scholarly journals Extracellular Atp Inhibits the Small-Conductance K Channel on the Apical Membrane of the Cortical Collecting Duct from Mouse Kidney

2000 ◽  
Vol 116 (2) ◽  
pp. 299-310 ◽  
Author(s):  
Ming Lu ◽  
Gordon G. MacGregor ◽  
Wenhui Wang ◽  
Gerhard Giebisch
Keyword(s):  
Protein Kinase ◽  
Apical Membrane ◽  
Purinergic Receptor ◽  
Collecting Duct ◽  
Extracellular Atp ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Inhibitory Effects ◽  
Small Conductance

We have used the patch-clamp technique to study the effects of changing extracellular ATP concentration on the activity of the small-conductance potassium channel (SK) on the apical membrane of the mouse cortical collecting duct. In cell-attached patches, the channel conductance and kinetics were similar to its rat homologue. Addition of ATP to the bathing solution of split-open single cortical collecting ducts inhibited SK activity. The inhibition of the channel by ATP was reversible, concentration dependent (Ki = 64 μM), and could be completely prevented by pretreatment with suramin, a specific purinergic receptor (P2) blocker. Ranking of the inhibitory potency of several nucleotides showed strong inhibition by ATP, UTP, and ATP-γ-S, whereas α, β-Me ATP, and 2-Mes ATP failed to affect channel activity. This nucleotide sensitivity is consistent with P2Y2 purinergic receptors mediating the inhibition of SK by ATP. Single channel analysis further demonstrated that the inhibitory effects of ATP could be elicited through activation of apical receptors. Moreover, the observation that fluoride mimicked the inhibitory action of ATP suggests the activation of G proteins during purinergic receptor stimulation. Channel inhibition by ATP was not affected by blocking phospholipase C and protein kinase C. However, whereas cAMP prevented channel blocking by ATP, blocking protein kinase A failed to abolish the inhibitory effects of ATP. The reduction of K channel activity by ATP could be prevented by okadaic acid, an inhibitor of protein phosphatases, and KT5823, an agent that blocks protein kinase G. Moreover, the effect of ATP was mimicked by cGMP and blocked by L-NAME (NG-nitro-l-arginine methyl ester). We conclude that the inhibitory effect of ATP on the apical K channel is mediated by stimulation of P2Y2 receptors and results from increasing dephosphorylation by enhancing PKG-sensitive phosphatase activity.

Mineralocorticoids decrease the activity of the apical small-conductance K channel in the cortical collecting duct

2005 ◽  
Vol 289 (5) ◽  
pp. F1065-F1071 ◽  
Author(s):  
Yuan Wei ◽  
Elisa Babilonia ◽  
Hyacinth Sterling ◽  
Yan Jin ◽  
Wen-Hui Wang
Keyword(s):  
Protein Tyrosine Kinase ◽  
Collecting Duct ◽  
K Channel ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Herbimycin A ◽  
Sk Channel ◽  
Small Conductance

We used the patch-clamp technique to examine the effect of DOCA treatment (2 mg/kg) on the apical small-conductance K (SK) channels, epithelial Na channels (ENaC), and the basolateral 18-pS K channels in the cortical collecting duct (CCD). Treatment of rats with DOCA for 6 days significantly decreased the plasma K from 3.8 to 3.1 meq and reduced the activity of the SK channel, defined as NPo, from 1.3 in the CCD of control rats to 0.6. In contrast, DOCA treatment significantly increased ENaC activity from 0.01 to 0.53 and the basolateral 18-pS K channel activity from 0.67 to 1.63. Moreover, Western blot analysis revealed that DOCA treatment significantly increased the expression of the nonreceptor type of protein tyrosine kinase (PTK), cSrc, and the tyrosine phosphorylation of ROMK in the renal cortex and outer medulla. The possibility that decreases in apical SK channel activity induced by DOCA treatment were the result of stimulation of PTK activity was further supported by experiments in which inhibition of PTK with herbimycin A significantly increased NPo from 0.6 to 2.1 in the CCD from rats receiving DOCA. Also, when rats were fed a high-K (10%) diet, DOCA treatment did not increase the expression of c-Src and decrease the activity of the SK channel in the CCD. We conclude that DOCA treatment decreased the apical SK channel activity in rats on a normal-K diet and that an increase in PTK expression may be responsible for decreased channel activity in the CCD from DOCA-treated rats.

scholarly journals Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na(+)-K(+)-ATPase.

