The small-conductance K+ channel (SK) in the apical membrane of the cortical-collecting duct (CCD) is regulated by adenosine triphosphate (ATP) and phosphorylation-dephosphorylation processes. When expressed in Xenopus oocytes, ROMK, a cloned K+ channel similar to the native SK channel, can be stimulated by phosphatidylinositol bisphosphate (PIP2), which is produced by phosphoinositide kinases from phosphatidylinositol. However, the effects of PIP2 on SK channel activity are not known. In the present study, we investigated the mechanism by which hydrolyzable ATP prevented run-down of SK channel activity in excised apical patches of principal cells from rat CCD. Channel run-down was significantly delayed by pretreatment with hydrolyzable Mg-ATP, but ATPγS and AMP-PNP had no effect. Addition of alkaline phosphatase also resulted in loss of channel activity. After run-down, SK channel activity rapidly increased upon addition of PIP2. Exposure of inside-out patches to phosphoinositide kinase inhibitors (LY294002, quercetin or wortmannin) decreased channel activity by 74% in the presence of Mg-ATP. PIP2 added to excised patches reactivated SK channels in the presence of these phosphoinositide kinase inhibitors. The protein kinase A inhibitor, PKI, reduced channel activity by 36% in the presence of Mg-ATP. PIP2 was also shown to modulate the inhibitory effects of extracellular and cytosolic ATP. We conclude that both ATP-dependent formation of PIP2 through membrane-bound phosphoinositide kinases and phosphorylation of SK by PKA play important roles in modulating SK channel activity.

Introduction

The cortical-collecting duct (CCD)* is an important site for potassium secretion (Wang et al., 1997; Giebisch, 1999). The secretory process is confined to principal cells and initiated by active uptake of potassium across the basolateral membrane through the activity of Na-K-ATPase. This is followed by passive diffusion along a favorable electrochemical gradient across the apical membrane through potassium channels. A small-conductance potassium channel (SK) in the apical membrane of CCD is the major pathway for this potassium secretion (Wang et al., 1997). SK channels in principal cells of the mammalian nephron are regulated by pH, phosphorylation and dephosphorylation processes, cell messengers, and the cytoskeleton (Wang et al., 1992, 1997; Kubokawa et al., 1995b; Palmer, 1999; Schulte and Fakler, 2000).

A distinguishing feature of the SK channel is the modulation of activity by adenosine triphosphate (ATP) (Wang and Giebisch, 1991a), typical of other ATP-sensitive (KATP or Kir6.x) K+ channels (Ashcroft and Ashcroft, 1990; Yokoshiki et al., 1998; Ashcroft, 2000). Low concentrations of ATP in the presence of protein kinase A (PKA) often restore the activity of run-down SK channels in CCD, whereas higher concentrations of ATP inhibit channel activity (Wang and Giebisch, 1991a). The former is thought to involve several distinct phosphorylation processes, whereas the latter may be due to direct binding of ATP to the channel as in other KATP channels (Tanabe et al., 1999, 2000; Ashcroft, 2000; MacGregor et al., 2001). ADP also affects SK channel activity by antagonizing the inhibitory effect of ATP (Wang and Giebisch, 1991b).

Phosphatidylinositol phosphates (PIPs) like PIP2 provide membrane signals (Martin, 1998; Corvera et al., 1999) that stimulate both KATP (Kir6.x) channels (Fan and Makielski, 1997; Baukrowitz et al., 1998; Huang et al., 1998; Shyng and Nichols, 1998; Shyng et al., 2000b) and other nonnucleotide-gated inward rectifier K+ channels (Huang et al., 1998; Zhang et al., 1999). The concentration of these PIPs in the membrane is regulated by phospholipases (like phospholipase C, PLC), phospholipid phosphatases, and phosphoinositide kinase (PI kinase). Hydrolyzable ATP and/or PIP2 have been shown to reactivate run-down Kir6.2 channels and the effect of Mg-ATP, but not PIP2, is abolished by wortmannin, a PI kinase inhibitor (Xie et al., 1999b). Since protein kinase inhibitors did not block the ability of Mg-ATP to reactivate these Kir6.2 channels, this suggests that the Mg-ATP–dependent channel recovery is mediated by membrane lipid kinases rather than protein kinases. However, another study has suggested that protein kinase–mediated phosphorylation of Kir6.2 maintains responsiveness to PIP2 (Ribalet et al., 2000). A role for PI kinases in regulating KATP channel activity is also supported by the modulation of ATP sensitivity of KATP channels in COSm6 cells transfected with PI kinase (Shyng et al., 2000a). In addition, the hydrolysis of PIP2 by phospholipases such as PLC leads to the formation of inositol triphosphate and diacyl glycerol and loss of the stimulatory effect of PIP2 on Kir channels (Xie et al., 1999a; Kobrinsky et al., 2000).

ROMK, cloned from the outer medulla of rat kidney (Ho et al., 1993; Zhou et al., 1994), shares many properties with the native SK channel (Palmer et al., 1997; Wang et al., 1997). ROMK exists in three isoforms, ROMK1, 2, and 3 (Boim et al., 1995; Lee and Hebert, 1995). In situ hybridization has shown expression of ROMK1 and ROMK2 mRNA in principal cells (Lee and Hebert, 1995), and immunohistochemistry has identified ROMK in apical membranes of principal cells in CCD (Lee and Hebert, 1995; Kohda et al., 1998). Thus, ROMK is believed to encode the SK channel in apical membranes of principal cells. ROMK1 channels expressed in oocytes can be activated by PIP2 (Huang et al., 1998) and this effect is modulated by PKA-dependent phosphorylation of ROMK (Liou et al., 1999), as well as by changes in cytosolic-side pH (Leung et al., 2000).

