Enhancement of BKCa channel activity induced by hydrogen peroxide: Involvement of lipid phosphatase activity of PTEN

Large-conductance calcium and voltage-dependent potassium (BK_Ca) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H₂O₂) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hβ1 were cotransfected with the PTEN_wt, PTEN_C124S, PTEN_G129E in HEK 293 cells. Typical BK_Ca channel currents could be recorded in cell-attached configurations. We found that PTEN_wt reduced the H₂O₂-induced BK_Ca channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTEN_C124S/PTEN_G129E mutants that lack lipid phosphatase activity produced no regulation on the H₂O₂-induced BK_Ca channel activation. These results demonstrated that PTEN regulated the H₂O₂-induced BK_Ca channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H₂O₂-induced BK_Ca channel activation was attenuated when cells were treated with H₂O₂ at concentrations higher than 100 μM or at 100 μM for long-term treatment. In addition, the p-AKT expression level in PTEN_wt overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca²⁺]_i) induced by H₂O₂ was also inhibited. These findings may elucidate a new mechanism for H₂O₂-induced BK_Ca channel activation and provide some evidences for the role of PTEN on vasodilation induced by H₂O₂.     

Apelin-13 Inhibits Large-Conductance Ca2+-Activated K+ Channels in Cerebral Artery Smooth Muscle Cells via a PI3-Kinase Dependent Mechanism

Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM) cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BK_Ca channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 μM) caused concentration-dependent inhibition of BK_Ca in VSM cells. Apelin-13 (0.1 µM) significantly decreased BK_Ca current density from 71.25±8.14 pA/pF to 44.52±7.10 pA/pF (n=14 cells, P<0.05). This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM) decreased the open-state probability (NP_o) of BK_Ca channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1µM) did not alter the NP_o of BK_Ca channels, suggesting that the inhibitory effect of apelin-13 on BK_Ca is not mediated by a direct action on BK_Ca. In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BK_Ca current density. In addition, treatment of arteries with apelin-13 (0.1 µM) significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BK_Ca channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone.     

Potentiation of large-conductance calcium-activated potassium (BK(Ca)) channels by a specific isoform of protein kinase C

The phosphorylation state of large-conductance calcium-activated potassium (BK_Ca) channels regulates their activity and is dynamically regulated by protein phosphatases and kinases, including protein kinase C (PKC). In this study, we showed that PKC activators up-regulate the activity of the BK_Ca channel alpha (α)-subunit, Slo1, in cell-attached patches of transfected COS7 cells. In an immune complex kinase assay, BK_Ca channels isolated from rat brain were phosphorylated in the presence of PKC activators, without the addition of exogenous PKC, which suggests that PKC and BK_Ca channels functionally interact in vivo. Four different PKC isozymes, including PKCδ, phosphorylated the C-terminus of Slo1 and the addition of purified PKCδ-activated BK_Ca channels in excised patches of transfected HEK293 cells. Our results demonstrate that PKC up-regulates BK_Ca channels and that PKCδ may functionally interact with BK_Ca channel complexes in vivo.     

Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.     

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca²⁺-activated K⁺ channel (BK_Ca) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BK_Ca channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K⁺ currents (STOC, a component pattern of BK_Ca currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BK_Ca currents) via an IP3 receptor- and sarcoplasmic Ca²⁺ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BK_Ca channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BK_Ca channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BK_Ca channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca²⁺ sensitivity of the channels.     

Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210

Abstract

Background. Insulin protects cardiomyocytes from reactive oxygen species (ROS)-induced apoptosis after ischemic/reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from ROS injury. Methods. Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H₂O₂) or insulin at various concentrations for various periods of time, or with insulin and H₂O₂ for various periods of time. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results. The percentage of viable cells was significantly and inversely associated with H₂O₂ concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H₂O₂ or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H₂O₂-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion. Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H₂O₂/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.     

