Transient outwardly rectifying potassium channel in the rabbit corneal endothelium

Summary Ionic currents from freshly dissociated rabbit corneal endothelial cells were examined using patch-clamp technology and a perforated patch technique. Whole-cell current recordings revealed a transient outward K⁺-selective current that was blockable in a dose-dependent manner by 4-aminopyridine (4-AP) and quinidine. This current is similar to the A-type current present in many excitable cells and is the first reported instance of such a current in any epithelial cell type. In addition to the transient current, an outwardly rectifying nonselective cation current was also observed. This current is also blocked by quinidine.To examine the possible role of these currents in the stromal volume regulatory function of the endothelium, corneas were perfused under a specular microscope with a glutathionebicarbonate Ringer's solution (GBR) or GBR plus either 1 mM quinidine or 10 mM 4-AP. For quinidine perfusions, control corneas swelled at a rate of 6 m/hr, while quinidine-perfused corneas swelled at a rate of 48 m/hr. For 4-AP perfusions, control corneas deswelled at a rate of –2 m/hr, while 4-AP perfused corneas swelled at a rate of 24 m/hr. One possible mechanism of the stromal swelling induced by these K⁺ channel blockers may be the result of loss of the K⁺ recycling pathway necessary for proper Na⁺/K⁺ ATPase function.We would like to thank Dr. William Bourne for the use of his specular microscopy corneal perfusion apparatus and Helen Hendrickson for her technical assistance. This work was supported by NIH grants EY06206, EY03282, EY06005, and an unrestricted award from Research to Prevent Blindness.     

Genistein inhibits the inward rectifying potassium current in guinea pig ventricular myocytes

Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. Previous studies have shown that genistein has additional effects, among which the direct blocking effects on various ionic channels have recently been disclosed. Using whole-cell voltage clamp and current clamp techniques, we demonstrate that micromolar concentrations of genistein dose-dependently and reversibly inhibit the inward rectifying K(+) current, and depolarize the resting membrane potential, resulting in abnormal automaticity in guinea pig ventricular myocytes. Interestingly, another potent tyrosine kinase inhibitor, tyrphostin 51, did not produce the same inhibitory effect, while the inactive analogue of genistein, daidzein, had a similar blocking effect. We suggest that genistein directly blocks the inward rectifying K(+) current in ventricular myocytes, and one should be cautious of its pro-arrhythmic effect in clinical use.     

组胺拮抗剂对小鼠ATP-敏感性钾离子通道的影响

目的观察比较3种组胺拮抗剂对缺血性心肌细胞的ATP-敏感性钾离子通道中的影响。方法利用急性酶解法分离小鼠心室肌细胞。结果组胺拮抗剂pyrilamine、chlorpheniramine及diphenhydramine均可抑制ATP-敏感性钾离子通道的活性,抑制程度为pyrilamine〉chlorpheniramine〉diphenhydramine。组胺对KATP通道活性无影响。结论第一代的组胺拮抗剂（pyrilamine、chlorpheniramine及diphenhydramine）对KATP通道活性有抑制作用,其抑制作用与膜上H1受体无关。     

TIMP-1 inhibits apoptosis in breast carcinoma cells via a pathway involving pertussis toxin-sensitive G protein and c-Src

In addition to inhibiting matrix metalloproteinases, tissue inhibitor of metalloproteinase-1 (TIMP-1) is involved in the regulation of cell growth and survival. To determine its mechanism of action, we investigated effects of TIMP-1 on cell proliferation and survival and signaling pathways induced by TIMP-1 in the human breast carcinoma T-47D cell line. Treatment of T-47D cells with TIMP-1 strongly inhibited apoptosis induced by serum deprivation, but did not affect cell proliferation. TIMP-1 induced phosphorylation of Akt and extracellular signal-regulated protein kinases (ERKs), but pertussis toxin and specific inhibitors of Src family tyrosine kinases, protein tyrosine kinases, and phosphatidylinositol-3 kinase (PI3 kinase) blocked the ability of TIMP-1 to activate Akt and ERKs as well as the anti-apoptotic effect of TIMP-1. We found that TIMP-1 enhanced the kinase activities of c-Src and PI3 kinase and that this enhancement was inhibited by pertussis toxin. Inhibition of ERK activation, however, resulted in a slight decrease of the TIMP-1-induced anti-apoptotic effect. These findings demonstrate that the ability of TIMP-1 to inhibit apoptosis in T-47D cells is mediated by the sequential activation of pertussis toxin-sensitive G protein, c-Src, PI3 kinase, and Akt.     