Although the studies of PIP2 effects on ROMK1 channels suggest a potential role for PIPs in regulating the native SK channel, there is currently no information about the effects of these phospholipids or PI kinases on SK channel activity in the mammalian CCD. In the present study we investigated the possible roles of ATP, PI kinase, PIP2, and PKA in modulating SK channel activity in principal cells from rat kidney. We found that hydrolyzable ATP can restore the low SK channel activity that follows excision of patches through a mechanism that involves membrane-bound kinases. Full channel activation requires both PI kinases increasing PIPs (like PIP2) and PKA phosphorylation of the SK channels.

Materials And Methods

Preparation of CCD

Sprague-Dawley rats (80–100 g) were purchased from Taconic Farms, Inc. and kept on normal K-containing diet for 7–10 d before being killed. Left kidneys were removed and cut into two coronary slices for manual dissection. CCD were isolated using forceps under microscopic view in ice-cold bath solution (∼4°C) as previously described (Wang and Giebisch, 1991b). One or two single CCD segments were placed on Cell-Tak (Becton Dickinson) –coated cover glass (2.5 × 2.5 mm) to immobilize them, and transferred to the experimental chamber mounted on the stage of an inverted microscope (IMT-2; Olympus). CCD tubules were opened with a glass pipette to expose the lumen surface for patch-clamping of the apical membrane. Only principal cells, identified by their large flat surface and hexagonal shape, were chosen and all experiments were conducted at room temperature (22–24°C). The time between sacrificing the animal to opening the tubules was 5–10 min.

Solutions and Chemicals

Control bath solutions contained (mM): 140 NaCl, 5 KCl, 1.8 MgCl2, 1.8 CaCl2, and 10 HEPES. Ca2+-free bath solution was the same except that it also contained (mM): 0 CaCl2, 0.8 MgCl2, and 1 EGTA. Ca2+/Mg2+-free bath solution contained (mM): 140 NaCl, 5 KCl, 1 EGTA, 1 EDTA, and 10 HEPES. All solutions (pH adjusted to 7.4 with NaOH) were kept in the refrigerator and used within 3 d. ATP-containing bath solutions were freshly prepared every day. Pipette solution contained (in mM): 140 KCl, 1.8 MgCl2, and 10 HEPES (pH adjusted to 7.4 with KOH). Adenosine 5′-triphosphate (magnesium salt, Mg-ATP), adenosine 5′-[γ-thio]triphosphate (tetralithium salt, ATPγS), 5′-adenylylimido-diphosphate (AMP-PNP), wortmannin, 2-deoxy-D-glucose, antimycin A, and DL-dithiothreitol (DTT) were purchased from Sigma-Aldrich. Alkaline phosphatase from calf intestine (cAP) was purchased from New England Biolabs, Inc. LY294002, protein kinase A inhibitor (PKI), and quercetin were purchased from Calbiochem-Novabiochem. L-α-phosphatidylinositol-4, 5-bisphosphate (PIP2) was purchased from Roche Diagnostics Corp. Phosphatidylinositol 4,5-bisphosphate antiserum (anti-PIP2) was purchased from Assay Designs, Inc. Stock solutions of PIP2 were sonicated in a cold bath for 30 min and kept in the freezer (−70°C). After sonicating for 1 min, PIP2 was added directly to the perfusion chamber by hand using a small pipette. All chemical solutions were freshly made before each experiment. Agents were dissolved in either DMSO or ethanol with final concentrations in bath solutions of <0.1%. These solvents had no effects on SK channel activity.

Patch-clamp Technique

Microelectrode pipettes were pulled from borosilicate glass capillaries (Dagan Corp.) using a two-step Narishige PP83 puller (Narishige Scientific Instrument Laboratory) and polished using a Narishige microforge (MF-83). The pipette resistances were 6–8 MΩ after filling with pipette solution. Single channel recordings were obtained using an EPC-7 amplifier (List Electronics) and filtered at 1 kHz by a Bessel filter (902 LPF; Frequency Devices, Inc.). Currents were digitized at a sampling rate of 4 kHz using a Digidata 1200 interface (Axon Instruments, Inc.) and stored in a PC (E-3100, Gateway 2000) for further statistical analysis and printing. NPo represents the channel activity, in which N is the number of channels in the patch and Po is the single channel open probability, calculated at a modified filter frequency of 500 Hz by using pCLAMP software (version 6.0.4 of Fetchan and pSTAT; Axon Instruments, Inc.). The changes of channel activity were determined when a steady-state was reached and calculated over a 30s interval before and after changing bath solutions or additions of agents. Statistical data are shown as mean ± SEM and P <0.05 was considered significant. Mean paired differences were calculated by Student's t test.

Results

SK Channel Activity Rapidly Decreases When Patches Are Excised into Bath Solutions Lacking ATP

Fig. 1 shows a representative single channel recording upon changing from a cell-attached to an inside-out patch configuration on the apical membrane of a principal cell. With the cell-attached configuration and Ringer's solution in the bath, channel conductance was 38.3 ± 1.2 pS (calculated at pipette holding potential [−Vpipette] between 0 and −80 mV). The I-V curve showed slight inward rectification (35.4 ± 0.8 pS, −Vpipette between 20 and −20 mV; 31.2 ± 1.5 pS, −Vpipette between 40 and 0 mV, n = 13). In the inside-out patch configuration with symmetrical 140 mM KCl solutions, the channel conductance was 31.3 ± 0.6 pS (−Vpipette between 20 and −80 mV, n = 4). With bath solution containing 140 mM NaCl and 5 mM KCl, the channel showed inward rectification with a conductance of 26.0 ± 0.3 pS, −Vpipette between 0 and −80 mV (n = 6). The latter conductance is typical of the SK conductance under these conditions (Frindt and Palmer, 1989; Wang et al., 1990). In all remaining experiments, inside-out patches were exposed to 140 mM NaCl and 5 mM KCl bath solutions. Channel kinetics calculated at a pipette holding potential (−Vpipette) of −40 mV were typical of the SK channels in mouse (Lu et al., 2000) and rat (Wang and Giebisch, 1991a,b): one open time (To = 37.6 ± 0.2 ms) and one closed time (Tc = 1.4 ± 0.3 ms); open probability (Po) was 0.89 ± 0.17. Consistent with previous observations (Wang and Giebisch, 1991b; Kubokawa et al., 1995a), channel activity progressively decreased in inside-out patches and channel activity disappeared completely within 1.4 ± 0.4 min (n = 21; Fig. 1).

