiments at the respective clamp voltage.

Since the actions of natriuretic peptides are classically displayed via the cGMP signalling pathway and 8-Br-cGMP markedly reduced Po in the cell-attached recordings, we also tested possible direct cGMP effects at the single channel level in excised inside-out membranes. cGMP at a concentration of 10−6 M significantly reduced Po by 22 ± 7 % (n = 6), 10−5 M cGMP reduced it by 27 ± 4 % (n = 7) and 10−4 M cGMP reduced Po by 34 ± 5 % (n = 15). Mg-ATP (10−4 M) together with cGMP (10−4 M), which would allow the activation of a membrane-bound protein kinase, did not increase the inhibitory effect (30 ± 7 %, n = 14). Original current traces of the cGMP effect and the effect of cGMP together with Mg-ATP on Po of these K+ channels are shown in Fig. 7. Besides cGMP, cAMP (10−4 M), GTP (10−4 M), Mg-ATP (10−4 M) and Na-ATP (10−4 M) were also tested on Po of this K+ channel in excised inside-out membrane patches. cAMP (-4 ± 3 %, n = 5) as well as GTP (-7 ± 4 %, n = 5) had no significant effect on Po. There was no significant difference between the inhibitory effects of Mg-ATP (n = 7) and Na-ATP (n = 7), and therefore the data were pooled. ATP (10−4 M) reduced Po slightly by 17 ± 5 % (n = 14). Lower concentrations of ATP (10−5 and 10−6 M) did not significantly alter Po of this K+ channel. A summary of the effects induced by the tested nucleotides is shown in Fig. 8. KT5823 (1 μm), a specific inhibitor of the cGMP-dependent protein kinase, did not significantly change Po of the channel (39 ± 12 %, n = 3, with KT5823 and 34 ± 5 %, n = 15, without KT5823) when blocked by cGMP; therefore, an indirect effect of cGMP via a protein kinase can be excluded.

Figure 7. Effects of cGMP and cGMP in combination with Mg-ATP (10−4 M each) on intermediate conductance K+ channels of IHKE-1 cells in an excised inside-out membrane patch.

Figure 7

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cGMP alone (upper panel) reduced Po of this K+ channel by 50 %. There was no additional inhibitory effect when cGMP was given in combination with Mg-ATP (lower panel). C → indicates the closed state (zero current level) of the K+ channels. The Po of each trace was analysed over a period of at least 60 s.

Figure 8. Summary of the effects of nucleotides on Po of the intermediate conductance K+ channel in excised inside-out membrane patches of IHKE-1 cells.

Figure 8

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cGMP inhibited Po of the K+ channel in a concentration-dependent manner, displaying greatest effects at a concentration of 10−4 M. A combination of cGMP and Mg-ATP did not increase the inhibitory potency of the substances. cAMP and GTP had no significant effects on Po of the K+ channel. ATP at 10−5 and 10−6 M had no significant effect on Po of the K+ channel. Only ATP at 10−4 M reduced Po significantly. There was no significant difference between the effects of Mg-ATP and Na-ATP. Therefore, the data were pooled. The numbers in parentheses refer to the number of experiments. * Statistically significant effect.

Besides the cGMP-regulated K+ channel three more K+ channels were detected. A small approximately 10 pS and a small 22 pS K+ channel were mostly seen in cell-attached membrane experiments. After excision the latter K+ channel rapidly inactivated. In a few experiments Po could be partly recovered by addition of 0.1 mM Mg-ATP. From these observations and the I-V curve we concluded that this K+ channel might be a ROMK-like channel, as described for the collecting duct (Wang et al. 1997). The third, large conductance K+ channel was never seen in the cell-attached configuration but was highly active in excised membrane patches. After reducing the Ca2+ concentration in the bath solution for inside-out experiments, Po rapidly decreased. This channel showed typical characteristics of a maxi-K channel.

DISCUSSION

K+ channels play an important role in renal transport. In the proximal tubule they provide the negative membrane potential as part of the driving force for substrate transport, are involved in cell volume regulation, and are partially responsible for NH4+ transport (Lang & Rehwald, 1992; Wang et al. 1992; Simon et al. 1992; Giebisch, 1995). In the proximal tubule K+ reabsorption occurs via the paracellular pathway (Kibble et al. 1995).

Here we describe a novel intermediate conductance K+ channel present in human kidney cells originating from proximal tubules which shows a reasonable open probability and opens in response to membrane depolarization or increases in intracellular Ca2+ concentration. In view of its voltage dependence and its activity under physiological conditions this K+ channel might be involved in the stabilization of the membrane voltage, especially in the repolarization of the luminal membrane after depolarizations due to activation of Na+-dependent substrate transport. The channel may also provide a pathway for NH4+ transport, especially since it is capable of conducting NH4+ (Lang et al. 1986; Lang & Rehwald, 1992; Simon et al. 1992).