Activation of the endothelin receptor inhibits the G protein-coupled inwardly rectifying potassium channel by a phospholipase A2-mediated mechanism

To develop a malleable system to model the well-described, physiological interactions between Gq/11 - coupled receptor and Gi/o-coupled receptor signaling, we coexpressed the endothelin A receptor, the mu-opioid receptor, and the G protein-coupled inwardly rectifying potassium channel (Kir 3) heteromultimers in Xenopus laevis oocytes. Activation of the Gi/o-coupled mu-opioid receptor strongly increased Kir 3 channel current, whereas activation of the Gq/11-coupled endothelin A receptor inhibited the Kir 3 response evoked by mu-opioid receptor activation. The magnitude of the inhibition of Kir 3 was channel subtype specific; heteromultimers composed of Kir 3.1 and Kir 3.2 or Kir 3.1 and Kir 3.4 were significantly more sensitive to the effects of endothelin-1 than heteromultimers composed of Kir 3.1 and Kir 3.5. The difference in sensitivity of the heteromultimers suggests that the endothelin-induced inhibition of the opioid- activated current was caused by an effect at the channel rather than at the opioid receptor. The endothelin-1-mediated inhibition was mimicked by arachidonic acid and blocked by the phospholipase A2 inhibitor arachidonoyl trifluoromethyl ketone. Consistent with a possible phospholipase A2-mediated mechanism, the endothelin-1 effect was blocked by calcium chelation with BAPTA-AM and was not affected by kinase inhibition by either staurosporine or genistein. The data suggest the hypothesis that Gq/11-coupled receptor activation may interfere with Gi/o-coupled receptor signaling by the activation of phospholipase A2 and subsequent inhibition of effector function by a direct effect of an eicosanoid on the channel.     

Decrease in acetylcholine-induced current by neomycin in PC12 cells

The effects of neomycin, one of the aminoglycoside antibiotics, on the acetylcholine (ACh)-induced current (I(ACh)) were studied in pheochromocytoma cells by using the whole-cell clamp technique. The I(ACh) proved to be generated through neuronal nicotinic receptor. ACh (30 microM) induced an inward current at a holding potential of -80 mV. When cells were treated with neomycin (0.01-1 mM) and ACh (30 microM) simultaneously, an inhibitory effect of neomycin on the peak of I(ACh) was found. This effect was fast, reversible, and concentration dependent. Pretreatment with neomycin for 3-8 min had no effect on the inhibition of I(ACh) induced by neomycin. External application of 0.1 mM neomycin neither shifted the dose-response curve of the peak I(ACh) to the right (dissociation constant (K(d)) = 16.5 microM) nor affected its coefficient (1.8) but inhibited the curve amplitudes by approximately 33%. Stimulated protein kinase C activation by using an exogenous activator produced inhibition of I(ACh), while using protein kinase C inhibitor (PKCI 19-31) had no effect on the inhibition of I(ACh) induced by neomycin. These results suggest that neomycin has an inhibitory effect on I(ACh) without the involvement of phospholipase C. It indicates that neomycin binds to a specific site on the cell membrane, probably on the neuronal nicotinic receptor-coupled channel, and inhibits the I(ACh) in a noncompetitive manner, thus controlling the immediate catecholamine release from the sympathetic cells.     

新霉素抑制PC12细胞烟碱受体介导的反应

新霉素是一种氨基甙类抗生素,在细胞水平可以抑制磷脂酶C介质的信号转导系统,本研究采用全细胞膜片钳技术,以大鼠肾上腺嗜铬细胞瘤细胞（PC12)为标本,观察了新霉素参考书国酰胆碱诱发电流（IACh)的影响,药理学鉴定表明,PC12细胞上的IACh是通过ACh激动烟碱受体引起的,钳制电压为－80mV时,ACh(30umol/L)诱发一内向电流;细胞外同时给予新霉素（0．01－1mmol/L)和ACh(30μmol/L)可显著抑制IACh峰值,此抑制作用迅速,可逆,呈浓度依赖性,用新霉素预处理细胞3－8min不影响其对IACh的抑制作用,用外源性蛋白激酶C（PKC）激剂激活PKC,同样可抑制IACh,而细胞内透析PKC抑制剂（PKCI19-31,0.1-5μmol/L）不影响新霉素对IACh的抑制作用,以上结果提示,新霉对PC12细胞的IACh的有抑制作用,这是一种与磷脂酶C阻断无关的药理学效应。     

