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Protein Kinase C Inhibits ROMK1 Channel Activity via a Phosphatidylinositol 4,5-Bisphosphate-dependent Mechanism

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m300619200

ISSN

1083-351X

Autores

Wei-Zhong Zeng, Xin-Ji Li, Donald W. Hilgemann, Chou-Long Huang,

Tópico(s)

Cardiac electrophysiology and arrhythmias

Resumo

The activity of apical K+channels in cortical collecting duct (CCD) is stimulated and inhibited by protein kinase A (PKA) and C (PKC), respectively. Direct interaction between phosphatidylinositol 4,5-bisphosphate (PIP2) and the cloned CCD K+ channel, ROMK1, is critical for channel opening. We have found previously that phosphorylation of ROMK1 by PKA increases affinity of the channel for PIP2 and mutation of PKA sites reduces the affinity of ROMK1 for PIP2. In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP2 affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopusoocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cell-attached recordings, application of PIP2 to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP2 content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP2 content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K+ secretion in CCD by hormones that activate PKC. The activity of apical K+channels in cortical collecting duct (CCD) is stimulated and inhibited by protein kinase A (PKA) and C (PKC), respectively. Direct interaction between phosphatidylinositol 4,5-bisphosphate (PIP2) and the cloned CCD K+ channel, ROMK1, is critical for channel opening. We have found previously that phosphorylation of ROMK1 by PKA increases affinity of the channel for PIP2 and mutation of PKA sites reduces the affinity of ROMK1 for PIP2. In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP2 affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopusoocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cell-attached recordings, application of PIP2 to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP2 content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP2 content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K+ secretion in CCD by hormones that activate PKC. thick ascending limb phosphatidylinositol 4,5-bisphosphate cortical collecting duct cyclic AMP-dependent protein kinase A protein kinase C high performance liquid chromatography phorbol 12-myristate 13-acetate 1-oleoyl-2-acetyl-sn-glycerol phospholipase C type 1 muscarinic receptor carbachol G protein-coupled inward rectifying K+ channels inward rectifying K+ channels The physiological roles of potassium (K+) channels in the regulation of water and electrolyte transport in kidney are well known (1Wright F.S. Giebisch G. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. Second Ed. Lippincott Williams ' Wilkins, Philadelphia1992: 2209-2246Google Scholar). In the thick ascending limb (TAL)1 of Henle's loop, the recycling of K+ ions across the low-conductance apical K+ channels is essential for NaCl reabsorption through the apical Na-K-2Cl cotransporter (1Wright F.S. Giebisch G. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. Second Ed. Lippincott Williams ' Wilkins, Philadelphia1992: 2209-2246Google Scholar). The importance of the apical K+ channels for NaCl reabsorption in TAL is underscored by the genetic disease, Bartter's syndrome, in which loss-of-function mutations of the channels result in impairment of NaCl transport in this nephron segment (2Simon D.B. Karet F.E. Rodriguez-Soriano J. Hamdan J.H. DiPietro A. Trachtman H. Sanjad S.A. Lifton R.P. Nat. Genet. 1996; 14: 152-156Crossref PubMed Scopus (731) Google Scholar). In the cortical collecting ducts (CCDs), secretion of K+ into urinary space is mediated by active transport of K+ into the cell through basolateral Na+-K+-ATPase, followed by passive movement of K+ into the tubular fluid through apical K+channels (1Wright F.S. Giebisch G. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. Second Ed. Lippincott Williams ' Wilkins, Philadelphia1992: 2209-2246Google Scholar). cDNAs for ROMK1 and its isoforms ROMK2 and -3 have been isolated (3Ho K. Nichols C.G. Lederer W.J. Lytton J. Vassilev P.M. Kanazirska M.V. Hebert S.C. Nature. 