scholarly journals Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD

2006 ◽  
Vol 290 (4) ◽  
pp. F806-F812 ◽  
Author(s):  
Dimin Li ◽  
Yuan Wei ◽  
Elisa Babilonia ◽  
Zhijian Wang ◽  
Wen-Hui Wang
Keyword(s):  
Collecting Duct ◽  
Sk Channels ◽  
Channel Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Patch Clamp Technique ◽  
Α Subunit ◽  
Sk Channel ◽  
Low K ◽  
Small Conductance ◽  
Phosphatidylinositol 3

We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa α-subunit (p110α) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels ( 2 ), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H2O2 stimulates the expression of p110α in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.

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 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.

scholarly journals EF hands at the N-lobe of calmodulin are required for both SK channel gating and stable SK–calmodulin interaction

2009 ◽  
Vol 134 (4) ◽  
pp. 281-293 ◽  
Author(s):  
Weiyan Li ◽  
David B. Halling ◽  
Amelia W. Hall ◽  
Richard W. Aldrich
Keyword(s):  
Modular Design ◽  
Binding Capacity ◽  
Current Model ◽  
Sk Channels ◽  
Channel Activity ◽  
Wild Type ◽  
Sk Channel ◽  
Ef Hand ◽  
Ef Hands ◽  
Small Conductance

Small conductance calcium-activated potassium (SK) channels respond to intracellular Ca2+ via constitutively associated calmodulin (CaM). Previous studies have proposed a modular design for the interaction between CaM and SK channels. The C-lobe and the linker of CaM are thought to regulate the constitutive binding, whereas the N-lobe binds Ca2+ and gates SK channels. However, we found that coexpression of mutant CaM (E/Q) where the N-lobe has only one functional EF hand leads to rapid rundown of SK channel activity, which can be recovered with exogenously applied wild-type (WT), but not mutant, CaM. Our results suggest that the mutation at the N-lobe EF hand disrupts the stable interaction between CaM and SK channel subunits, such that mutant CaM dissociates from the channel complex when the inside of the membrane is exposed to CaM-free solution. The disruption of the stable interaction does not directly result from the loss of Ca2+-binding capacity because SK channels and WT CaM can stably interact in the absence of Ca2+. These findings question a previous conclusion that CaM where the N-lobe has only one functional EF hand can stably support the gating of SK channels. They cannot be explained by the current model of modular interaction between CaM and SK channels, and they imply a role for N-lobe EF hand residues in binding to the channel subunits. Additionally, we found that a potent enhancer for SK channels, 3-oxime-6,7-dichloro-1H-indole-2,3-dione (NS309), enables the recovery of channel activity with CaM (E/Q), suggesting that NS309 stabilizes the interaction between CaM and SK channels. CaM (E/Q) can regulate Ca2+-dependent gating of SK channels in the presence of NS309, but with a lower apparent Ca2+ affinity than WT CaM.

scholarly journals PKC expression is regulated by dietary K intake and mediates internalization of SK channels in the CCD

2004 ◽  
Vol 286 (6) ◽  
pp. F1072-F1078 ◽  
Author(s):  
Hyacinth Sterling ◽  
Dao-Hong Lin ◽  
Yu-Jung Chen ◽  
Yuan Wei ◽  
Zhi-Jian Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Renal Cortex ◽  
Collecting Duct ◽  
Control Diet ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Outer Medulla ◽  
Protein Tyrosine ◽  
Pkc Isoforms ◽  
Low K

We have used Western blot analysis and immunocytochemistry to determine the effect of dietary K intake on the expression of protein kinase C (PKC) isoforms in the kidney. Western blot has demonstrated that conventional PKC isoforms (α and β), novel PKC isoforms (δ, ε, and η), and atypical PKC isoforms (ζ) are expressed in the renal cortex and outer medulla. Moreover, a low K intake significantly increases the expression of PKC-ε in the renal cortex and outer medulla but does not change the expression of PKC-α, PKC-β, PKC-δ, PKC-η, and PKC-ζ. Also, immunocytochemistry shows that PKC-ε isoform is expressed in the cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) and that the intensity of PKC-ε staining is higher in the kidney from rats on a K-deficient diet than those on a control diet. Also, we used the patch-clamp technique to study the role of PKC in mediating internalization of ROMK (Kir 1.1)-like small-conductance K (SK) channels induced by phenylarsine oxide (PAO), an agent that inhibits protein tyrosine phosphatase and has been shown to stimulate the internalization of the SK channel in the CCD (Sterling H, Lin DH, Qu RM, Dong K, Herbert SC, and Wang WH. J Biol Chem 277: 4317–4323, 2002). Inhibition of PKC with calphostin C and GF-109203x had no significant effect on channel activity but abolished the inhibitory effect of PAO on SK channels. In conclusion, a low K intake increases the expression of PKC-ε isoform in the renal cortex and outer medulla, and PKC is involved in mediating the internalization of SK channels in the CCD induced by stimulation of protein tyrosine kinase activity.

scholarly journals Molecular overlap in the regulation of SK channels by small molecules and phosphoinositides