The Ca2+ dependence of this K+ channel is significantly steeper than that of e.g. the maxi-K channel, which we also found in IHKE-1 cells. Under physiological cytosolic Ca2+ concentration the channel has an open probability of 0.4 in excised membranes which fits the data obtained from cell-attached membrane patches. This Ca2+ dependence explains why this novel K+ channel unlike the maxi-K channel is active under resting conditions with low cytosolic Ca2+ concentration.

This K+ channel is most probably responsible for the K+ conductance that was shown by us recently to be regulated by natriuretic peptides (Hirsch et al. 1999). All natriuretic peptides inhibited this ion channel in the cell-attached configuration by up to 50 %; inhibition by 8-Br-cGMP as the membrane-permeable second messenger reached 70 %. In a few experiments the inhibition of Po by natriuretic peptides even reached 80-90 % (see also Fig. 2). Thus, inhibition is mediated via cGMP except in the case of CNP which uses a different pathway, most probably via tyrosine kinase activity of a truncated natriuretic peptide receptor-B (NPR-B) (Hirsch et al. 1999). This strong inhibition of Po by 8-Br-cGMP on the cell cannot necessarily be compared quantitatively with the inhibition in excised membranes, since several physiological parameters are different, such as membrane potential, electrical field, Ca2+ concentration and cytoskeletal components. And this might explain the reduced effect of cGMP on K+ channel Po in excised membrane patches.

It has been shown before that mammalian K+ conductances or K+ channels are regulated by natriuretic peptides (White et al. 1993; Wei et al. 1994; Ganz et al. 1994; Cermak et al. 1996; Stockand & Sansom, 1996; Sansom & Stockand, 1996). In all these cases a K+ conductance or K+ channel was activated. Only one of the above-mentioned studies clarified the signalling pathway of the natriuretic peptides and their action on K+ channels. White et al. 1993 showed that maxi-K channels in GH4C1 cells of rat are activated via a cGMP-dependent dephosphorylation. In the cortical collecting duct of rat kidney we demonstrated that the K+ channel of the basolateral membrane is upregulated by a cGMP-dependent protein kinase (Hirsch & Schlatter, 1995a,b). However, the cGMP increase in these collecting duct cells is not mediated by natriuretic peptides (Schlatter et al. 1996).

An inhibitory effect of natriuretic peptides on Na+-coupled substrate transport in the proximal tubule was postulated previously (Hammond et al. 1985; Harris et al. 1987; Reddy et al. 1994; Eitle et al. 1998; Jacobs et al. 1999). Besides the possibility of a direct effect of ANP on Na+-coupled transport proteins, a second possibility would be a decrease in the driving force for Na+-coupled transport by inhibition of K+ conductances. Therefore, the inactivation of this K+ channel by natriuretic peptides would fit this second hypothesis since the inhibition of the K+ channel prevents the repolarization of the luminal membrane leading to a reduction in the driving force for Na+ uptake and consequent Na+ loss. The surprising finding in the present study was that the inhibition of this K+ channel of IHKE-1 cells by natriuretic peptides is apparently not mediated via cGMP-dependent phosphorylation or dephosphorylation. Rather, this effect is directly mediated by cGMP as it is also present in excised membranes in the absence of Mg-ATP. Addition of Mg-ATP did not increase the inhibitory effect as would have been expected if a membrane-bound cGMP-dependent protein kinase was involved in this regulation. Furthermore, KT5823, a specific inhibitor of the cGMP-dependent protein kinase, did not reverse the inhibition by cGMP. So far, a regulatory effect of cGMP on mammalian K+ channels has only been shown once, for the cyclic nucleotide-gated K+ (KCN) channel. This putative cGMP-gated K+ channel has a permeability ratio of K+/Na+ of only 4:1 which makes this channel rather a non-selective cation channel (Yao et al. 1995). The K+ channel described here, like most selective K+ channels, does not have a significant Na+ permeability. But the most striking difference between the KCN and the K+ channel described here is the fact that the KCN channel needs cGMP to be active while the K+ channel described in this study was directly inhibited by cGMP.

In this study we present the first electrophysiological characterization of a mammalian K+ channel from human proximal tubule cells that is directly regulated by cGMP and that might be involved in the hyperpolarization of the apical membrane and the maintenance of electrogenic transport such as Na+-coupled substrate transport in the proximal tubule. This K+ channel is responsible for the decrease in K+ conductance seen with natriuretic peptides in these cells.

Acknowledgments

The authors thank Marion Knollmann and Joachim Windau for excellent technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft DFG Schl 277/5-1 to 5-3, the Alexander von Humboldt Foundation, and the Zentrum für Innovative Medizinische Forschung (IMF) Münster (Hi-1-1-II/96-7).

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