1993; 362: 31-38Crossref PubMed Scopus (833) Google Scholar, 4Hebert S.C. Kidney Int. 1995; 48: 1010-1016Abstract Full Text PDF PubMed Scopus (89) Google Scholar). Based on the distribution of mRNA and proteins, and biophysical characterization, it is known that ROMK1 and ROMK2 encode the low-conductance secretory K+ channels in CCDs and TALs, respectively (4Hebert S.C. Kidney Int. 1995; 48: 1010-1016Abstract Full Text PDF PubMed Scopus (89) Google Scholar). The apical K+ channels in CCDs and TALs are regulated by multiple signaling pathways, including protein kinase A (PKA), protein kinase C (PKC), cGMP, calcium-calmodulin activated kinase II, intracellular pH, arachidonic acid, etc. (5Giebisch G. Wang W. Acta Physiol. Scand. 2000; 170: 153-173Crossref PubMed Google Scholar). These signaling pathways play important roles in hormonal regulation of the K+channels. The PKA pathway is important for the regulation of the K+ channels in TALs and CCDs by vasopressin (6Cassola A.C. Giebisch G. Wang W. Am. J. Physiol. 1993; 264: F502-F509PubMed Google Scholar, 7Wang W.H. Am. J. Physiol. 1994; 267: F599-F605PubMed Google Scholar). The importance of direct phosphorylation by PKA for channel function is further supported by the finding that one of the mutations in Bartter's syndrome is at a PKA phosphorylation site (2Simon D.B. Karet F.E. Rodriguez-Soriano J. Hamdan J.H. DiPietro A. Trachtman H. Sanjad S.A. Lifton R.P. Nat. Genet. 1996; 14: 152-156Crossref PubMed Scopus (731) Google Scholar). The PKC pathway is likely important for regulation of K+ transport in CCDs by many PLC-activating hormones and growth factors, including prostaglandin E2, bradykinin, and epidermal growth factor (8Sugimoto Y. Namba T. Shigemoto R. Negishi M. Ichikawa A. Narumiya S. Am. J. Physiol. 1994; 266: F823-F828PubMed Google Scholar, 9Figueroa C.D. Gonzalez C.B. Grigoriev S. Alla A.A. Haasemann M. Jarnagin K. Muller-Esterl W. J. Histochem. Cytochem. 1995; 43: 137-148Crossref PubMed Scopus (121) Google Scholar, 10Ankorina-Stark I. Haxelmans S. Schlatter E. Cell Calcium. 1997; 22: 269-275Crossref PubMed Scopus (22) Google Scholar, 11Hays S.R. Baum M. Kokko J.P. J. Clin. Invest. 1987; 80: 1561-1570Crossref PubMed Scopus (50) Google Scholar). Activation of PKC by phorbol 12-myristate 13-acetate (PMA) inhibits apical K+ channels in CCDs (11Hays S.R. Baum M. Kokko J.P. J. Clin. Invest. 1987; 80: 1561-1570Crossref PubMed Scopus (50) Google Scholar, 12Wang W. Giebisch G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9722-9725Crossref PubMed Scopus (141) Google Scholar). Recently, we and others reported a novel mechanism for regulation of ROMK and other inward rectifying K+ channels via direct interaction with membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) (13Hilgemann D.W. Ball R. Science. 1996; 273: 956-959Crossref PubMed Scopus (560) Google Scholar, 14Fan Z. Makielski J.C. J. Biol. Chem. 1997; 272: 5388-5395Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar). This direct interaction occurs between PIP2 and several positively charged residues in the proximal C termini of the inward rectifying K+channels (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar) and likely regulates channel opening by stabilizing the structure of the cytoplasmic entrance of the pore. The importance of PIP2 for inward rectifier K+ channels is further supported by many studies showing that PIP2influences the regulation of the channels by other signaling or gating molecules. PIP2 modulates the regulation of GIRK channels by Gऔγ and intracellular Na+ and Mg2+ ions (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar, 16Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1307-1312Crossref PubMed Scopus (213) Google Scholar), the regulation of KATP by intracellular ATP (17Baukrowitz T. Schulte U. Oliver D. Herlitze S. Krauter T. Tucker S.J. Ruppersberg J.P. Fakler B. Science. 