1993 ◽  
Vol 101 (5) ◽  
pp. 673-694 ◽  
Author(s):  
W H Wang ◽  
J Geibel ◽  
G Giebisch
Keyword(s):  
Protein Kinase ◽  
Collecting Duct ◽  
Inhibitory Effect ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Dependent Protein Kinase ◽  
Bath Solution ◽  
Pump Activity ◽  
Dependent Protein

The effects of inhibition of the basolateral Na(+)-K(+)-ATPase (pump) on the apical low-conductance K+ channel of principal cells in rat cortical collecting duct (CCD) were studied with patch-clamp techniques. Inhibition of pump activity by removal of K+ from the bath solution or addition of strophanthidin reversibly reduced K+ channel activity in cell-attached patches to 36% of the control value. The effect of pump inhibition on K+ channel activity was dependent on the presence of extracellular Ca2+, since removal of Ca2+ in the bath solution abolished the inhibitory effect of 0 mM K+ bath. The intracellular [Ca2+] (measured with fura-2) was significantly increased, from 125 nM (control) to 335 nM (0 mM K+ bath) or 408 nM (0.2 mM strophanthidin), during inhibition of pump activity. In contrast, cell pH decreased only moderately, from 7.45 to 7.35. Raising intracellular Ca2+ by addition of 2 microM ionomycin mimicked the effect of pump inhibition on K+ channel activity. 0.1 mM amiloride also significantly reduced the inhibitory effect of the K+ removal. Because the apical low-conductance K channel in inside-out patches is not sensitive to Ca2+ (Wang, W., A. Schwab, and G. Giebisch, 1990. American Journal of Physiology. 259:F494-F502), it is suggested that the inhibitory effect of Ca2+ is mediated by a Ca(2+)-dependent signal transduction pathway. This view was supported in experiments in which application of 200 nM staurosporine, a potent inhibitor of Ca(2+)-dependent protein kinase C (PKC), markedly diminished the effect of the pump inhibition on channel activity. We conclude that a Ca(2+)-dependent protein kinase such as PKC plays a key role in the downregulation of apical low-conductance K+ channel activity during inhibition of the basolateral Na(+)-K(+)-ATPase.

Regulation of ATP-sensitive K+ channel by membrane-bound protein phosphatases in rat principal tubule cell

1995 ◽  
Vol 269 (3) ◽  
pp. F355-F362 ◽  
Author(s):  
M. Kubokawa ◽  
C. M. McNicholas ◽  
M. A. Higgins ◽  
W. Wang ◽  
G. Giebisch
Keyword(s):  
Katp Channel ◽  
Collecting Duct ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Inhibitory Effects ◽  
Patch Clamp Technique ◽  
Cytosolic Side ◽  
Membrane Bound

The role of membrane-bound protein serine/threonine phosphatases (PP) in modulating the renal ATP-sensitive K+ (KATP) channel was examined using the patch-clamp technique in principal cells of rat cortical collecting duct. In the absence of ATP, channel activity rapidly (11.2 s) declines (channel "rundown") upon excision of the membrane patches into control bath solutions (1 mM Mg2+, Ca2+ free). Both orthovanadate (5 mM), a broad-spectrum inhibitor of phosphatases except for Ca(2+)-dependent PP (PP-2B), and okadaic acid (OA, 1 microM), a potent inhibitor of PP types 1 and 2A (PP-1 and PP-2A), significantly slowed channel rundown. Removal of Mg2+ from the bath also slowed the rundown process. Incubation of cells with OA in the absence of Mg2+ or with orthovanadate in ATP-free solution maintained channel activity at levels of approximately 70% of control values for 3 min after membrane excision. In contrast, Ca2+ (0.1 mM) and calmodulin (1 microM) in the presence of 1 mM Mg2+, a condition in which PP-2B is stimulated, had no significant effect on the channel activity that persisted in the presence of OA and orthovanadate. Application of exogenous PP-2A (1 U/ml) to the cytosolic side of membrane in inside-out patches significantly inhibited channel activity to 35.0% of control, but the inhibitory-effects of PP-1 (1 U/ml) and PP-2B (20 micrograms/ml) were minor. These results suggest that rundown of the renal KATP channel after membrane excision results mainly from dephosphorylation of the channel or an associated protein by membrane-bound phosphatases.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Hydrolyzable ATP and PIP2 Modulate the Small-conductance K+ Channel in Apical Membranes of Rat Cortical-Collecting Duct (CCD)