2015 ◽  
Vol 1 (6) ◽  
pp. e1500008 ◽  
Author(s):  
Miao Zhang ◽  
Xuan-Yu Meng ◽  
Ji-fang Zhang ◽  
Meng Cui ◽  
Diomedes E. Logothetis
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Critical Element ◽  
Sk Channels ◽  
Channel Activity ◽  
Sk Channel ◽  
Small Molecule Drugs ◽  
Close Proximity ◽  
Channel Modulator ◽  
Small Conductance

Phosphatidylinositol 4,5-bisphosphate (PIP2) directly interacts with the small-conductance Ca2+-activated K+ 2-a (SK2-a) channel/calmodulin complex, serving as a critical element in the regulation of channel activity. We report that changes of protein conformation in close proximity to the PIP2 binding site induced by a small-molecule SK channel modulator, NS309, can effectively enhance the interaction between the protein and PIP2 to potentiate channel activity. This novel modulation of PIP2 sensitivity by small-molecule drugs is likely not to be limited in its application to SK channels, representing an intriguing strategy to develop drugs controlling the activity of the large number of PIP2-dependent proteins.

Role of the cytoskeleton in mediating effect of vasopressin and herbimycin A on secretory K channels in CCD

2002 ◽  
Vol 282 (4) ◽  
pp. F680-F686 ◽  
Author(s):  
Yuan Wei ◽  
Wen-Hui Wang
Keyword(s):  
Collecting Duct ◽  
Cytochalasin D ◽  
Mediating Effect ◽  
Microtubule Assembly ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Herbimycin A

We have previously demonstrated that inhibiting protein tyrosine kinase (PTK) and stimulating protein kinase A (PKA) increase the activity of the small-conductance K (SK) channel in the cortical collecting duct (CCD) of rat kidneys (Cassola AC, Giebisch G, and Wang WH. Am J Physiol Renal Fluid Electrolyte Physiol 264: F502–F509, 1993; Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165–F171, 2000). In the present study, we used the patch-clamp technique to study the role of the cytoskeleton in mediating the effect of herbimycin A, an inhibitor of PTK, and vasopressin on the SK channels in the CCD. The addition of colchicine, an inhibitor of microtubule assembly, or taxol, an agent that blocks microtubule reconstruction, had no significant effect on channel activity. However, colchicine and taxol treatment completely abolished the stimulatory effect of herbimycin A on the SK channels in the CCD. Removal of the microtubule inhibitors restored the stimulatory effect of herbimycin A. In contrast, treatment of the tubules with either taxol or colchicine did not block the stimulatory effect of vasopressin on the SK channels. Moreover, the effect of herbimycin A on the SK channels was also absent in the CCDs treated with either cytochalasin D or phalloidin. In contrast, the stimulatory effect of vasopressin was still observed in the tubules treated with phalloidin. However, cytochalasin D treatment abolished the effect of vasopressin on the SK channels. Finally, the effects of vasopressin and herbimycin A are additive because inhibiting PTK can still increase the channel activity in CCD that has been challenged by vasopressin. We conclude that an intact cytoskeleton is required for the effect on the SK channels of inhibiting PTK and that the SK channels that are activated by inhibiting PTK were differently regulated from those stimulated by vasopressin.

scholarly journals Effect of hydrogen peroxide on ROMK channels in the cortical collecting duct

2007 ◽  
Vol 292 (4) ◽  
pp. F1151-F1156 ◽  
Author(s):  
Yuan Wei ◽  
ZhiJian Wang ◽  
Elisa Babilonia ◽  
Hyacinth Sterling ◽  
Peng Sun ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Inhibitory Effect ◽  
Mitogen Activated Protein Kinase ◽  
Hek293 Cells ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Direct Oxidation ◽  
Patch Clamp Technique ◽  
Sk Channel

We used the patch-clamp technique to study the effect of H2O2 on the apical ROMK-like small-conductance K (SK) channel in the cortical collecting duct (CCD). The addition of H2O2 decreased the activity of the SK channels and the inhibitory effect of H2O2 was larger in the CCD from rats on a K-deficient diet than that from rats on a normal-K or a high-K diet. However, application of H2O2 did not inhibit the SK channels in inside-out patches. This suggests that the H2O2-mediated inhibition of SK channels was not due to direct oxidation of the SK channel protein. Because a previous study showed that H2O2 stimulated the expression of Src family protein tyrosine kinase (PTK) which inhibited SK channels ( 3 ), we explored the role of PTK in mediating the effect of H2O2 on SK channels. The application of H2O2 stimulated the activity of endogenous PTK in M-1 cells and increased tyrosine phosphorylation of ROMK in HEK293 cells transfected with GFP-ROMK1 and c-Src. However, blockade of PTK only attenuated but did not completely abolish the inhibitory effect of H2O2 on SK channels. Since H2O2 has also been demonstrated to activate mitogen-activated protein kinase, P38, and ERK ( 3 ), we examined the role of P38 and ERK in mediating the effect of H2O2 on SK channels. Similar to blockade of PTK, suppression of P38 and ERK did not completely abolish the H2O2-induced inhibition of SK channels. However, combined use of ERK, P38, and PTK inhibitors completely abolished the effect of H2O2 on SK channels. Also, treatment of the CCDs with concanavalin A, an agent which has been shown to inhibit endocytosis ( 19 ), abolished the inhibitory effect of H2O2. We conclude that addition of H2O2 inhibited SK channels by stimulating PTK activity, P38, and ERK in the CCD and that H2O2 enhances the internalization of the SK channels.