1998; 282: 1141-1144Crossref PubMed Scopus (441) Google Scholar, 18Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (486) Google Scholar), and the regulation of ROMK by PKA (19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar) and pHi (20Leung Y.M. Zeng W.Z. Liou H.H. Solaro C.R. Huang C.L. J. Biol. Chem. 2000; 275: 10182-10189Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). We found that phosphorylation of ROMK by PKA does not directly activate ROMK1 channels in membranes that are depleted of PIP2 (19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar). Rather, it lowers the concentration of PIP2 necessary for activation of the channels, suggesting that PKA activates ROMK1 by enhancing PIP2-channel interaction. The molecular mechanism by which PKC inhibits the activity of ROMK channels remains poorly understood. It was reported previously that PKA antagonizes PKC inhibition of K+ channels in CCD (12Wang W. Giebisch G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9722-9725Crossref PubMed Scopus (141) Google Scholar). In the present study, we examine the hypothesis that PKC inhibits ROMK1 channels via a PIP2-dependent mechanism. Wild type ROMK1 cDNA was in the pSPORT plasmid (3Ho K. Nichols C.G. Lederer W.J. Lytton J. Vassilev P.M. Kanazirska M.V. Hebert S.C. Nature. 1993; 362: 31-38Crossref PubMed Scopus (833) Google Scholar). Site-directed mutagenesis of ROMK1 was performed using a commercial mutagenesis kit (QuikChange from Stratagene, La Jolla, CA), and confirmed by nucleotide sequencing as previously described (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar, 19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar, 20Leung Y.M. Zeng W.Z. Liou H.H. Solaro C.R. Huang C.L. J. Biol. Chem. 2000; 275: 10182-10189Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). mCAP cRNAs of the wild type and mutant ROMK1 channels were transcribed in vitro using T7 RNA polymerase (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar, 19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar, 20Leung Y.M. Zeng W.Z. Liou H.H. Solaro C.R. Huang C.L. J. Biol. Chem. 2000; 275: 10182-10189Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Xenopus laevis oocytes were prepared as previously described (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar,19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar, 20Leung Y.M. Zeng W.Z. Liou H.H. Solaro C.R. Huang C.L. J. Biol. Chem. 2000; 275: 10182-10189Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Oocytes were injected with cRNA for wild type or mutant ROMK1. Current-voltage (I-V) relationships (−100 to +100 mV, in 25 mV steps) were measured in oocytes at ∼23 °C by two-electrode voltage-clamp using an OC-725C oocyte clamp amplifier (Warner Instrument), pCLAMP7 software, and Digidata 1200A digitizer (Axon Instrument). The resistance of current and voltage microelectrodes (filled with 3m KCl solution) was 1–2 MΩ. The bath solution contained (in mm) 96 KCl, 1 MgCl2, 1 CaCl2, 5 Hepes (pH 7.5 by KOH). Patch clamp pipettes (pulled from borosilicate glass, Warner Instrument Co., Hamden, CT) were filled with solutions containing (in mm): 100 KCl, 1 MgCl2, 2 CaCl2, 5 Hepes (pH 7.4 with KOH). Pipette tip resistance ranged from 3 to 5 megaohms. For cell-attached recordings, bath solution contained 100 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes (pH 7.4 with KOH). For inside-out recordings, we used the Mg2+-free bath solution containing 100 KCl, 5 EDTA, and 5 Hepes (to prevent fast run-down) (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar, 19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar, 20Leung Y.M. Zeng W.Z. Liou H.H. Solaro C.R. Huang C.L. J. Biol. Chem. 2000; 275: 10182-10189Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In the experiments applying PKC in inside-out membranes (Fig. 7), purified rat brain PKC (from Calbiochem; 1 unit/ml) was applied in a Mg2+-free bath solution containing 500 nm Ca2+ (4.86 mm CaCl2 and 5 mm EDTA) and 50 ॖm 1-oleoyl-2-acetyl-sn-glycerol (OAG). Single-channel currents were recorded with an Axopatch 200B patch clamp amplifier (Axon Instruments, Foster City, CA), low-pass filtered at 1 kHz using an 8-pole Bessel filter, sampled every 0.1 ms (10 kHz) with Digidata-1200A interface and stored directly onto the computer hard disk (100 GB) using pCLAMP8 software. Data were transferred to CD for long-term storage. For analysis, event list files were generated using the Fetchan program and analyzed for open probability, amplitude, and dwell-time histograms using pCLAMP6 pSTAT (version 6.0.5, Axon Instruments). Open probability (NPo) was analyzed on segments of continuous recording (duration as indicated, respectively) as previously described (20Leung Y.M. Zeng W.Z. Liou H.H. Solaro C.R. Huang C.L. J. Biol. Chem. 2000; 275: 10182-10189Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 21Zeng W.