2002 ◽  
Vol 120 (5) ◽  
pp. 603-615 ◽  
Author(s):  
Ming Lu ◽  
Steven C. Hebert ◽  
Gerhard Giebisch
Keyword(s):  
Kinase Inhibitors ◽  
Collecting Duct ◽  
K Channel ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Sk Channel ◽  
Phosphoinositide Kinase ◽  
Excised Patches ◽  
Small Conductance

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.

Involvement of actin cytoskeleton in modulation of apical K channel activity in rat collecting duct

1994 ◽  
Vol 267 (4) ◽  
pp. F592-F598 ◽  
Author(s):  
W. H. Wang ◽  
A. Cassola ◽  
G. Giebisch
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Collecting Duct ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Channel Inhibition ◽  
Inside Out

We have employed the patch-clamp technique to investigate the role of the actin cytoskeleton in the modulation of the low-conductance K+ channel in the apical membrane of the rat cortical collecting duct (CCD). This K+ channel is inactivated by application of cytochalasin B or D, both compounds known to disrupt actin filaments. The effect of both cytochalasins, B and D, was fully reversible in cell-attached patches, but channel activity could not be fully restored in excised membrane patches. The effect of cytochalasins on channel activity was specific and resulted from depolymerization of the actin cytoskeleton, since application of 10 microM chaetoglobosin C, a cytochalasin analogue that does not depolymerize the actin filaments, had no effect on channel activity in inside-out patches. Addition of either actin monomers or of the polymerizing actin filaments in inside-out patches to the cytosolic medium had no effect on channel activity. This suggests that cytochalasin B- or D-induced inactivation of apical K+ channels is not caused by obstruction of the channel pore by actin. We also observed that channel inhibition by cytochalasin B or D could be blocked by pretreatment with 5 microM phalloidin, a compound that stabilizes actin filaments. We conclude that apical K+ channel activity depends critically on the integrity of the actin cytoskeleton.

Role of Ca2+/CaMK II in Ca(2+)-induced K+ channel inhibition in rat CCD principal cell

1995 ◽  
Vol 268 (2) ◽  
pp. F211-F219 ◽  
Author(s):  
M. Kubokawa ◽  
W. Wang ◽  
C. M. McNicholas ◽  
G. Giebisch
Keyword(s):  
Protein Kinase ◽  
Collecting Duct ◽  
Inhibitory Effect ◽  
K Channel ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Pkc Inhibitor ◽  
Control Value ◽  
Gf 109203X

The apical low-conductance K+ channel of rat cortical collecting duct (CCD) is inhibited by increased intracellular Ca2+ concentrations. This effect has been shown to be mediated at least in part by activation of protein kinase C (PKC). In the present study, we used the patch-clamp technique to examine the role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) in mediating the Ca(2+)-induced inhibitory effect. In cell-attached patches of principal cells of rat tubules, clamping of intracellular Ca2+ concentration at 400 nM by using 1 microM ionomycin reduced channel activity to 26.5% of the control value. A further reduction in channel activity, to 8.8% of the control value, was observed following the addition of phorbol 12-myristate 13-acetate (PMA), an agent known to activate PKC. Pretreatment of cells with KN-62 (CaMK II inhibitor) or GF-109203X (PKC inhibitor) attenuated the inhibitory effect of Ca2+ on K+ channel activity (83.2 and 50.7% of the control value, respectively). Even in the presence of KN-62, addition of 10 microM PMA significantly decreased channel activity to 57.2% of the control value. The Ca(2+)-induced inhibition was completely abolished by simultaneous incubation with both KN-62 and GF-109203X. In inside-out patches, addition of 20 micrograms/ml CaMK II in the presence of a PKC inhibitor reduced channel activity to 66.2% of control values. It is concluded that CaMK II is involved in mediating the Ca(2+)-induced inhibition of the activity of the apical K+ channel of rat CCD.