ATP-dependent Phosphorylation Processes Are Required in Intact CCD Principal Cells for SK Channel Activity

The concentration of ATP on the cytosolic face of principal cells plays an important role in modulating SK channel activity: in excised patches sub-mM concentrations of ATP can reactivate run-down SK channels whereas mM concentrations of Mg-ATP reduce channel activity (Wang and Giebisch, 1991a). Antimycin A, an inhibitor of mitochondrial electron transfer, and 2-deoxy-D-glucose, a glycolysis inhibitor, deplete cellular ATP stores in cultured renal epithelial cells (Mandel et al., 1994). We used these metabolic inhibitors to assess effects of depletion of intracellular ATP on SK channel activity. Fig. 2 is a representative recording from a cell-attached patch showing that SK channel activity decreased following exposure to 20 μM antimycin A and 20 mM 2-deoxy-D-glucose. NPo decreased from a control value of 4.7 ± 0.5 to 0.9 ± 0.3 (n = 3) within 7 min. The inhibitory effect of antimycin A + 2-deoxy-D-glucose was partially reversible. Thus, ATP depletion in principal cells reduces SK channel activity.

To investigate further the effects of ATP on SK channel activity, we assessed the ability of Mg-ATP or ATP analogs (ATPγS or ANP-PNP) on reactivation of run-down SK channels. ATPγS has been reported to be a substrate for some protein kinases (Eckstein, 1985; Jeong and Nikiforov, 1999) but not for lipid kinases in oocytes (Liou et al., 1999) while AMP-PNP is a nonhydrolyzable ATP analogue. Fig. 3 shows the effects of Mg-ATP on SK channel run-down. Fig. 3 A shows the decline of channel activity following exposure to Ca2+/Mg2+-free and Mg-ATP-free bath solutions. Additions of 0.5 mM Mg-ATP (n = 6) reactivated SK channels following run-down. Fig. 3 B shows that ATPγS (n = 11) and AMP-PNP (unpublished data; n = 3) failed to maintain channel activity, whereas addition of 0.5 mM Mg-ATP reactivated channels after run-down in each condition. To provide additional evidence that the activating effect of Mg-ATP was due to phosphorylation processes, we exposed excised patches to calf intestinal alkaline phosphatase (cAP, a broad-spectrum phosphatase) in the presence of 0.5 mM Mg-ATP. Fig. 4 shows a representative recording demonstrating that cAP abolished SK channel activity even in the presence of Mg-ATP (from an NPo = 3.3 ± 0.3 to an NPo = 0.2 ± 0.1; n = 16). This effect of cAP was reversible (NPo = 2.9 ± 0.6; 88 ± 11% of control after 5 min of cAP wash-out), likely due to both reestablishment of PKA-dependent phosphorylation of proteins (including the SK channel) and lipid kinase production of PIP2.

In rat principal cells, phosphorylation processes have been associated with both channel activation (PKA) and inhibition (PKC or Ca2+-dependent calmodulin-activated kinase II [CaMK II]). Increasing cytosolic Ca2+ reduces SK channel activity in cell-attached patches by activating PKC (Wang and Giebisch, 1991b; Wang et al., 1993) and/or CaMK II (Kubokawa et al., 1995a,b). However, addition of bath Ca2+ in excised patches did not reduce channel activity in the absence of exogenous PKC (Wang et al., 1990). Moreover, Mg2+ can activate membrane-bound phosphatases and reduce SK channel activity in excised patches (Kubokawa et al., 1995a). To test whether Ca2+ and/or Mg2+ affect channel run-down after patch excision, experiments were performed in bath solutions lacking Ca2+ or both Ca2+ and Mg2+. There was no difference in time to run-down between experiments using Ca2+-free (Mg2+-containing) solutions or Ca2+/Mg2+-free solutions (1.8 ± 0.3 min [n = 6] vs. 1.8 ± 0.6 min [n = 5], respectively). These times to run-down were also not different from the time of run-down in control Ca2+/Mg2+-containing solutions (Figs. 1 and 3 A). Thus, these kinase inhibitors appear to play no significant role in our present experiments with rats fed a normal K diet. Although in a previous study in rats fed a high K diet, the time to run-down was significantly prolonged in the absence of Ca2+ and Mg2+ (Kubokawa et al., 1995a), it is not clear whether this difference was due to K in the diet or to other undetermined factors.

Phosphatidylinositol-4, 5-bisphosphate (PIP2) Modulates SK Channel Activity

In further experiments, we examined the role of PI kinases in mediating the Mg-ATP–dependent phosphorylation effects on maintaining SK channel activity (Figs. 57). Two experimental approaches were used to test whether PIP2 affects SK channel activity. First, we investigated the effects of PIP2 applied to the cytosolic face of excised membrane patches in which activity had declined after exposure to solutions free of Mg-ATP and Ca2+. As shown in Fig. 5 A, a significant increase of NPo was produced by addition of 50 μM PIP2 (NPo from 0.9 ± 0.3 to 3.7 ± 0.7; n = 8). This result is similar to the increase in the activity of ROMK1 produced by PIP2 in oocytes (Huang et al., 1998).

