WNK1 activates large-conductance Ca2+-activated K+ channels through modulation of ERK1/2 signaling.
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ABSTRACT:
SUBMITTER: Liu Y
PROVIDER: S-EPMC4378107 | biostudies-literature | 2015 Apr
REPOSITORIES: biostudies-literature
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Project description:Small-conductance Ca2+-activated K+ (SK) channels are widely expressed in neuronal tissues where they underlie post-spike hyperpolarizations, regulate spike-frequency adaptation, and shape synaptic responses. SK channels constitutively interact with calmodulin (CaM), which serves as Ca2+ sensor, and with protein kinase CK2 and protein phosphatase 2A, which modulate their Ca2+ gating. By recording coupled activities of Ca2+ and SK2 channels, we showed that SK2 channels can be inhibited by neurotransmitters independently of changes in the activity of the priming Ca2+ channels. This inhibition involvesSK2-associated CK2 and results from a 3-fold reduction in the Ca2+ sensitivity of channel gating. CK2phosphorylated SK2-bound CaM but not KCNQ2-bound CaM, thereby selectively regulating SK2 channels. We extended these observations to sensory neurons by showing that noradrenaline inhibits SK current and increases neuronal excitability in aCK2-dependent fashion. Hence, neurotransmitter-initiated signaling cascades can dynamically regulate Ca2+ sensitivity of SK channels and directly influence somatic excitability.
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Project description:Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction is likely due to activation of inward currents in smooth muscle cells, and prolonged relaxation may be due to activation of small-conductance Ca(2+)-activated K(+) (SK) channels via P2Y1 receptors expressed by detrusor PDGF receptor (PDGFR)?(+) cells. We investigated whether other subtypes of P2Y receptors are involved in the activation of SK channels in PDGFR?(+) cells of detrusor muscles. Quantitative analysis of transcripts revealed that P2ry2, P2ry4, and P2ry14 are expressed in PDGFR?(+) cells of P2ry1-deficient/enhanced green fluorescent protein (P2ry1(-/-)/eGFP) mice at similar levels as in wild-type mice. UTP, a P2Y2/P2Y4 agonist, activated large outward currents in detrusor PDGFR?(+) cells. SK channel blockers and an inhibitor of phospholipase C completely abolished currents activated by UTP. In contrast, UTP activated nonselective cation currents in smooth muscle cells. Under current-clamp (current = 0), UTP induced significant hyperpolarization of PDGFR?(+) cells. MRS2500, a selective P2Y1 antagonist, did not affect UTP-activated outward currents in PDGFR?(+) cells from wild-type mice, and activation of outward currents by UTP was retained in P2ry1(-/-)/eGFP mice. As a negative control, we tested the effect of MRS2693, a selective P2Y6 agonist. This compound did not activate outward currents in PDGFR?(+) cells, and currents activated by UTP were unaffected by MRS2578, a selective P2Y6 antagonist. The nonselective P2Y receptor blocker suramin inhibited UTP-activated outward currents in PDGFR?(+) cells. Our data demonstrate that P2Y2 and/or P2Y4 receptors function, in addition to P2Y1 receptors, in activating SK currents in PDGFR?(+) cells and possibly in mediating purinergic relaxation responses in detrusor muscles.
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Project description:Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms.
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Project description:Silver has been widely used in various medical products because of its antibacterial properties. However, there is only limited information concerning silver-related cytotoxicity. In this study we show that Ag(+) at low nanomolar concentrations (<10 nM) strongly inhibits the activity of large-conductance Ca(2+)-activated K(+) channels (BK) (Slo1), a widely expressed and physiologically important potassium channel. The Ag(+) inhibition is caused by irreversible modification on cytosolically accessible parts of the BK channel. At least four intracellular cysteines are involved in this process. In addition, at least one of these key cysteines is not accessible to the bulkier thiolate-active reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide. One of the cysteine-less constructs generated in this study shows gating properties similar to wild-type BK channel but with much lower Ag(+) sensitivity, in which the Ag(+) modification rate was decreased by approximately 20-fold. The results from the present study suggest a possible contribution of BK channel inhibition to the cytotoxicity of Ag(+) in humans and other species.
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Project description:Carbon monoxide (CO) is an endogenous paracrine and autocrine gaseous messenger that regulates physiological functions in a wide variety of tissues. CO induces vasodilation by activating arterial smooth muscle large-conductance Ca2+-activated potassium (BK(Ca)) channels. However, the mechanism by which CO activates BK(Ca) channels remains unclear. Here, we tested the hypothesis that CO activates BK(Ca) channels by binding to channel-bound heme, a BK(Ca) channel inhibitor, and altering the interaction between heme and the conserved heme-binding domain (HBD) of the channel alpha subunit C terminus. Data obtained using thin-layer chromatography, spectrophotometry, mass spectrometry (MS), and MS-MS indicate that CO modifies the binding of reduced heme to the alpha subunit HBD. In contrast, CO does not alter the interaction between the HBD and oxidized heme (hemin), to which CO cannot bind. Consistent with these findings, electrophysiological measurements of native and cloned (cbv) cerebral artery smooth muscle BK(Ca) channels show that CO reverses BK(Ca) channel inhibition by heme but not by hemin. Site-directed mutagenesis of the cbv HBD from CKACH to CKASR abolished both heme-induced channel inhibition and CO-induced activation. Furthermore, on binding CO, heme switches from being a channel inhibitor to an activator. These findings indicate that reduced heme is a functional CO receptor for BK(Ca) channels, introduce a unique mechanism by which CO regulates the activity of a target protein, and reveal a novel process by which a gaseous messenger regulates ion channel activity.
