Ca2+-activated K+ channel-associated phosphatase and kinase activities during development

2005 ◽  
Vol 289 (1) ◽  
pp. H414-H425 ◽  
Author(s):  
Mike T. Lin ◽  
Lawrence D. Longo ◽  
William J. Pearce ◽  
David A. Hessinger
Keyword(s):  
Protein Kinase ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Developmentally Regulated ◽  
Ion Channel Regulation ◽  
Differential Phosphorylation

In ovine basilar arterial smooth muscle cells (SMCs), the fetal “big” Ca2+-activated K+ (BK) channel activity is significantly greater and has a lower Ca2+ setpoint than BK channels from adult cells. In the present study, we tested the hypothesis that these differences result from developmentally regulated phosphorylation of these channels. Using the patch-clamp technique and a novel in situ enzymological approach, we measured the rates and extents of changes in BK channel voltage activation from SMC inside-out patch preparations in response to selective activation and inhibition of channel-associated protein phosphatases and kinases (CAPAKs). We show that BK channel activity is modulated during development by differential phosphorylation and that the activities of CAPAKs change substantially during development. In particular, excised membrane patches from adult SMCs exhibited greater protein kinase A activity than those from a fetus. In contrast, fetal SMCs exhibited greater protein kinase G activity and phosphatase activity than adult SMCs. These findings extend our previous observation that the BK channel Ca2+ setpoint differs significantly in adult and fetal cerebrovascular myocytes and suggest a biochemical mechanism for this difference. In addition, these findings suggest that the functional stoichiometry of CAPAKs varies significantly during development and that such variation may be a hitherto unrecognized mechanism of ion channel regulation.

scholarly journals Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

2014 ◽  
Vol 306 (5) ◽  
pp. C460-C470 ◽  
Author(s):  
Kiril L. Hristov ◽  
Amy C. Smith ◽  
Shankar P. Parajuli ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Regulation ◽  
Pkc Activation

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility.

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.

scholarly journals OSR1 and SPAK Sensitivity of Large-Conductance Ca2+ Activated K+ Channel

2016 ◽  
Vol 38 (4) ◽  
pp. 1652-1662 ◽  
Author(s):  
Bernat Elvira ◽  
Yogesh Singh ◽  
Jamshed Warsi ◽  
Carlos Munoz ◽  
Florian Lang
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Cell Volume Regulation ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Channel Activity ◽  
Wild Type ◽  
Constitutively Active

Background/Aims: The oxidative stress-responsive kinase 1 (OSR1) and the serine/threonine kinases SPAK (SPS1-related proline/alanine-rich kinase) are under the control of WNK (with-no-K [Lys]) kinases. OSR1 and SPAK participate in diverse functions including cell volume regulation and neuronal excitability. Cell volume and neuronal excitation are further modified by the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels). An influence of OSR1 and/or SPAK on BK channel activity has, however, never been shown. The present study thus explored whether OSR1 and/or SPAK modify the activity of BK channels. Methods: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively active T185EOSR1, catalytically inactive D164AOSR1, constitutively active T233ESPAK or catalytically inactive D212ASPAK. K+ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly decreased by co-expression of OSR1 or SPAK. The effect of wild-type OSR1/SPAK was mimicked by T185EOSR1 and T233ESPAK, but not by D164AOSR1 or D212ASPAK. Conclusions: OSR1 and SPAK suppress BK channels, an effect possibly contributing to cell volume regulation and neuroexcitability.

scholarly journals 14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel

2020 ◽  
Vol 319 (1) ◽  
pp. F52-F62
Author(s):  
Shan Chen ◽  
Xiuyan Feng ◽  
Xinxin Chen ◽  
Zhizhi Zhuang ◽  
Jia Xiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Specific Inhibitor ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Protein Ubiquitination ◽  
293 Cells

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal 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.