-Z. Liou H.-H. Krishna U.M. Falck J.R. Huang C.-L. Am. J. Physiol. 2002; 282: F826-F834Crossref PubMed Scopus (33) Google Scholar). 32P-Labeled PIP2 in oocyte membrane was measured by TLC as described previously for cultured cells (22Yamamoto M. Hilgemann D.H. Feng S. Bito H. Ishihara H. Shibasaki Y. Yin H.L. J. Cell Biol. 2001; 152: 867-876Crossref PubMed Scopus (104) Google Scholar, 23Wei Y.J. Sun H.Q. Yamamoto M. Wlodarski P. Kunii K. Martinez M. Barylko B. Albanesi J.P. Yin L.Y. J. Biol. Chem. 2002; 277: 46586-46593Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) with minor modifications. Briefly, PIP2 in oocytes (∼10 each group) were labeled by incubating in ND96 (1 ml) containing [32P]PO4 (40–50 ॖCi) for 4 h. Oocytes were further incubated in the isotope-free ND96 for 2 h before treatments by Me2SO (vehicle), PMA and/or calphostin-C as indicated. Oocytes were homogenized in a buffer containing 1 ml of 57 trichloroacetic acid and 1 mm EDTA and spun in a microcentrifuge. Pellets were extracted in a buffer containing CHCl3, MeOH, 10 n HCl (20:20:0.2). The organic (lower) phase was collected, dried, and dissolved in 20 ॖl of CHCl3. Samples (5 ॖl) and lipid standards were resolved by TLC using 1-propanol, H2O, NH4OH (65:15:20). 32P-Labeled lipids were detected by autoradiography. Lipid standards were detected with iodine vapor. PIP2 mass was measured by separation of deacylated phospholipids using anion-exchange HPLC as previously described (24Nasuhoglu C. Feng S. Mao J. Yamamoto M. Yin H.L. Earnes S. Barylko B. Albanesi J.P. Hilgemann D.W. Anal. Biochem. 2002; 301: 243-254Crossref PubMed Scopus (112) Google Scholar, 25Nasuhoglu C. Feng S. Mao Y. Shammat I. Yamamoto M. Earnest S. Lemmon M. Hilgemann D.W. Am. J. Physiol. Cell Physiol. 2002; 283: C223-C234Crossref PubMed Scopus (74) Google Scholar). Briefly, oocytes with or without PMA treatment were homogenized and phospholipids were extracted in 1 ml of cold CHCl3, MeOH, 10 n HCl (20:40:1). Phospholipids were deacylated with monomethylamine and the head groups were separated by anion-exchange HPLC (Ionpac AS-11-HC2 mm column and AG11-HC2 mm guard column) using a three-stage NaOH gradient from 10 to 80 mm. Elution of the deacylated phospholipids (in comparison with the standards) was monitored by detection of the glycerol head group by suppressed conductivity. To test the hypothesis that PIP2 plays an important role in modulating PKC inhibition of ROMK1 channels, we compare the effects of PKC activators, PMA and OAG, on wild type and mutant ROMK1 channels with reduced PIP2 affinity. We have previously shown that mutation of a PKA consensus site, serine 219, to alanine (S219A) reduces the affinity of ROMK1 for PIP2 (19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar). As shown in Fig. 1B, the activity of S219A mutant was partially inhibited by addition of PMA (300 nm) to the extracellular bath solution. The decrease in channel activity was apparent within 10 min after administration of PMA and leveled at ∼607 inhibition by 30 min. Pretreatment with PKC inhibitor calphostin-C (0.5 ॖm) prevented the inhibition of channels by PMA (Fig. 1C). Another PKC activator OAG inhibited S219A channel activity similarly (Fig. 1D). As reported previously by others (26Henry P. Pearson W.L. Nichols C.G. J. Physiol. 1996; 495: 681-688Crossref PubMed Scopus (74) Google Scholar), activation of PKC by PMA did not cause a significant inhibition of wild type ROMK channels expressed in oocytes (Fig. 1A). These results suggest that reduction of PIP2-ROMK1 interaction increases the sensitivity of channels to inhibition by PKC. Lack of inhibition by PKC on the wild type channel in oocytes is likely because of that ROMK1 has a higher baseline affinity for PIP2 (see "Discussion"). The role of PIP2-channel interaction in modulating PKC inhibition of ROMK1 is further supported by the following studies using additional mutants with reduced PIP2 affinity. Arginine 188 of ROMK1 is critical for electrostatic interaction with PIP2 (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar, 19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar). PIP2-channel interaction is reduced by neutralization of arginine 188 to glutamine (R188Q), but not by a conserved charge substitution by lysine (R188K) (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar, 19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar). Mutation of another PKA phosphorylation site, serine 313, to alanine (S313A) also reduces PIP2 affinity (19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar). As shown in Fig.2, addition of PMA caused inhibition on S313A and R188Q mutants but not on R188K mutant. Pretreatment with calphostin-C prevented the inhibition (Fig. 2, A–C). OAG caused a similar inhibition on S313A and R188Q (not shown). We performed cell-attached patch clamp recording to examine whether PKC affects single channel conductance and/or open probability (NPo) of the channel. In oocytes expressing S219A mutants, PMA caused a reduction in NPo (Fig.3A). TheNPo of S219A was reduced to 25 ± 117 in 30 min (Fig. 4A). PMA did not alter the current-voltage relationship (not shown) or unitary conductance of single channels (36 ± 2.7 pS versus35 ± 1.9 pS; between −50 and −100 mV). In time control experiments, Me2SO (vehicle) had no effect on the channel (not shown). Pretreatment with calphostin-C prevented the reduction ofNPo caused by PMA (Fig. 3B). The effect of PMA to reduce NPo was further confirmed on S313A and R188Q mutants. As shown in Fig. 4, B and C, PMA reduced NPo of S313A and R188Q by 65 and 687, respectively.Figure 4Effect of PMA on the activity of S219A (panel A), S313A (panel B), and R188Q (panel C) mutant in cell-attached recording.n = 5–7 for each experimental condition. The activity of channel (measured as NPo) after 10 min addition of PMA was normalized to the pre-PMA level (1007).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The activity of S219A mutant channels was first recorded in on-cell patch clamp recording. As shown earlier, addition of PMA caused a reduction of NPo of channel in ∼10 min (Fig. 5A). After channel inhibition by PMA, inside-out membrane patches were then excised and PIP2 (50 ॖm) was applied to the cytoplasmic face. Application of PIP2 to the cytoplasmic face of the excised inside-out membrane increased NPo of the channel. These results suggest that PMA inhibits the activity of the S219A mutant ROMK by reducing PIP2-channel interaction. After recovery of channel activity by PIP2, further application of PMA to the excised patch did not cause inhibition of the channel (Fig. 5A). The lack of effect for the second application of PMA in the inside-out membrane is not because of the fact that PKC was not present in the excised membranes (see Fig. 7, below). Fig. 5B summaries the results of five similar experiments. As shown, PMA decreased NPo to 15 ± 47 of the control level (Fig. 5B, time point2). Application of PIP2 to the inside-out membranes increased NPo to 68 ± 187 of the control level (time point 3), which was not affected by re-application of PMA to the excised membranes (63 ± 117; time point 4). ROMK channels run down slowly in the excised membranes in Mg2+-free bath solutions (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar, 19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar, 20Leung Y.M. Zeng W.Z. Liou H.H. Solaro C.R. Huang C.L. J. Biol. Chem. 2000; 275: 10182-10189Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The incomplete recovery by PIP2 in the excised membranes is likely because of run-down of channels over time. The reduction of PIP2-ROMK interaction by PKC may be because of reduction of membrane PIP2 content. We examined the effect of PKC activation on PIP2 content in oocyte membrane. Phospholipids in oocyte membranes were labeled by incubating with [32P]PO4, extracted, and resolved by thin layer chromatography. As shown in Fig.6A, PMA caused a reduction of32P-labeled PIP2 and PIP. Pretreatment by calphostin-C prevented the reduction. The average reductions of32P-labeled PIP2 and PIP by PMA were 57 (p < 0.05; Fig. 6B) and 627 (not shown), respectively. To assure that the effect of PMA is not because of alteration of the kinetics of labeling by [32P]PO4, we also measured PIP2and PIP mass using anion exchange high pressure liquid chromatography (24Nasuhoglu C. Feng S. Mao J. Yamamoto M. Yin H.L. Earnes S. Barylko B. Albanesi J.P. Hilgemann D.W. Anal. Biochem. 2002; 301: 243-254Crossref PubMed Scopus (112) Google Scholar, 25Nasuhoglu C. Feng S. Mao Y. Shammat I. Yamamoto M. Earnest S. Lemmon M. Hilgemann D.W. Am. J. Physiol. Cell Physiol. 