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Project description:The cellular events that cause ischemic neurological damage following aneurysmal subarachnoid hemorrhage (SAH) have remained elusive. We report that subarachnoid blood profoundly impacts communication within the neurovascular unit-neurons, astrocytes, and arterioles-causing inversion of neurovascular coupling. Elevation of astrocytic endfoot Ca(2+) to ?400 nM by neuronal stimulation or to ?300 nM by Ca(2+) uncaging dilated parenchymal arterioles in control brain slices but caused vasoconstriction in post-SAH brain slices. Inhibition of K(+) efflux via astrocytic endfoot large-conductance Ca(2+)-activated K(+) (BK) channels prevented both neurally evoked vasodilation (control) and vasoconstriction (SAH). Consistent with the dual vasodilator/vasoconstrictor action of extracellular K(+) ([K(+)](o)), [K(+)](o) <10 mM dilated and [K(+)](o) >20 mM constricted isolated brain cortex parenchymal arterioles with or without SAH. Notably, elevation of external K(+) to 10 mM caused vasodilation in brain slices from control animals but caused a modest constriction in brain slices from SAH model rats; this latter effect was reversed by BK channel inhibition, which restored K(+)-induced dilations. Importantly, the amplitude of spontaneous astrocytic Ca(2+) oscillations was increased after SAH, with peak Ca(2+) reaching ?490 nM. Our data support a model in which SAH increases the amplitude of spontaneous astrocytic Ca(2+) oscillations sufficiently to activate endfoot BK channels and elevate [K(+)](o) in the restricted perivascular space. Abnormally elevated basal [K(+)](o) combined with further K(+) efflux stimulated by neuronal activity elevates [K(+)](o) above the dilation/constriction threshold, switching the polarity of arteriolar responses to vasoconstriction. Inversion of neurovascular coupling may contribute to the decreased cerebral blood flow and development of neurological deficits that commonly follow SAH.
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Project description:Zinc is an essential trace element and plays crucial roles in normal development, often as an integral structural component of transcription factors and enzymes. Recent evidence suggests that intracellular Zn(2+) functions as a signaling molecule, mediating a variety of important physiological phenomena. However, the immediate effectors of intracellular Zn(2+) signaling are not well known. We show here that intracellular Zn(2+) potently and reversibly activates large-conductance voltage- and Ca(2+)-activated Slo1 K(+) (BK) channels. The full effect of Zn(2+) requires His(365) in the RCK1 (regulator of conductance for K(+)) domain of the channel. Furthermore, mutation of two nearby acidic residues, Asp(367) and Glu(399), also reduced activation of the channel by Zn(2+), suggesting a possible structural arrangement for Zn(2+) binding by the aforementioned residues. Extracellular Zn(2+) activated Slo1 BK channels when coexpressed with Zn(2+)-permeable TRPM7 (transient receptor potential melastatin 7) channels. The results thus demonstrate that Slo1 BK channels represent a positive and direct effector of Zn(2+) signaling and may participate in sculpting cellular response to an increase in intracellular Zn(2+) concentration.
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Project description:The Ca2+-dependent gating mechanism of large-conductance calcium-activated K+ (BK) channels from cultured rat skeletal muscle was examined from low (4 microM) to high (1,024 microM) intracellular concentrations of calcium (Ca2+i) using single-channel recording. Open probability (Po) increased with increasing Ca2+i (K0. 5 11.2 +/- 0.3 microM at +30 mV, Hill coefficient of 3.5 +/- 0.3), reaching a maximum of approximately 0.97 for Ca2+i approximately 100 microM. Increasing Ca2+i further to 1,024 microM had little additional effect on either Po or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca2+i (>100 microM), compared with three to four open and five to seven closed states at lower Ca2+i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca2+i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca2+i. Kinetic schemes drawn from Eigen's general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca2+i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca2+i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca2+i. A model that assumed that the apparent Ca2+-binding steps can reach a maximum rate at high Ca2+i could also approximate the gating from low to high Ca2+i. The considered models can serve as working hypotheses for the gating of BK channels.
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Project description:The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.
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Project description:Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K(+) channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca(2+) -activated K(+) channels (BK(Ca)). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K(+) currents carried by BK(Ca) channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC(50) 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K(+) currents (~45% blockage) in which, under our working conditions, BK(Ca) is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK(Ca) channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.
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