β1-Subunits are required for regulation of coupling between Ca2+ transients and Ca2+-activated K+ (BK) channels by protein kinase C

2003 ◽  
Vol 285 (5) ◽  
pp. C1270-C1280 ◽  
Author(s):  
Brian M. Hagen ◽  
Orline Bayguinov ◽  
Kenton M. Sanders
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Coupling Strength ◽  
Bk Channels ◽  
Bk Channel ◽  
Cation Channels ◽  
Patch Clamp Technique ◽  
Kinase C ◽  
Nonselective Cation Channels ◽  
Gf 109203X

Colonic myocytes have spontaneous, localized, Ins ( 1 , 4 , 5 ) trisphosphate (IP3) receptor-dependent Ca2+ transients that couple to the activation of Ca2+-dependent K+ channels and spontaneous transient outward currents (STOCs). We previously reported that the coupling strength between spontaneous Ca2+ transients and large conductance Ca2+ activated K+ (BK) channels is regulated by Ca2+ influx through nonselective cation channels and activation of protein kinase C (PKC). Here, we used confocal microscopy and the patch-clamp technique to further investigate the coupling between localized Ca2+ transients and STOCs in colonic myocytes from animals lacking the regulatory β1-subunit of BK channels. Myocytes from β1-knockout (β1-/-) animals loaded with fluo 4 showed typical localized Ca2+ transients, but the STOCs coupled to these events were of abnormally low amplitude. Reduction in external Ca2+ or application of inhibitors of nonselective cation channels (SKF-96365) caused no significant change in the amplitude or frequency of STOCs. Likewise, an inhibitor of PKC, GF 109203X, had no significant effect on STOCs. Single-channel recording from BK channels showed that application of an activator (PMA) and an inhibitor (GF 109203X) of PKC did not affect BK channel openings in myocytes of β1-/- mice. These data show that PKC-dependent regulation of coupling strength between Ca2+ transients and STOCs in colonic myocytes depends upon the interaction between α- and β1-subunits.

scholarly journals Up-Regulation of the Large-Conductance Ca2+-Activated K+ Channel by Glycogen Synthase Kinase GSK3β

2016 ◽  
Vol 39 (3) ◽  
pp. 1031-1039 ◽  
Author(s):  
Myriam Fezai ◽  
Musaab Ahmed ◽  
Zohreh Hosseinzadeh ◽  
Florian Lang
Keyword(s):  
Cell Volume ◽  
Glycogen Synthase ◽  
Negative Control ◽  
Bk Channels ◽  
Bk Channel ◽  
Glycogen Synthase Kinase ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Channel Activity ◽  
Wild Type

Background/Aims: The pleotropic functions of the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels) include regulation of neuronal excitation and cell volume. Kinases participating in those functions include the glycogen synthase kinase GSK3 ß which is under negative control of protein kinase B (PKB/Akt). GSK3ß is inhibited by the antidepressant Lithium. The present study thus explored whether GSK3ß modifies the activity of BK channels. Methods: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type GSK3ß, inactive K85RGSK3ß, or wild-type GSK3ß with wild-type PKB. K+ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly increased by co-expression of GSK3ß, but not by co-expression of K85RGSK3ß. The effect of wild type GSK3ß was abrogated by additional co-expression of wild-type PKB and by 24 hours incubation with Lithium (1 mM). Disruption of channel insertion into the cell membrane by brefeldin A (5 µM) was followed by a decline of the current to a similar extent in oocytes expressing BK and GSK3ß and in oocytes expressing BK alone. Conclusion: GSK3ß may up-regulate BK channels, an effect disrupted by Lithium or additional expression of PKB and possibly participating in the regulation of cell volume and excitability.