Second, we also investigated the effect of PIP2 antibodies (anti-PIP2). As shown in Fig. 5 B, the addition of anti-PIP2 to the cytosolic face of excised membrane patches led to a prompt decline of SK channel activity in the presence of Mg-ATP–containing bath solution. NPo decreased from 5.6 ± 1.2 to 0.1 ± 0.1 (n = 10). Fig. 5 C shows that the inhibition of channels could not be reversed by repeated wash-out with anti-PIP2–free and Mg-ATP–containing/Ca2+-free bath solutions. However, adding DL-dithiothreitol (DTT, 100 μM) to the bath restored channel activity to 88 ± 9% of control values (NPo = 4.9 ± 0.9, n = 5). These results are consistent with those of Huang and coworkers in ROMK1 expressed in oocytes showing that the reversal of the PIP2 antibody effect also required exposure to reducing agents like DTT (Liou et al., 1999). This effect of DTT may be due reduction of bonds in the anti-PIP2 immunoglobulin molecule (Liou et al., 1999). In another study, the low pH–inhibited ROMK1 channels expressed in oocytes required reducing agents for reactivation after returning pH to control (Schulte et al., 1998).

In further experiments we investigated the relationship between hydrolyzable ATP and PI kinases. The effect of LY294002 on SK channel activity is shown in Fig. 6. Although Fig. 6 A shows that LY294002 abolished SK channel activity in this patch, this was not always the case (Fig. 6 B). On average, SK channel activity was reversibly diminished to 17 ± 3% of control by 20 μM LY294002, a selective inhibitor of PI kinase (NPo decreased from 4.3 ± 0.6 to 0.7 ± 0.3 and recovered to 3.2 ± 0.6; n = 14; Figs. 6 A and 7). This inhibition was also rapidly reversed in the continued presence of LY294002 by addition of 50 μM PIP2 (Fig. 6 B; NPo decreased from 3.1 ± 0.1 to 0.4 ± 0.1 and then increased to 2.6 ± 0.5 after addition of PIP2; n = 7).

Experiments were also performed using two additional PI kinases inhibitors: quercetin (60 μM), a nonselective inhibitor of PI kinase, and wortmannin (20 μM), a specific covalent inhibitor of PI kinase. Results of experiments using these two PI kinase inhibitors are summarized in Fig. 7. NPo was reduced to 37 ± 4% of control by quercetin (n = 5) or 25 ± 7% of control by wortmannin (n = 6). The average reduction by all PI kinase inhibitors was to 26 ± 6% of control, a value significantly different from zero. Thus, none of the PI kinase inhibitors completely abolished channel activity. PIP2 reversed channel activity in the presence of quercetin and wortmannin (Fig. 7). Similar reactivation was also observed by addition of PIP3 (20μM, n = 3; unpublished data).

Full Activation of SK Channels Requires PKA-dependent Phosphorylation

In Xenopus oocytes, PKA phosphorylation of ROMK has several effects that enhance channel activity. Phosphorylation of specific sites on ROMK modulates either Po or channel density, NPo (MacGregor et al., 1998). In addition, the stimulation of channel activity by PIP2 is enhanced by PKA phosphorylation of ROMK1 (Liou et al., 1999). To investigate the interaction of PKA and PIP2 in the native SK channel, two approaches were used.

Fig. 8 A summarizes the effect of blocking PKA activity by protein kinase A inhibitor (PKI) in the presence of 0.5 mM Mg-ATP. In control experiments NPo was maintained over 10 min after patch excision in the absence of PKI (Fig. 8 A, solid circles). SK channel activity declined to 64 ± 13% of control (Fig. 8 A, open circles) with 20 μM PKI in the presence of Mg-ATP. This 36% reduction in SK channel activity is consistent with our previous observations of the activating effect of PKA phosphorylation on SK channel activity (Wang and Giebisch, 1991a,b). In the presence of PKI (Fig. 8 A), Mg-ATP would be expected to maintain the concentration of PIPs in the membrane. Thus, PKA phosphorylation appears to be necessary for full activation of SK channels even in the presence of PIPs.

To further investigate the interplay of PKA phosphorylation processes and PIPs, we took advantage of the ability of cAP to abolish channel activity in the presence of Mg-ATP, as shown in Fig. 4. Fig. 8 B shows the run-down of channel activity by cAP in the presence of both 0.5 mM Mg-ATP and 20 μM PKI. Following removal of cAP, but in the continued presence of Mg-ATP and PKI, channel activity was only partially restored by endogenous production of PIP2 even after prolonged observation (∼10 min; 68 ± 7% recovery; n = 7). These observations support that PKA phosphorylation is necessary for full PIP2 responsiveness, and are consistent with observations in oocytes that PKA phosphorylation reduced the concentration of PIP2 necessary for activation of ROMK1 channels (Liou et al., 1999). A similar conclusion regarding the necessity for protein phosphorylation of Kir6.2 in maintaining responsiveness to PIP2 had also been reached (Ribalet et al., 2000).

ATP-PIP2 Interactions in Regulating SK Channel Activity

To begin to understand the relevance of ATP–PIP2 interactions in regulating SK channel activity, we examined the effects of PIP2 in modulating both extracellular and intracellular ATP-mediated channel inhibition. We showed previously that extracellular ATP could reduce SK channel activity in mouse principal cells by stimulated purinergic (P2Y2) receptors (Lu et al., 2000). This effect of extracellular ATP on SK channel activity was due to enhanced phosphatase activity as it could be prevented by inhibiting phosphatases by okadaic acid, activating PKA with cyclic AMP, or blocking protein kinase G with KT5823.