Inhibition of Na+ and Ca2+ reabsorption by P2u purinoceptors requires PKC but not Ca2+ signaling

1996 ◽  
Vol 270 (1) ◽  
pp. F53-F60 ◽  
Author(s):  
H. P. Koster ◽  
A. Hartog ◽  
C. H. van Os ◽  
R. J. Bindels
Keyword(s):  
Inhibitory Concentration ◽  
Purinergic Receptor ◽  
Rank Order ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Inhibitory Effects ◽  
Maximal Inhibition ◽  
Kinase C ◽  
Collecting Duct Cells ◽  
Dose Dependent

Rabbit connecting tubule and cortical collecting duct cells were isolated by immunodissection and cultured to confluence on permeable filters and on glass coverslips. Extracellular ATP dose-dependently reduced transcellular Na+ and Ca2+ transport (half-maximal inhibitory concentration, IC50, of 0.5 +/- 0.2 and 3.2 +/- 0.5 microM), with a maximal inhibition of 57 +/- 5 and 43 +/- 4%, respectively. Purinergic receptor agonists inhibited transport with the following rank order of potency: UTP = ATP > ADP; this suggests involvement of P2u purinoceptors. ATP also caused a dose-dependent (50% effective dose, EC50, of 1.5 +/- 0.2 microM) transient increase in intracellular Ca2+ concentration ([Ca2+]i), which decreased to a sustained elevated level. In the absence of extracellular Ca2+, a similar Ca2+ transient occurred, but the sustained response was abolished. Preloading the cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) completely prevented the ATP-induced Ca2+ transients, but not the ATP-induced inhibition of Na+ and Ca2+ absorption. Activation of protein kinase C (PKC) by the cell-permeable diacylglycerol analogue, 1,2-dioctanoyl-en-glycerol, mimicked ATP-induced inhibition of Na+ and Ca2+ absorption. The inhibitory effects of ATP were no longer observed after culturing cells in the presence of phorbol ester (12-O-tetradecanoylphorbol-13-acetate) for 5 days, which resulted in downregulation of cellular PKC activity.

Arachidonic acid inhibits activity of cloned renal K+ channel, ROMK1

1996 ◽  
Vol 271 (3) ◽  
pp. F588-F594 ◽  
Author(s):  
C. M. Macica ◽  
Y. Yang ◽  
S. C. Hebert ◽  
W. H. Wang
Keyword(s):  
Arachidonic Acid ◽  
Membrane Fluidity ◽  
Collecting Duct ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Lipoxygenase Inhibitor ◽  
Open Probability ◽  
Apical Membranes

Arachidonic acid (AA) has been shown to inhibit the activity of the low-conductance ATP-sensitive K+ channel in the apical membrane of the cortical collecting duct [W. Wang, A. Cassola, and G. Giebisch. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F554-F559, 1992]. ROMK1, a K+ channel derived from the rat renal outer medulla, shares many biophysical properties of the native low-conductance K+ channel, which is localized to the apical membranes of the cortical collecting duct and thick ascending limb. This study was designed to determine whether the ROMK channel maintains the property of AA sensitivity of the native low-conductance K+ channel. Experiments were conducted in Xenopus oocytes injected with cRNA encoding the ROMK1 channel by use of patch-clamp techniques. We have confirmed previous reports that the cloned ROMK1 has similar channel kinetics, high open probability, and inward slope conductance as the native low-conductance K+ channel, respectively. Addition of 5 microM AA to an inside-out patch resulted in reversible inhibition of channel activity at a concentration similar to the inhibitor constant for AA on the native K+ channel. The effect of AA on channel activity was preserved in the presence of 10 microM indomethacin, a cyclooxygenase inhibitor, 4 microM cinnamyl-3,4-dihydroxycyanocinnamate, a lipoxygenase inhibitor, and 4 microM 17-octadecynoic acid, an inhibitor of cytochrome P-450 monooxygenases, thus indicating that the effect of AA was not mediated by metabolites of AA. The effect did not appear to be the result of changes in membrane fluidity, since 5 microM eicosatetraynoic acid, an AA analogue that is a potent modulator of membrane fluidity, had no effect. Furthermore, the addition of AA to the outside of the patch also had no effect on channel activity. These results indicate that, like the native low-conductance channel, AA is able to directly inhibit ROMK1 channel activity.