Inhibition of slow-wave repolarization and Ca(2+)-activated K+ channels by quaternary ammonium ions

1993 ◽  
Vol 264 (3) ◽  
pp. C625-C631 ◽  
Author(s):  
A. Carl ◽  
B. W. Frey ◽  
S. M. Ward ◽  
K. M. Sanders ◽  
J. L. Kenyon
Keyword(s):  
Smooth Muscle ◽  
Binding Site ◽  
Slow Wave ◽  
Voltage Dependence ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Circular Smooth Muscle

We studied the effects of the K+ channel blocker tetrapentylammonium (TPeA) on the electrical activity of intact circular smooth muscle from canine colon. TPeA (10 and 20 microM) increased slow-wave duration and "locked" the membrane potential around -30 mV plateau potential after several minutes of application, suggesting that K+ channels are essential for termination of colonic slow waves. Repolarization and normal slow-wave activity resumed after 20-30 min of washout. The patch-clamp technique was used to study the block of large-conductance Ca(2+)-activated K+ channels (BK channels) by TPeA and tetraethylammonium (TEA) in excised and cell-attached patches from isolated colonic smooth muscle cells. Channel block was characterized by a voltage-dependent dissociation constant [Kd(V)] for the binding of TEA and TPeA to a blocking site located a fraction of the distance across the membrane field (delta). The extracellular TEA binding site had a Kd(0) of 0.33 mM and a delta of 0.23. The extracellular TPeA binding site had a Kd(0) of 2.2 mM but showed significantly less voltage dependence (delta = 0.02). The intracellular binding site for TEA was of low affinity [Kd(0) = 76 mM]. Intracellular TPeA was the most potent blocker of BK channel current [Kd(0) = 11.7 microM]. The voltage dependence of block by intracellular TPeA (delta = -0.21) was not significantly different from that of intracellular TEA (delta = -0.3). Internal TPeA (10 microM) also blocked a 70-pS K+ channel and a 23-pS K+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca2+-activated K+ channel

2012 ◽  
Vol 302 (9) ◽  
pp. C1361-C1370 ◽  
Author(s):  
Wenkuan Xin ◽  
Qiuping Cheng ◽  
Rupal P. Soder ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
Dependent Manner ◽  
Detrusor Smooth Muscle ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Pde Inhibition

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca2+-activated K+ (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ∼3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ∼2-fold and hyperpolarized the DSM cell resting membrane potential by ∼6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca2+ sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.

Modulation of BK channel calcium affinity by differential phosphorylation in developing ovine basilar artery myocytes

2006 ◽  
Vol 291 (2) ◽  
pp. H732-H740 ◽  
Author(s):  
Mike T. Lin ◽  
David A. Hessinger ◽  
William J. Pearce ◽  
Lawrence D. Longo
Keyword(s):  
Basilar Artery ◽  
Dissociation Constants ◽  
Age Groups ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Fetal Sheep ◽  
Calcium Affinity ◽  
Differential Phosphorylation ◽  
Voltage Activation

Large-conductance Ca2+-sensitive K+ (BK) channel activity is greater in basilar artery smooth muscle cells (SMCs) of the fetus than the adult, and this increased activity is associated with a lower BK channel Ca2+ set point (Ca0). Associated PKG activity is three times greater in BK channels from fetal than adult myocytes, whereas associated PKA activity is three times greater in channels from adult than fetal myocytes. We hypothesized that the change in Ca0 during development results from different levels of channel phosphorylation. In inside-out membrane patch preparations of basilar artery SMCs from adult and fetal sheep, we measured BK channel activity in four states of phosphorylation: native, dephosphorylated, PKA phosphorylated, and PKG phosphorylated. BK channels from adult and fetus exhibited similar voltage-activation curves, Ca0 values, and Ca2+ dissociation constants ( Kd) for the dephosphorylated, PKA phosphorylated, and PKG phosphorylated states. However, voltage-activation curves of native fetal BK channels shifted significantly to the left of those of the adult, with Ca0 and Kd values half those of the adult. For the two age groups at each of the phosphorylation states, Ca0 and Kd produced linear relations when plotted against voltage at half-maximal channel activation. We conclude that the Ca0 and Kd values of the BK channel can be modulated by differential channel phosphorylation. Lower Ca0 and Kd values in BK channels of fetal myocytes can be explained by a greater extent of channel phosphorylation of fetal than adult myocytes.

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