In the representative experiments shown in Fig. 9, cell-attached apical membrane patches were obtained, extracellular 0.2 mM Mg-ATP added to the bath, and then the patch was excised in the inside-out configuration. Extracellular Mg-ATP inhibited SK channel activity in 1–3 min in cell-attached patches, confirming our previous observations in the mouse (Lu et al., 2000). As expected, no SK channel activity was restored after the patch was excised into Ca2+-free and Mg-ATP–free bath solution (Fig. 9 A). However, patch excision in a bath containing 0.5 mM Mg-ATP (Fig. 9 B) led to 48 ± 7% recovery of SK channel activity in 10 of 12 patches: NPo changed from 6.68 ± 0.92 (cell-attached) to 3.17 ± 0.68 (inside-out). If PIP2 (50 μM) alone was present in the bath upon patch excision, SK channel activity could be restored to 56 ± 9% of control: NPo changed from 9.79 ± 1.25 (cell-attached) to 5.45 ± 0.82 (inside-out); n = 3 (unpublished data). Further addition of 50 U of PKA to the 0.5 mM Mg-ATP-containing bath restored channel activity to 81 ± 10% of that in the cell-attached configuration: NPo changed from 6.53 ± 2.14 (cell-attached) to 5.28 ± 1.30 (inside-out); n = 4; Fig. 9 B. Thus, channel recovery requires both PIP2 and PKA-dependent phosphorylation processes.

Since the intracellular ATP concentration is in the range of 2–4 mM in rat principal cells and 1 mM Mg-ATP in the bath inhibits SK channel activity in inside-out patches (Wang and Giebisch, 1991a), mechanisms must exist in intact cells to maintain K+ secretion through SK channels. One possibility is PIP2 competition of Mg-ATP binding to the SK channel. PIP2 has been shown to modulate the ATP sensitivity of the pancreatic β-cell KATP channel, Kir6.2 (Shyng and Nichols, 1998; Enkvetchakul et al., 2000; Loussouarn et al., 2001) and to directly compete with ATP for binding to the COOH-termini of Kir1.1 (ROMK) and Kir6.1 channels (MacGregor et al., 2001).

To examine the interactions of Mg-ATP and PIP2 in rat principal cells, we assessed the Mg-ATP concentration dependence of SK channel activity in the presence of 50 μM PIP2 in excised patches (Fig. 10). SK channel activity was completely inhibited by 1 mM Mg-ATP in the absence of added PIP2 (Fig. 10 B, solid circle), confirming our previous observation in rat SK channels in principal cells (Wang and Giebisch, 1991a). Mg-ATP sensitivity is considerably lower in ROMK channels expressed in X. laevis oocytes (EC50 ∼2–3 mM; McNicholas et al., 1996), but similar to native SK channels when ROMK is coexpressed with CFTR (Ruknudin et al., 1998). A representative experiment showing the concentration dependence of Mg-ATP inhibition in the presence of PIP2 is shown in Fig. 10 A. The patch was excised into a Ca2+-free and Mg-ATP–free bath solution containing 50 μM PIP2 that maintained SK channel activity for 2 min. Addition of 1 mM Mg-ATP had almost no effect on SK channel activity: control NPo 3.12 ± 0.66 versus 2.90 ± 0.62 (n = 9). Increasing Mg-ATP led to a concentration-dependent inhibition of SK channel activity with 3 mM Mg-ATP abolishing activity (Fig. 10 B). PIP2 reduced the sensitivity of SK channels to Mg-ATP: EC50 for Mg-ATP was 1.80 ± 0.02 mM. Thus, PIP2 reduced the Mg-ATP affinity of SK channels to a value close to that of the normal cell ATP concentration.

Discussion

The present study provides evidence that both PI kinase and PKA play important roles in the regulation of the native SK channels in principal cells from rat kidney. Several lines of evidence support this conclusion. First, hydrolyzable ATP (Mg-ATP) is required to maintain channel activity or to reactivate channels after run-down through the endogenous PKA and PIP2 (Figs. 3 and 4). This effect of Mg-ATP depends on phosphorylation reactions since exposure of SK channels to alkaline phosphatase in the presence of Mg-ATP nearly abolished channel activity. Second, stimulation of channel activity by Mg-ATP is mediated, at least in part, by generation of PIP2 through endogenous PI kinases. This is supported by our observations that declining channel activity after excision of membrane patches is mitigated by directly applied PIP2, that PIP2 antibodies inhibit channel activity in the presence of Mg-ATP (Fig. 5, A and B), and that Mg-ATP or PIP2 partially reactivated SK channels inhibited by extracellular Mg-ATP (Fig. 9). It should be noted in this regard that ATPγS does not interact with PI kinases to generate PIP2 (Liou et al., 1999). This explains our observation that ATPγS does not sustain channel activity (Fig. 3 B). Moreover, inhibition of PI kinases reduced SK channel activity and attenuated Mg-ATP effects on maintenance of channel activity (Figs. 6 and 7). Third, processes involving PKA phosphorylation provide an additional mechanism for modulating SK channel activity by Mg-ATP. Maximal PIP2 effects require PKA phosphorylation (Figs. 8 and 9 B).

PI kinases are involved in the synthesis of PIP2 and several isoforms have been identified (Anderson et al., 1999). Together with membrane phosphatases and phospholipases, PI kinases are regulated by several hormones and growth factors (Martin, 1998; Leevers et al., 1999). PIP2 has been shown to be an important regulator of a number of ion channels and transporters (Hilgemann et al., 2001). Our present results add the SK channel in principal cells to this growing list of PIP2-regulated ion channels. However, the cellular distribution of PI kinases between membranes and the cytosol, and their regulation in principal cells has not been explored.