Ginsenoside Rg<Subscript>3</Subscript> enhances large conductance Ca<Superscript>2+</Superscript>-activated potassium channel currents: A role of Tyr360 residue

Ginsenosides, active ingredients of Panax ginseng, are known to exhibit neuroprotective effects. Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are key modulators of cellular excitability of neurons and vascular smooth muscle cells. In the present study, we examined the effects of ginsenosides on rat brain BK_Ca (rSlo) channel activity heterologously expressed in Xenopus oocytes to elucidate the molecular mechanisms how ginsenoside regulates the BK_Ca channel activity. Ginsenoside Rg₃ (Rg₃) enhanced outward BK_Ca channel currents. The Rg₃-enhancement of outward BK_Ca channel currents was concentration-dependent, voltage-dependent, and reversible. The EC₅₀ was 15.1 ± 3.1 μM. Rg₃ actions were not desensitized by repeated treatment. Tetraetylammonium (TEA), a K⁺ channel blocker, inhibited BK_Ca channel currents. We examined whether extracellular TEA treatment could alter the Rg₃ action and vice versa. TEA caused a rightward shift of the Rg₃ concentration-response curve (i.e., much higher concentration of Rg₃ is required for the activation of BK_Ca channel compared to the absence of TEA), while Rg₃ caused a rightward shift of the TEA concentration-response curve in wild-type channels. Mutation of the extracellular TEA binding site Y360 to Y360I caused a rightward shift of the TEA concentration-response curve and almost abolished both the Rg₃ action and Rg₃-induced rightward shift of TEA concentration-response curve. These results indicate that Tyr360 residue of BK_Ca channel plays an important role in the Rg₃-enhancement of BK_Ca channel currents.     

Ca2+ dependence and pharmacology of large-conductance K+ channels in nonlabor and labor human uterine myocytes

Two populations,Ca²⁺-dependent(BK_Ca) andCa²⁺-independentK⁺ (BK) channels of largeconductance were identified in inside-out patches of nonlabor and laborfreshly dispersed human pregnant myometrial cells, respectively.Cell-attached recordings from nonlabor myometrial cells frequentlydisplayed BK_Ca channel openings characterized by a relatively low open-state probability, whereas similar recordings from labor tissue displayed either no channel openings or consistently high levels of channel activity that oftenexhibited clear, oscillatory activity. In inside-out patch recordings,Ba²⁺ (2-10 mM),4-aminopyridine (0.1-1 mM), andShaker B inactivating peptide("ball peptide") blocked theBK_Ca channel but were much lesseffective on BK channels. Application of tetraethylammonium toinside-out membrane patches reduced unitary current amplitude ofBK_Ca and BK channels, withdissociation constants of 46 mM and 53 µM, respectively.Tetraethylammonium applied to outside-out patches decreased the unitaryconductance of BK_Ca and BKchannels, with dissociation constants of 423 and 395 µM,respectively. These results demonstrate that the properties of humanmyometrial large-conductance K⁺channels in myocytes isolated from laboring patients are significantly different from those isolated from nonlaboring patients.

     

Liposomal quercetin potentiates maxi-K channel openings in smooth muscles and restores its activity after oxidative stress

The effects of quercetin-loaded liposomes (PCL-Q) and their constituents, that is, free quercetin (Q) and ‘empty’ phosphatidylcholine vesicles (PCL), on maxi-K channel activity were studied in single mouse ileal myocytes before and after H₂O₂-induced oxidative stress. Macroscopic Maxi-K channel currents were recorded using whole-cell patch clamp techniques, while single BK_Ca channel currents were recorded in the cell-attached configuration. Bath application of PCL-Q (100?μg/ml of lipid and 3?μg/ml of quercetin) increased single Maxi-K channel activity more than threefold, from 0.010?±?0.003 to 0.034?±?0.004 (n?=?5; p?<?0.05), whereas single-channel conductance increased non-significantly from 138 to 146?pS. In the presence of PCL-Q multiple simultaneous channel openings were observed, with up to eight active channels in the membrane patch. Surprisingly, ‘empty’ PCL (100?μg/ml) also produced some channel activation, although it was less potent compared to PCL-Q, that is, these increased NPo from 0.010?±?0.003 to 0.019?±?0.003 (n?=?5; p?<?0.05) and did not affect single-channel conductance (139?pS). Application of PCL-Q restored macroscopic Maxi-K currents suppressed by H₂O₂-induced oxidative stress in ileal smooth muscle cells. We conclude that PCL-Q can activate Maxi-K channels in ileal myocytes mainly by increasing channel open probability, as well as maintain Maxi-K-mediated whole-cell current under the conditions of oxidative stress. While fusion of the ‘pure’ liposomes with the plasma membrane may indirectly activate Maxi-K channels by altering channel’s phospholipids environment, the additional potentiating action of quercetin may be due to its better bioavailability.     