2002; 283: C223-C234Crossref PubMed Scopus (74) Google Scholar). In agreement with the results from measurement of32P-labeled PIP2 and PIP, we found that PMA reduced PIP2 and PIP mass in oocyte membranes by 43 and 647, respectively. The effect of PKC on S219A channels was further studied using purified PKC in the excised inside-out patches. As shown in Fig. 7, application of purified PKC (1 units/ml) to the cytoplasmic face of the membrane did not cause inhibition of the channel over 10 min. The activity of channel in the membrane patches after application of purified PKC (Fig.7B, open square) was not different from that in the time-control experiments (closed circle). These results suggest that the pathway(s) involved in inhibition of channels by PKC is disrupted in the excised membranes. Finally, we examined the role of PKC in the regulation of ROMK channel by phospholipase C (PLC)-coupled receptors. Type 1 muscarinic receptor (M1R) activates PLC via Gq (27Kobrinsky E. Mirshahi T. Zhang H. Jin T. Logothetis D.E. Nat. Cell Biol. 2000; 2: 507-514Crossref PubMed Scopus (203) Google Scholar). In many cell types including Xenopus oocytes, elevation of intracellular Ca2+ following receptor activation of PLC increases outwardly rectifying Cl− currents by activating the endogenous Ca2+-dependent Cl−channels (27Kobrinsky E. Mirshahi T. Zhang H. Jin T. Logothetis D.E. Nat. Cell Biol. 2000; 2: 507-514Crossref PubMed Scopus (203) Google Scholar). Addition of carbachol (indicated by the upward arrow in the left panel of Fig.8A) caused an immediate increase in outwardly rectifying Cl− currents, confirming the expression of M1 receptors in oocytes. In oocytes co-expressing M1R and S219A, carbachol inhibited the activity of the channel over 7–10 min (Fig. 8B). Compared with the time course of hydrolysis of PIP2 by PLC (based on the rise in the intracellular Ca2+ and activation of outwardly rectifying Cl− currents in Fig. 8A), the time course of inhibition of S219A channel was much slower. This delayed inhibition of channel by carbachol correlated with the effect observed for activation of PKC by PMA. In support of the interpretation that carbachol inhibits the channel via activation of PKC, pretreatment with calphostin-C prevented the inhibition (not shown). As in the experiments using PMA, carbachol did not affect wild type ROMK1 channel co-expressed with M1 muscarinic receptor in oocytes (not shown). The effect of carbachol on PIP2 content was examined (Fig.8C). Expression of M1R by itself (without addition of carbachol) did not alter [32P]PIP2 content in oocytes (labeled M1R; 95 ± 77 of control). Application of carbachol reduced [32P]PIP2content to 65 ± 137 of the control level at 10 min (labeledM1R+CCh; p < 0.05 versus Control). The reduction of [32P]PIP2 content by carbachol at 10 min was prevented by pretreatment with calphostin-C (labeled M1R+Cal-C +CCh; 109 ± 157 of the control level). The complete reversal of PIP2 to the control level by calphostin-C is consistent with the notion that reduction of PIP2 by PLC-mediated hydrolysis is transient (lasting <5 min) (28Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar). Together, these results suggest that hydrolysis of PIP2 by phospholipase C by itself is not sufficient to cause inhibition of the channel and that activation of PKC is necessary. PKC inhibits the activity of K+ channels in rat CCDs (12Wang W. Giebisch G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9722-9725Crossref PubMed Scopus (141) Google Scholar), but the molecular mechanism for this regulation is not known. We have shown previously that PIP2 is critical for the activity of ROMK channel (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar, 21Zeng W.-Z. Liou H.-H. Krishna U.M. Falck J.R. Huang C.-L. Am. J. Physiol. 2002; 282: F826-F834Crossref PubMed Scopus (33) Google Scholar) and the regulation of ROMK by other pathways such as PKA (19Liou H.H. Zhou S.S. Huang C.