PIPs have thus emerged as important regulators of inward rectifier K+ channels (Hilgemann, 1997; Huang et al., 1998; Hilgemann et al., 2001), including ROMK. The generation of PIP2 by ATP-dependent PI kinases has been shown to activate ROMK1, and this effect is enhanced by PKA phosphorylation of the channel protein (Liou et al., 1999). Our observations in principal cells support this view by demonstrating that PIP2 activates the SK channel and that PKA phosphorylation processes are required to give full SK channel activation by PIPs (Figs. 79).

Given that ATPγS is a substrate for PKA phosphorylation of ROMK1 channels (Jeong and Nikiforov, 1999; Liou et al., 1999), the inability of ATPγS to maintain channel activity in the absence of Mg-ATP indicates that PKA phosphorylation of SK is insufficient to sustain channel activity in the absence of PIP2. In addition, PKI reduced channel activity in the presence of Mg-ATP (Fig. 8 A) where PI kinases would be active and generating PIP2. This latter observation is consistent with the modulation of PIP2 sensitivity of ROMK1 by PKA in oocytes (Liou et al., 1999). Moreover, PIP2 antibodies abolished channel activity in the presence of a concentration of Mg-ATP sufficient to sustain PKA phosphorylation processes. Furthermore, after dephosphorylation by cAP, Mg-ATP did not fully reactivate channel activity in the presence of PKI. Together, our observations on SK channels strongly support the notions that PIP2 can reactivate channels in the absence of PKA, but the sensitivity of PIP2-mediated reactivation is modulated by PKA phosphorylation. Thus, our observations in SK channels are fully consistent with the interactions of PIP2 and PKA in the regulation of ROMK1 expressed in oocytes. However, we were unable to consistently fully inhibit channel activity by any one of several PI kinase inhibitors. It is possible that the inhibitor concentrations were insufficient to completely abolish PI kinase activity or that insufficient phosphatase or phospholipase activity was present so that a low level of PIPs remained in the membrane even in the presence of the PI kinase inhibitors.

Although the physiological actions of PIPs in the regulation of SK channel activity are incompletely understood, we have shown two circumstances where PIPs can modulate Mg-ATP inhibition. First, extracellular Mg-ATP can inhibit SK channels in intact principal cells by activation of P2Y2 receptors in mouse (Lu et al., 2000) and rat (Fig. 9). This appears to be due to P2Y2-mediated activation of phosphatases (Lu et al., 2000). The partial reactivation of the purinergic receptor-inhibited SK channels by bath Mg-ATP in excised patches (Fig. 9 B) is likely due to the action of PI kinase, since a similar degree of reactivation was observed with PIP2 in the absence of Mg-ATP. These latter observations would be consistent with P2Y2-activated phosphatases reducing the concentration of PIPs in the plasma membrane. The further reactivation of these channels by addition of PKA suggests that the P2Y2-activated phosphatases also dephosphorylate ROMK, which is then rephosphorylated by addition of PKA. Furthermore, increase in SK channel activity by PKA is consistent with the necessity of PKA phosphorylation process for full activity of PIP2.

Second, while low concentrations of ATP (<1 mM; Kd ≈ 0.5 mM) are important for channel activation by phosphorylation processes (via PI kinase, PKA, and potentially other kinases), SK channels are inhibited by higher concentrations of ATP (>1 mM; Wang and Giebisch, 1991a). The activity of SK channels in vivo and in intact cells in vitro is clearly maintained under conditions in which the cytosolic ATP is 2–4 mM, a concentration higher than that known to block SK channels in excised membrane patches. The data presented in Fig. 10 demonstrate that PIP2 shifts the ATP sensitivity in SK channels to the right and thereby help to maintain native channel activity at the relatively high cell ATP concentrations. PIPs also modulate the ATP sensitivity of the pancreatic β-cell KATP channel, Kir6.2 (Shyng and Nichols, 1998; Enkvetchakul et al., 2000; Loussouarn et al., 2001). Recently, we have shown that PIPs compete with ATP for binding to the carboxy terminus of ROMK and Kir6.1 channels (MacGregor et al., 2001) and this could provide a mechanism for PIPs maintaining SK channels in the presence of high cytosolic concentrations of ATP. Finally, given the recent observations by Marx and coworkers (Marx et al., 2002) of a macromolecular signaling complex for the sympathetic regulation of outward K+ current in heart, it is possible that all of the proteins regulating SK channel activity (PKA, lipid kinases, phosphatases) are similarly compartmentalized into a macromolecular complex.

Acknowledgments

The authors thank Dr. Chou-Long Huang for constructive suggestions.

This work was supported by National Institutes of Health grants DK54999 (S.C. Herbert) and DK54998 (G. Giebisch).

*

Abbreviations used in this paper: ATP, adenosine triphosphate; cAP, calf intestinal alkaline phosphatase; CCD, cortical-collecting duct; PIP, phosphatidylinositol phosphate; PKA, protein kinase A; SK, small-conductance K+ channel.