Canonical Transient Receptor Potential 6 (TRPC6), a Redox-regulated Cation Channel

This study examined the effect of H₂O₂ on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H₂O₂ significantly stimulated Ca²⁺ entry in a dose-dependent manner. Electrophysiological experiments showed that H₂O₂ significantly increased TRPC6 channel open probability and whole-cell currents. H₂O₂ also evoked a robust inward current in A7r5 vascular smooth muscle cells, which was nearly abolished by knockdown of TRPC6 using a small interfering RNA. Catalase substantially attenuated arginine vasopressin (AVP)-induced Ca²⁺ entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H₂O₂ response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide- and H₂O₂-induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-sn-glycerol effect on TRPC6 was significantly greater in the presence of H₂O₂. Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H₂O₂. Similarly, in cells transfected with TRPC6-EGFP, confocal microscopy showed a significant increase in fluorescence intensity in the region of the cell membrane and adjacent to the membrane. AVP also increased the fluorescence intensity on the surface of the cells co-transfected with TRPC6-EGFP and V1 receptor, and this response was inhibited by catalase. These data indicate that H₂O₂ activates TRPC6 channels via modification of thiol groups of intracellular proteins. This cysteine oxidation-dependent pathway not only stimulates the TRPC6 channel by itself but also sensitizes the channels to diacylglycerol and promotes TRPC6 trafficking to the cell surface.     

III. Ion Channel Expression in PMA-differentiated Human THP-1 Macrophages

Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K⁺ currents, I _DR, present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K⁺ channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K⁺ channel (I _IR) and a Ca²⁺-activated maxi-K channel (I _BK). I _IR was a classical inward rectifier, conducting large inward currents negative to E _K and very small outward currents. I _IR was blocked in a voltage-dependent manner by Cs⁺, Na⁺, and Ba²⁺, block increasing with hyperpolarization. Block by Na⁺ and Ba²⁺ was time-dependent, whereas Cs⁺ block was too fast to resolve. Rb⁺ was sparingly permeant. In cell-attached patches with high [K⁺] in the pipette, the single I _IR channel conductance was ∼30 pS and no outward current could be detected. I _BK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the conductance was ∼200–250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was observed, suggesting block by intracellular Na⁺. I _BK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship was shifted to more negative potentials by increased [Ca²⁺]. Macroscopic I _BK was blocked by external TEA⁺ with half block at 0.35 mm. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K⁺ channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold. Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed. Received: 19 September 1995/Revised: 14 March 1996     

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK_Ca channel regulation by IP₃ and IP₃Rs in rat and mouse cerebral artery smooth muscle cells. IP₃ activated BK_Ca channels both in intact cells and in excised inside-out membrane patches. IP₃ caused concentration-dependent BK_Ca channel activation with an apparent dissociation constant (K_d) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca²⁺ concentration ([Ca²⁺]_i; 10 µM). IP₃ also caused a leftward-shift in BK_Ca channel apparent Ca²⁺ sensitivity and reduced the K_d for free [Ca²⁺]_i from ∼20 to 12 µM, but did not alter the slope or maximal P_o. BAPTA, a fast Ca²⁺ buffer, or an elevation in extracellular Ca²⁺ concentration did not alter IP₃-induced BK_Ca channel activation. Heparin, an IP₃R inhibitor, and a monoclonal type 1 IP₃R (IP₃R1) antibody blocked IP₃-induced BK_Ca channel activation. Adenophostin A, an IP₃R agonist, also activated BK_Ca channels. IP₃ activated BK_Ca channels in inside-out patches from wild-type (IP₃R1^+/+) mouse arterial smooth muscle cells, but had no effect on BK_Ca channels of IP₃R1-deficient (IP₃R1^−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP₃R1 is located in close spatial proximity to BK_Ca α subunits. The IP₃R1 monoclonal antibody coimmunoprecipitated IP₃R1 and BK_Ca channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP₃R1 activation elevates BK_Ca channel apparent Ca²⁺ sensitivity through local molecular coupling in arterial smooth muscle cells.     