scholarly journals Activity of the p110-α subunit of phosphatidylinositol-3-kinase is required for activation of epithelial sodium transport

2008 ◽  
Vol 295 (3) ◽  
pp. F843-F850 ◽  
Author(s):  
Jian Wang ◽  
Zachary A. Knight ◽  
Dorothea Fiedler ◽  
Olusegun Williams ◽  
Kevan M. Shokat ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Collecting Duct ◽  
Phosphatidylinositol 3 Kinase ◽  
Α Subunit ◽  
Akt Phosphorylation ◽  
Pi3k Inhibitors ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Renal Collecting Duct ◽  
Phosphatidylinositol 3

The pathways implicated in the control of epithelial Na+ channel (ENaC)-dependent Na+ transport in renal collecting duct cells share substantial parallels with those implicated in insulin-regulated glucose metabolism. Notably, both are inhibited by wortmannin and LY294002 and signal through phosphatidylinositol-3-kinase (PI3K)-dependent kinases SGK1 and Akt. The inhibitor pattern is thought to reflect dependence on PI3K activity since wortmannin and LY294002 are both effective inhibitors of this kinase. However, these inhibitors block a variety of kinases from different families and lack specificity within the PI3K family. To begin to dissect more precisely the pathways required for signaling and for control of Na+ transport in renal collecting duct cells, we have examined the effect of a set of PI3K inhibitors, which selectively block distinct subsets of PI3K catalytic subunit isoforms. We have found that ENaC-dependent Na+ transport was blocked by inhibitors of the p110-α isoform of PI3K, but not by inhibitors of p110-β, -γ, or -δ. Inhibitors that block Na+ current also blocked SGK1 and Akt phosphorylation. In contrast to insulin-stimulated glucose uptake in muscle cells, p110-β inhibition did not enhance sensitivity to p110-α inhibition. These data support the conclusion that ENaC-dependent Na+ current is controlled exclusively by p110-α, the same isoform that is the principal mediator of insulin effects on glucose metabolism, and lacks any dependence on p110-β. These findings further underscore the extent to which Na+ and glucose regulation are intertwined and provide additional insight into the interconnections between diabetes and hypertension.

scholarly journals Cardiac small-conductance calcium-activated potassium channels in health and disease

2021 ◽  
Vol 473 (3) ◽  
pp. 477-489 ◽  
Author(s):  
Xiao-Dong Zhang ◽  
Phung N. Thai ◽  
Deborah K. Lieu ◽  
Nipavan Chiamvimonvat
Keyword(s):  
Ventricular Myocytes ◽  
Pulmonary Veins ◽  
Differential Sensitivity ◽  
Sk Channels ◽  
Sk Channel ◽  
Ventricular Cells ◽  
Intracellular Ca ◽  
Life Threatening ◽  
Health And Disease ◽  
Small Conductance

AbstractSmall-conductance Ca2+-activated K+ (SK, KCa2) channels are encoded by KCNN genes, including KCNN1, 2, and 3. The channels play critical roles in the regulation of cardiac excitability and are gated solely by beat-to-beat changes in intracellular Ca2+. The family of SK channels consists of three members with differential sensitivity to apamin. All three isoforms are expressed in human hearts. Studies over the past two decades have provided evidence to substantiate the pivotal roles of SK channels, not only in healthy heart but also with diseases including atrial fibrillation (AF), ventricular arrhythmia, and heart failure (HF). SK channels are prominently expressed in atrial myocytes and pacemaking cells, compared to ventricular cells. However, the channels are significantly upregulated in ventricular myocytes in HF and pulmonary veins in AF models. Interests in cardiac SK channels are further fueled by recent studies suggesting the possible roles of SK channels in human AF. Therefore, SK channel may represent a novel therapeutic target for atrial arrhythmias. Furthermore, SK channel function is significantly altered by human calmodulin (CaM) mutations, linked to life-threatening arrhythmia syndromes. The current review will summarize recent progress in our understanding of cardiac SK channels and the roles of SK channels in the heart in health and disease.

scholarly journals The Class II Phosphatidylinositol 3 kinase C2β Is Required for the Activation of the K+ Channel KCa3.1 and CD4 T-Cells

2009 ◽  
Vol 20 (17) ◽  
pp. 3783-3791 ◽  
Author(s):  
Shekhar Srivastava ◽  
Lie Di ◽  
Olga Zhdanova ◽  
Zhai Li ◽  
Santosha Vardhana ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Critical Role ◽  
Class Ii ◽  
K Channel ◽  
Channel Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

The Ca2+-activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2β (PI3K-C2β) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2β by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2β siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2β in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca2+ influx, whereas silencing of PI3K-C2β inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2β colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.

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