References

References
Anderson, R.A., I.V. Boronenkov, S.D. Doughman, J. Kunz, and J.C. Loijens.
1999
. Phosphatidylinositol phosphate kinases, a multifaceted family of signaling enzymes.
J. Biol. Chem.
274
:
9907
–9910.
Ashcroft, S.J.
2000
. The beta-cell K(ATP) channel.
J. Membr. Biol.
176
:
187
–206.
Ashcroft, S.J.H., and F.M. Ashcroft.
1990
. Properties and functions of ATP-sensitive K-channels.
Cell. Signal.
2
:
197
–214.
Baukrowitz, T., U. Schulte, D. Oliver, S. Herlitze, T. Krauter, S.J. Tucker, J.P. Ruppersberg, and B. Fakler.
1998
. PIP2 and PIP as determinants for ATP inhibition of KATP channels.
Science.
282
:
1141
–1144.
Boim, M.A., K. Ho, M.E. Shuck, M.J. Bienkowski, J.H. Block, J.L. Slightom, Y. Yang, B.M. Brenner, and S.C. Hebert.
1995
. The ROMK inwardly rectifying ATP-sensitive K+ channel. II. Cloning and intra-renal distribution of alternatively spliced forms.
Am. J. Physiol.
268
:
F1132
–F1140.
Corvera, S., A. D'Arrigo, and H. Stenmark.
1999
. Phosphoinositides in membrane traffic.
Curr. Opin. Cell Biol.
11
:
460
–465.
Eckstein, F.
1985
. Nucleoside phosphorothioates.
Annu. Rev. Biochem.
54
:
367
–402.
Enkvetchakul, D., G. Loussouarn, E. Makhina, S.L. Shyng, and C.G. Nichols.
2000
. The kinetic and physical basis of KATP channel gating: toward a unified molecular understanding.
Biophys. J.
78
:
2334
–2348.
Fan, Z., and J.C. Makielski.
1997
. Anionic phospholipids activate ATP-sensitive potassium channels.
J. Biol. Chem.
272
:
5388
–5395.
Frindt, G., and L.G. Palmer. 1989. Low-conductance K channels in apical membrane of rat cortical collecting tubule. Am. J. Physiol. 256-25:F143–F151.
Giebisch, G.
1999
. Physiological roles of renal potassium channels.
Semin. Nephrol.
19
:
458
–471.
Hilgemann, D.W.
1997
. Cytoplasmic ATP-dependent regulation of ion transporters and channels: mechanisms and messengers.
Annu. Rev. Physiol.
59
:
193
–220.
Hilgemann, D.W., S. Feng, and C. Nasuhoglu.
2001
. The complex and intriguing lives of PIP2 with ion channels and transporters.
Science's STKE.
111
:
RE19
.
Ho, K., C.G. Nichols, W.J. Lederer, J. Lytton, P.M. Vassilev, M.V. Kanazirska, and S.C. Hebert.
1993
. Cloning and expression of an inwardly rectifying ATP-regulated potassium channel.
Nature.
362
:
31
–38.
Huang, C.-L., S. Feng, and D.W. Hilgemann.
1998
. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gβγ.
Nature.
391
:
803
–806.
Jeong, S., and T.T. Nikiforov.
1999
. Kinase assay based on thiophosphorylation and biotinylation.
Biotechniques.
27
:
1232
–1238.
Kobrinsky, E., T. Mirshahi, H. Zhang, T. Jin, and D.E. Logothetis.
2000
. Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization.
Nat. Cell Biol.
2
:
507
–514.
Kohda, Y., W. Ding, E. Phan, I. Housini, J. Wang, R.A. Star, and C.L. Huang.
1998
. Localization of the ROMK potassium channel to the apical membrane of distal nephron in rat kidney.
Kidney Internat.
54
:
1214
–1223.
Kubokawa, M., C.M. McNicholas, M.A. Higgins, W. Wang, and G. Giebisch.
1995
a. Regulation of ATP-sensitive K+ channel by membrane-bound protein phosphatases in rat principal tubule cell.
Am. J. Physiol.
269
:
F355
–F362.
Kubokawa, M., W. Wang, C.M. McNicholas, and G. Giebisch.
1995
b. Role of Ca2+/CaMK II in Ca2+-induced K+ channel inhibition in rat CCD principal cell.
Am. J. Physiol.
268
:
F211
–F219.
Lee, W.-S., and S.C. Hebert.
1995
. The ROMK inwardly rectifying ATP-sensitive K+ channel. I. Expression in rat distal nephron segments.
Am. J. Physiol.
268
:
F1124
–F1131.
Leevers, S.J., B. Vanhaesebroeck, and M.D. Waterfield.
1999
. Signalling through phosphoinositide 3-kinases: the lipids take centre stage.
Curr. Opin. Cell Biol.
11
:
219
–225.
Leung, Y.M., W.Z. Zeng, H.H. Liou, C.R. Solaro, and C.L. Huang.
2000
. Phosphatidylinositol 4,5-bisphosphate and intracellular pH regulate the ROMK1 potassium channel via separate but interrelated mechanisms.
J. Biol. Chem.
275
:
10182
–10189.
Liou, H.H., S.S. Zhou, and C.L. Huang.
1999
. Regulation of ROMK1 channel by protein kinase A via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism.
Proc. Natl. Acad. Sci. USA.
96
:
5820
–5825.
Loussouarn, G., L.J. Pike, F.M. Ashcroft, E.N. Makhina, and C.G. Nichols.
2001
. Dynamic sensitivity of ATP-sensitive K(+) channels to ATP.
J. Biol. Chem.
276
:
29098
–29103.
Lu, M., G.G. MacGregor, W. Wang, and G. Giebisch.
2000
. Extracellular ATP inhibits the small-conductance K channel on the apical membrane of the cortical collecting duct from mouse kidney.
J. Gen. Physiol.
116
:
299
–310.
MacGregor, G.G., K. Dong, C.G. Vanoye, L. Tang, G. Giebisch, and S.C. Hebert. 2001. Nucleotides and phospholipids compete for binding to the C-terminus of KATP channels. Proc. Natl. Acad. Sci. USA. 99:2726–2731.