Interplay between Calmodulin and Phosphatidylinositol 4,5-Bisphosphate in Ca2+-induced Inactivation of Transient Receptor Potential Vanilloid 6 Channels

The epithelial Ca²⁺ channel transient receptor potential vanilloid 6 (TRPV6) undergoes Ca²⁺-induced inactivation that protects the cell from toxic Ca²⁺ overload and may also limit intestinal Ca²⁺ transport. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca²⁺, calmodulin (CaM), and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) in excised inside-out patches. The activity of TRPV6 strictly depended on the presence of PI(4,5)P₂, and Ca²⁺-CaM inhibited the channel at physiologically relevant concentrations. Ca²⁺ alone also inhibited TRPV6 at high concentrations (IC₅₀ = ∼20 μm). A double mutation in the distal C-terminal CaM-binding site of TRPV6 (W695A/R699E) essentially eliminated inhibition by CaM in excised patches. In whole cell patch clamp experiments, this mutation reduced but did not eliminate Ca²⁺-induced inactivation. Providing excess PI(4,5)P₂ reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P₂ did not inhibit binding of CaM to the C terminus of the channel. Overall, our data show a complex interplay between CaM and PI(4,5)P₂ and show that Ca²⁺, CaM, and the depletion of PI(4,5)P₂ all contribute to inactivation of TRPV6.     

Single Mechanosensitive and Ca2+-Sensitive Channel Currents Recorded from Mouse and Human Embryonic Stem Cells

Cell-attached and inside-out patch clamp recording was used to compare the functional expression of membrane ion channels in mouse and human embryonic stem cells (ESCs). Both ESCs express mechanosensitive Ca²⁺ permeant cation channels (MscCa) and large conductance (200 pS) Ca²⁺-sensitive K⁺ (BK_Ca2+) channels but with markedly different patch densities. MscCa is expressed at higher density in mESCs compared with hESCs (70 % vs. 3 % of patches), whereas the BK_Ca2+ channel is more highly expressed in hESCs compared with mESCs (~50 % vs. 1 % of patches). ESCs of both species express a smaller conductance (25 pS) nonselective cation channel that is activated upon inside-out patch formation but is neither mechanosensitive nor strictly Ca²⁺-dependent. The finding that mouse and human ESCs express different channels that sense membrane tension and intracellular [Ca²⁺] may contribute to their different patterns of growth and differentiation in response to mechanical and chemical cues.     

Mechanism of activation of K++ channels by minoxidil-sulfate in Madin-Darby canine kidney cells

Summary We studied the mechanism of K⁺⁺ channel activation by minoxidil-sulfate (MxSO₄) in fused Madin-Darby canine kidney (MDCK) cells. Patch-clamp techniques were used to assess single channel activity, and fluorescent dye techniques to monitor cell calcium. A Ca⁺²⁺-dependent inward-rectifying K⁺⁺ channel with slope conductances of 53±3 (negative potential range) and 20±3 pS (positive potential range) was identified. Channel activity is minimal in cell-attached patches. MxSO₄ initiated both transient channel activation and an increase of intracellular Ca⁺²⁺ (from 94.2±9.1 to 475±12.6 nmol/liter). The observation that K⁺⁺ channel activity of excised inside-out patches was detected only at Ca⁺²⁺ concentrations in excess of 10 mol/liter suggests the involvement of additional mechanisms during channel activation by MxSO₄.Transient K⁺⁺ channel activity was also induced in cell-attached patches by 10 mol/liter of the protein kinase C activator 1-oleoyl-2-acetyl-glycerol (OAG). OAG (10 mol/liter in the presence of 1.6 mmol/liter ATP) increased the Ca⁺² sensitivity of the K⁺ channel in inside-out patches significantly by lowering the K _mfor Ca⁺² from 100 mol/liter to 100 nmol/liter. The channel activation by OAG was reversed by the protein kinase inhibitor H8. Staurosporine, a PKC inhibitor, blocked the effect of MxSO₄ on K⁺ channel activation. We conclude that MxSO₄-induced K⁺ channel activity is mediated by the synergistic effects of an increase in intracellular Ca⁺² and a PKC-mediated enhancement of the K⁺ channel's sensitivity to Ca⁺².A. Schwab was recipient of a Feodor-Lynen-Fellowship from the Alexander von Humboldt-Stiftung. This work was supported by NIH grant DK 17433. The authors thank Nikon Instruments Partners in Research Program for their support and generous use of equipment during the course of this study. Minoxidil-sulfate was kindly provided by Upjohn, Kalamazoo, MI.     