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5820-5825Crossref PubMed Scopus (134) Google Scholar) and intracellular pH (20Leung Y.M. Zeng W.Z. Liou H.H. Solaro C.R. Huang C.L. J. Biol. Chem. 2000; 275: 10182-10189Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) interacts with PIP2 regulation of the channel. In the present study, we found that mutations of ROMK1 with reduction of affinity for PIP2 increase the sensitivity of the channel to inhibition by PKC. We also found that application of PIP2 to the cytoplasmic surface reverses PKC-mediated inhibition and that activation of PKC reduces membrane PIP2 content. Together, these results suggest that PKC inhibits ROMK channels by reducing membrane PIP2 content. Others have also reported that for other inward rectifier K+ channels mutation of the PIP2-binding site increases the sensitivity of channels to reduction in membrane PIP2 content. Kobrinsky et al. (27Kobrinsky E. Mirshahi T. Zhang H. Jin T. Logothetis D.E. Nat. Cell Biol. 2000; 2: 507-514Crossref PubMed Scopus (203) Google Scholar) reported that receptor-regulated PIP2 hydrolysis (via M1 muscarinic receptor) causes desensitization of K+ currents through G protein-coupled inward rectifying K+ channels (GIRK). Another inward rectifying K+ channel, IRK1, has a higher PIP2 affinity relative to the GIRK channel (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar). Kobrinskyet al. (27Kobrinsky E. Mirshahi T. Zhang H. Jin T. Logothetis D.E. Nat. Cell Biol. 2000; 2: 507-514Crossref PubMed Scopus (203) Google Scholar) reported that hydrolysis of PIP2under the same experimental conditions does not affect the activity of IRK1. They also found that mutations of PIP2-binding residues of IRK1 render the channel sensitive to receptor-regulated reduction of PIP2 in the membrane (27Kobrinsky E. Mirshahi T. Zhang H. Jin T. Logothetis D.E. Nat. Cell Biol. 2000; 2: 507-514Crossref PubMed Scopus (203) Google Scholar). The affinity of ROMK1 for PIP2 is the same as that of IRK1 (15Huang C.L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (765) Google Scholar). Thus, it is perhaps no surprise that a partial reduction of PIP2 by PMA does not affect the activity of the wild type ROMK1 channel in oocytes. Then, why does activation of PKC cause inhibition on K+channels in CCD (12Wang W. Giebisch G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9722-9725Crossref PubMed Scopus (141) Google Scholar) but not on the wild type ROMK1 channel expressed in Xenopus oocytes (see Ref. 26Henry P. Pearson W.L. Nichols C.G. J. Physiol. 1996; 495: 681-688Crossref PubMed Scopus (74) Google Scholar and Fig. 1Aalso)? Our study suggests that this difference may be explained by a lower baseline PIP2-K+ channel interaction in CCD than in Xenopus oocytes, which renders channels in CCDs more sensitive to PKC regulation. The lower baseline PIP2-K+ channel interaction in CCD may be because of lower PIP2 content in the apical membrane of CCD and/or that native K+ channels are partially phosphorylated by PKA and thus have a lower affinity for PIP2. It is difficult to directly compare PIP2 content in the apical membrane of CCD with that in oocyte membrane. Our finding that PKA site mutants S219A and S313A are sensitive to inhibition by PKC at least provides support for the latter possibility. Many hormones, including bradykinin, prostaglandin E2, and epidermal growth factor inhibit K+ transport in CCDs (8Sugimoto Y. Namba T. Shigemoto R. Negishi M. Ichikawa A. Narumiya S. Am. J. Physiol. 1994; 266: F823-F828PubMed Google Scholar, 9Figueroa C.D. Gonzalez C.B. Grigoriev S. Alla A.A. Haasemann M. Jarnagin K. 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Physiol. 1991; 260: F163-F169PubMed Google Scholar, 31Guan Y Zhang Y. Breyer R.M. Fowler B. Davis L. Hebert R.L. Breyer M.D. J. Clin. Invest. 1998; 102: 194-201Crossref PubMed Scopus (124) Google Scholar). These PLC-activating hormones may inhibit K+ transport through either hydrolyzing PIP2 and/or through activating PKC. However, some hormone receptors, such as epidermal growth factor receptors, are present only in the basolateral membrane (30Muto S. Furuya H. Tabei K. Asano Y. Am. J. Physiol. 