MacGregor, G.G., J. Xu, C.M. McNicholas, G. Giebisch, and S.C. Hebert.
1998
. Partially active channels produced by PKA site mutation of the cloned renal K+ channel ROMK2.
Am. J. Physiol.
275
:
F415
–F422.
Marx, S.O., J. Kurokawa, S. Reiken, H. Motoike, J. D'Armiento, A.R. Marks, and R.S. Kass.
2002
. Requirement of a macromolecular signaling complex for β adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel.
Science.
295
:
496
–499.
McNicholas, C.M., Y. Yang, G. Giebisch, and S.C. Hebert.
1996
. Molecular site for nucleotide binding on an ATP-sensitive renal K+ channel (ROMK2).
Am. J. Physiol.
40
:
F275
–F285.
Mandel, L.J., R.B. Doctor, and R. Bacallao. 1994. ATP depletion: a novel method to study junctional properties in epithelial tissues. II. Internalization of Na+,K+-ATPase and E- cadherin. J. Cell Sci. 107(Pt 12):3315–3324.
Martin, T.F.J.
1998
. Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking.
Annu. Rev. Cell Dev. Biol.
14
:
231
–264.
Palmer, L.G.
1999
. Potassium secretion and the regulation of distal nephron K channels.
Am. J. Physiol.
277
:
F821
–F825.
Palmer, L.G., H. Choe, and G. Frindt.
1997
. Is the secretory K channel in the rat CCT ROMK?
Am. J. Physiol.
273
:
F404
–F410.
Ribalet, B., S.A. John, and J.N. Weiss.
2000
. Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP2): a study of channel rundown and reactivation.
J. Gen. Physiol.
116
:
391
–410.
Ruknudin, A., D.H. Schulze, S.K. Sullivan, W.J. Lederer, and P.A. Welling.
1998
. Novel subunit composition of a renal epithelial KATP channel.
J. Biol. Chem.
273(23)
:
14165
–14171.
Schulte, U., and B. Fakler.
2000
. Gating of inward-rectifier K+ channels by intracellular pH.
Eur. J. Biochem.
267
:
5837
–5841.
Schulte, U., H. Hahn, H. Wiesinger, J.P. Ruppersberg, and B. Fakler.
1998
. pH-dependent gating of ROMK (Kir1.1) channels involves conformational changes in both N and C termini.
J. Biol. Chem.
273
:
34575
–34579.
Shyng, S., A. Barbieri, A. Gumusboga, C. Cukras, L. Pike, J.N. Davis, P.D. Stahl, and C.G. Nichols.
2000
a. Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase.
Proc. Natl. Acad. Sci. USA
.
97
:
937
–941.
Shyng, S.L., C.A. Cukras, J. Harwood, and C.G. Nichols.
2000
b. Structural determinants of PIP2 regulation of inward rectifier KATP channels.
J. Gen. Physiol.
116
:
599
–608.
Shyng, S.L., and C.G. Nichols.
1998
. Membrane phospholipid control of nucleotide sensitivity of KATP channels.
Science.
282
:
1138
–1141.
Tanabe, K., S.J. Tucker, F.M. Ashcroft, P. Proks, N. Kioka, T. Amachi, and K. Ueda.
2000
. Direct photoaffinity labeling of Kir6.2 by [γ-32P]ATP-[γ]4-azidoanilide.
Biochem. Biophys. Res. Commun.
272
:
316
–319.
Tanabe, T., S.J. Tucker, M. Matsuo, P. Proks, F.M. Ashcroft, S. Seino, T. Amachi, and K. Ueda.
1999
. Direct photoaffinity labeling of the Kir6.2 subunit of the ATP-sensitive K+ channel by 8-azido-ATP*.
J. Biol. Chem.
274
:
3931
–3933.
Wang, W., and G. Giebisch.
1991
a. Dual effect of adenosine triphosphate on the apical small conductance K+ channel of the rat cortical collecting duct.
J. Gen. Physiol.
98
:
35
–61.
Wang, W., and G. Giebisch.
1991
b. Dual modulation of renal ATP-sensitive K+ channels by protein kinase A and C.
Proc. Natl. Acad. Sci. USA.
88
:
9722
–9725.
Wang, W., S.C. Hebert, and G. Giebisch.
1997
. Renal K+ channels: structure and function.
Annu. Rev. Physiol.
59
:
413
–436.
Wang, W., H. Sackin, and G. Giebisch.
1992
. Renal potassium channels and their regulation.
Annu. Rev. Physiol.
54
:
81
–96.
Wang, W., A. Schwab, and G. Giebisch. 1990. Regulation of small-conductance K+ channel in apical membrane of rat cortical collecting duct. Am. J. Physiol. 259-28:F494–F502.
Wang, W.H., J. Geibel, and G. Giebisch.
1993
. Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na+-K+-ATPase.
J. Gen. Physiol
.
101
:
673
–694.
Xie, L.H., M. Horie, and M. Takano.
1999
a. Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism.
Proc. Natl. Acad. Sci. USA.
96
:
15292
–15297.
Xie, L.H., M. Takano, M. Kakei, M. Okamura, and A. Noma.
1999
b. Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP-dependent recovery of Kir6.2/SUR2A channels.
J. Physiol.
514
:
655
–665.
Yokoshiki, H., M. Sunagawa, T. Seki, and N. Sperelakis.
1998
. ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells.
Am. J. Physiol.
274
:
C25
–C37.
Zhang, H., C. He, X. Yan, T. Mirshahi, and D.E. Logothetis.
1999
. Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions.
Nat. Cell Biol.
1
:
183
–188.
Zhou, H., S.S. Tate, and L.G. Palmer. 1994. Primary structure and functional properties of an epithelial K channel. Am. J. Physiol. 266-35:C809–C824.