Structural and biophysical determinants of single CaV3.1 and CaV3.2 T-type calcium channel inhibition by N2O

We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels Ca_V3.1 and Ca_V3.2 by nitrous oxide (N₂O). To identify functionally important channel structures, chimeras with reciprocal exchange of the N-terminal domains I and II and C-terminal domains III and IV were examined. In whole-cell recordings N₂O significantly inhibited Ca_V3.2, and – less pronounced – Ca_V3.1. A Ca_V3.2-prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cell-attached patches containing ≤3 channels N₂O reduced average peak current of Ca_V3.2 by decreasing open probability and open time duration. Effects on Ca_V3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single Ca_V3.1 channels were significantly less active than Ca_V3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within Ca_V3.2 (H191), are responsible for the subtype-prevalent N₂O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N₂O on Ca_V3.2. Such a fingerprint of single channels can help identifying the molecular nature of native channels. This is exemplified by a characterization of single channels expressed in human hMTC cells as functional homologues of recombinant Ca_V3.1.     

Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation

We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPβS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca²⁺-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca²⁺ induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca²⁺-containing but not in a Ca²⁺-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y₂ receptors and PI3K/Akt pathway activation involving Ca²⁺, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca²⁺ influx/CaM pathway.     

Cloning of large-conductance Ca-activated K channel α-subunits in mouse cardiomyocytes

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are widely distributed in cellular membranes of various tissues, but have not previously been found in cardiomyocytes. In this study, we cloned a gene encoding the mouse cardiac BK_Ca channel α-subunit (mCardBKa). Sequence analysis of the cDNA revealed an open reading frame encoding 1154 amino acids. Another cDNA variant, identical in amino acid sequence, was also identified by sequence analysis. The nucleotide sequences of the two mCardBKa cDNAs, type 1 (mCardBKa1) and type 2 (mCardBKa2), differed by three nucleotide insertions and one nucleotide substitution in the N-terminal sequence. The amino acid sequence demonstrated that mCardBKa was a unique BK_Ca channel α-subunit in mouse cardiomyocytes, with amino acids 41-1153 being identical to calcium-activated potassium channel SLO1 and amino acids 1-40 corresponding to BK_Ca channel subfamily M alpha member 1. These findings suggest that a unique BK_Ca channel α-subunit is expressed in mouse cardiomyocytes.     

Activation of cGMP-dependent protein kinase stimulates cardiac ATP-sensitive potassium channels via a ROS/calmodulin/CaMKII signaling cascade

Background

Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (K_ATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue.

Methods and Findings

Single-channel recordings of cardiac K_ATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type K_ATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H₂O₂ scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H₂O₂ also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of K_ATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions.

Conclusion

The present study provides novel evidence that PKG exerts dual regulation of cardiac K_ATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H₂O₂ in particular), calmodulin and CaMKII, alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins. The novel cGMP/PKG/ROS/calmodulin/CaMKII signaling pathway may regulate cardiomyocyte excitability by opening K_ATP channels and contribute to cardiac protection against ischemia-reperfusion injury.     

Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K⁺ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC₅₀= 5.3 mm). Raising the internal Ca²⁺ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V _1/2) of the K⁺ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca²⁺-activated (BK _Ca ) K⁺ channel with a conductance of 296 pS (130 mm K⁺ at both sides of the patch). V _1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca²⁺, respectively, at the internal face of the patch. In cell-attached patches the open probability (P _o ) of BK _Ca channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K⁺ currents with 10 nm Ca²⁺ in the pipette. Application of 20 μm cytochalasin D increased P _o of BK _Ca channels in cell-attached patches within minutes. These data suggest that the activation of BK _Ca channels in meningioma cells does not only depend on voltage and internal Ca²⁺ but is also controlled by the cytoskeleton. Received 18 June 1999/Revised: 18 January 2000