1991; 260: F163-F169PubMed Google Scholar) in contrast to the localization of the K+ channel in the apical membrane of CCD. Furthermore, the reduction of PIP2in the plasma membranes caused by hydrolysis via PLC alone is generally limited in magnitude and duration (28Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar) and is probably not sufficient to cause a decrease in the activity of the channels with a high affinity for PIP2, such as ROMK. In this study, we found that hydrolysis of PIP2 following M1 receptor activation is not sufficient to cause inhibition of the S219A ROMK mutant (which has a lower affinity for PIP2 probably equivalent to the native channels) and activation of PKC is necessary for inhibition of the channel via stimulation of M1 receptors (Fig. 8). Activation of PKC likely does so by amplifying the reduction of PIP2initiated by hydrolysis by PLC. Thus, activation of PKC is likely the mechanism by which PLC-activating hormones inhibit K+channels in the apical membrane of CCD. Regulation of K+channels by these hormones is important in certain physiological and pathophysiological states. For example, inhibition of K+channels may help to prevent excess kaliuresis during natriuresis (occurs as a result of inhibition of Na+ reabsorption at the proximal sites). A recent review suggests that loss of this direct inhibitory action in K+ secretion by prostaglandin E2 may be helpful in mitigating hyperkalemia associated with administration of nonsteroidal anti-inflammatory drugs (29Breyer M.D. Breyer R.M. Am. J. Physiol. 2000; 279: F12-F23PubMed Google Scholar). One common mechanism for PKC to regulate protein function is by direct phosphorylation of target proteins. Indeed, direct phosphorylation is important for PKC regulation of some inward-rectifier K+channels (32Light P.E. Bladen C. Winkfein R.J. Walsh M.P. French R.J Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9058-9063Crossref PubMed Scopus (127) Google Scholar, 33Karle C.A. Zitron E. Zhang W. Wendt-Nordahl G. Kathofer S. Thomas D. Gut B. Scholz E. Vahl C.F. Katus H.A. Kiehn J. Circulation. 2002; 106: 1493-1499Crossref PubMed Scopus (64) Google Scholar). Our present study does not contradict with the possibility that phosphorylation of ROMK by PKC may be important for channel inhibition. Rather, it suggests that reduction of PIP2 content by PKC is important for inhibition of ROMK1 in oocytes. Hill and Peralta (34Hill J. Peralta E. J. Biol. Chem. 2001; 276: 5505-5510Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) recently reported that activation of PKC by PMA and M1 receptor inhibits GIRK channels expressed in oocytes. Interestingly, the authors found that mutation of potential PKC phosphorylation sites does not prevent the inhibition of GIRK channels by PKC. Our work opens new possibilities for regulation of PIP2-regulated ion channels and other proteins by PLC-activating hormones. Nasuhoglu et al. (25Nasuhoglu C. Feng S. Mao Y. Shammat I. Yamamoto M. Earnest S. Lemmon M. Hilgemann D.W. Am. J. Physiol. Cell Physiol. 2002; 283: C223-C234Crossref PubMed Scopus (74) Google Scholar) recently reported that PMA and diacylglycerol decrease membrane PIP2 content in guinea pig ventricles and in a mouse CCD cell line, but not in all cell types. The mechanism by which activation of PKC alters membrane PIP2content remain unknown. Potential mechanisms include regulation of PLC (35Boon A.M. Beresford B.J. Mellors A. Biochem. Biophys. Res. Commun. 1985; 129: 431-438Crossref PubMed Scopus (20) Google Scholar, 36Halenda S.P. Feinstein M.B. Biochem. Biophys. Res. Commun. 1984; 124: 507-513Crossref PubMed Scopus (73) Google Scholar), regulation of phosphatidylinositol transfer protein (37Van Tiel C.M. Westerman J. Paasman M. Wirtz K.W. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and regulation of activity and location of lipid phosphatases and/or lipid kinases (25Nasuhoglu C. Feng S. Mao Y. Shammat I. Yamamoto M. Earnest S. Lemmon M. Hilgemann D.W. Am. J. Physiol. Cell Physiol. 2002; 283: C223-C234Crossref PubMed Scopus (74) Google Scholar, 38Ooms L.M. McColl B.K. Wiradjaja F. Wijayaratnam A.P. Gleeson P. Gething M.J. Sambrook J. Mitchell C.A. Mol. Cell. Biol. 2000; 20: 9376-9390Crossref PubMed Scopus (31) Google Scholar). The molecular identity of lipid kinases and phosphatases regulating PIP2 metabolism in the cell surface remains controversial. Identification of the lipid kinases and phosphatases involved may help future studies to investigate the mechanism by which PKC alters PIP2 content in the cell surface. We thank Dr. R. Alpern for encouragement, Dr. S. Mumby for cDNA for M1 receptor, Y. Mao for technical assistance with HPLC analysis, and Drs. M. Yamamoto and H. L. Yin for help in setting up TLC assay.

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