scholarly journals Angiotensin II type 2 receptor regulates ROMK-like K+ channel activity in the renal cortical collecting duct during high dietary K+ adaptation

2014 ◽  
Vol 307 (7) ◽  
pp. F833-F843 ◽  
Author(s):  
Yuan Wei ◽  
Yi Liao ◽  
Beth Zavilowitz ◽  
Jin Ren ◽  
Wen Liu ◽  
...  
Keyword(s):  
Collecting Duct ◽  
No Synthase ◽  
K Channel ◽  
Ang Ii ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
High K ◽  
Pka Pathway

The kidney adjusts K+ excretion to match intake in part by regulation of the activity of apical K+ secretory channels, including renal outer medullary K+ (ROMK)-like K+ channels, in the cortical collecting duct (CCD). ANG II inhibits ROMK channels via the ANG II type 1 receptor (AT1R) during dietary K+ restriction. Because AT1Rs and ANG II type 2 receptors (AT2Rs) generally function in an antagonistic manner, we sought to characterize the regulation of ROMK channels by the AT2R. Patch-clamp experiments revealed that ANG II increased ROMK channel activity in CCDs isolated from high-K+ (HK)-fed but not normal K+ (NK)-fed rats. This response was blocked by PD-123319, an AT2R antagonist, but not by losartan, an AT1R antagonist, and was mimicked by the AT2R agonist CGP-42112. Nitric oxide (NO) synthase is present in CCD cells that express ROMK channels. Blockade of NO synthase with N-nitro-l-arginine methyl ester and free NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt completely abolished ANG II-stimulated ROMK channel activity. NO enhances the synthesis of cGMP, which inhibits phosphodiesterases (PDEs) that normally degrade cAMP; cAMP increases ROMK channel activity. Pretreatment of CCDs with IBMX, a broad-spectrum PDE inhibitor, or cilostamide, a PDE3 inhibitor, abolished the stimulatory effect of ANG II on ROMK channels. Furthermore, PKA inhibitor peptide, but not an activator of the exchange protein directly activated by cAMP (Epac), also prevented the stimulatory effect of ANG II. We conclude that ANG II acts at the AT2R to stimulate ROMK channel activity in CCDs from HK-fed rats, a response opposite to that mediated by the AT1R in dietary K+-restricted animals, via a NO/cGMP pathway linked to a cAMP-PKA pathway.

The cGMP-dependent protein kinase stimulates the basolateral 18-pS K channel of the rat CCD

2000 ◽  
Vol 278 (6) ◽  
pp. C1212-C1217 ◽  
Author(s):  
Wen Hui Wang
Keyword(s):  
Protein Kinase ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
K Channel ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

We used the patch-clamp technique to study the effect of cGMP on the 18-pS K channel in the basolateral membrane of the rat cortical collecting duct. Addition of 100 μM 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) increased the activity of the 18-pS K channel, defined by NP o, by 95%. In contrast, applying 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) has no effect on channel activity. The effect of 8-Br-cGMP was observed only in cell-attached but not in inside-out patches. Application of 1 μM KT-5823, an inhibitor of the cGMP-dependent protein kinase (PKG), not only reduced the channel activity, but also completely abolished the stimulatory effect of 8-Br-cGMP, suggesting that the 18-pS K channel is not a cGMP-gated K channel. Addition of H-89, an agent that also blocks the PKG, mimicked the effect of KT-5823. To examine the possibility that the effect of 8-Br-cGMP is the result of inhibiting cGMP-dependent phosphodiesterase (PDE) and, accordingly, increasing cAMP or cGMP levels, we explored the effect on the 18-pS K channel of IBMX, an agent that inhibits the PDE. The addition of 100 μM IBMX had no significant effect on channel activity in cell-attached patches. Moreover, in the presence of IBMX, 8-Br-cGMP increased the channel activity to the same extent as that observed in the absence of IBMX, suggesting that the effect of cGMP is not mediated by inhibiting the cGMP-dependent PDE. That the effect of cGMP is mediated by stimulating PKG was further indicated by experiments in which application of exogenous PKG restored the channel activity when it decreased after the excision of the patches. In contrast, adding exogenous cAMP-dependent protein kinase catalytic subunit failed to reactivate the run-down channels. We conclude that cGMP stimulates the 18-pS channel, and the effect of cGMP is mediated by PKG.

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