慢性间歇性低氧降低急性缺氧对大鼠肺动脉平滑肌细胞膜上电压门控性钾通道的抑制

为了从离子通道水平上探讨机体低氧适应的离子机制,本实验将雄性 SD 大鼠随机分为常氧对照组和慢性间歇性低氧组[氧浓度(10 ± 0.5) %, 间断缺氧每天 8 h]。用酶解法急性分离单个大鼠肺内动脉平滑肌细胞(pulmonary artery smoothmuscle cells, PASMCs),以全细胞膜片钳技术记录 PASMCs 膜上的电压门控性钾通道 (voltage-gated potassium channel, KV) 电流,观察急性缺氧对慢性间歇性低氧大鼠 PASMCs 的 KV 的影响, 为机体适应低氧能力提供实验依据。结果显示:⑴常氧对照组在电流钳下,急性缺氧可使膜电位明显去极化(由-47.2 ±2.6 mV 去极到 -26.7 ±1.2 mV ); 在电压钳下, 急性缺氧可显著抑制 KV电流( 60 mV 时, KV电流密度从 153.4 ± 9.5 pA/pF降到 70.1 ± 10.6 pA/pF), 峰电流的抑制率为(57.6 ± 3.3) %, 电流-电压关系曲线向右下移。⑵慢性间歇性低氧组KV电流密度随低氧时间延长而逐渐减少(慢性低氧10 d后就有显著性意义),电流- 电压关系曲线逐渐右下移。⑶急性缺氧对慢性间歇性低氧大鼠PASMCs KV电流的抑制作用随慢性间歇性低氧时间延长而逐渐减弱。上述观察结果提示慢性间歇性低氧减弱急性缺氧对 KV 的抑制, 这可能是机体低氧适应的一种重要机制。     

Activation of M3 muscarinic receptors inhibits T-type Ca(2+) channel currents via pertussis toxin-sensitive novel protein kinase C pathway in small dorsal root ganglion neurons

Cobrotoxin (CbT), a short-chain postsynaptic α-neurotoxin, has been reported to play a role in analgesia. However, to date, the detailed mechanisms still remain unknown. In the present study, we identify a novel functional role of CbT in modulating T-type Ca²⁺ channel currents (T-currents) in small dorsal root ganglia (DRG) neurons as well as pain behaviors in mice. We found that CbT inhibited T-currents in a dose-dependent manner. CbT at 1 μM reversibly inhibited T-currents by ~ 26.3%. This inhibitory effect was abolished by the non-selective muscarinic acetylcholine receptor (mAChR) antagonist atropine, or the selective M3 mAChR antagonist 4-DAMP, while naloxone, an opioid receptor antagonist had no effect. Intracellular infusion of GDP-β-S or pretreatment of the cells with pertussis toxin (PTX) completely blocked the inhibitory effects of CbT. Using depolarizing prepulse, we found the absence of direct binding between G-protein βγ subunits and T-type Ca²⁺ channels in CbT-induced T-current inhibition. CbT responses were abolished by the phospholipase C inhibitor U73122 (but not the inactive analog U73343). The classical and novel protein kinase C (nPKC) antagonist chelerythrine chlorid or GF109203X abolished CbT responses, whereas the classical PKC antagonist Ro31-8820 or inhibition of PKA elicited no such effects. Intrathecal administration of CbT (5 μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models. Moreover, CbT-induced antinociception could be abrogated by 4-DAMP. Taken together, these results suggest that CbT acting through M3 mAChR inhibits T-currents via a PTX-sensitive nPKC pathway in small DRG neurons, which could contribute to its analgesic effects in mice.     

Overexpression of Kir2.3 in PC12 cells resists rotenone-induced neurotoxicity associated with PKC signaling pathway

Parkinson’s disease (PD) can be triggered by genetic or environmental factors. Although the precise etiopathogenesis of the disease remains unknown, recent studies focusing on the K⁺ channel gene have uncovered the dysfunctions in various K⁺ channels (e.g., Kir2, Kv, K_ATP, and SKCa) that are involved in the pathological mechanisms underlying PD. Here we show that Kir2.3 overexpression can protect against rotenone-induced apoptosis in cell models in the neurodegenerative process, suggesting Kir2.3’s general neuroprotective function. The protection of Kir2.3 against neurodegeneration may be associated with the protein kinase C (PKC) pathway, as PKC is downregulated by Kir2.3 overexpression and the PKC activator can reduce the protective effect of Kir2.3. Our studies provide an entry point for understanding the novel roles of Kir2.3 in cell models of PD, and they offer clues for the common mechanisms underlying different neurodegenerative conditions.     

Endothelin-1 and insulin activate the steady-state voltage dependent R-type Ca2+ channel in aortic smooth muscle cells via a pertussis toxin and cholera toxin sensitive G-protein

In single rabbit aortic smooth muscle cells, and at a concentration known to induce a maximum sustained increase of intracellular Ca²⁺ via activation of the steady-state voltage dependent R-type Ca²⁺ channels, endothelin-1 (10^-7 M) and insulin (80 U/ml) were found to induce a sustained increase in cytosolic free Ca²⁺ ([Ca]_i) levels that was significantly attenuated by pre-treatment with either pertussis toxin (PTX), cholera toxin (CTX) or removal of extracellular Ca²⁺.However, both PTX and CTX failed to inhibit the sustained depolarization-evoked sustained Ca²⁺ influx and [Ca]_i elevation via activation of the R-type Ca²⁺ channels. Moreover, ET-1 and insulin-evoked sustained increases in Ca²⁺ influx were not attenuated by the selective PKC inhibitor, bisindolylmaleimide (BIS), or the specific L-type Ca²⁺ channel blocker, nifedipine, but were completely reversed by the R-type Ca²⁺ channel blocker, (-) PN 200-110 (isradipine). These data suggest that both insulin and ET-1 activate the nifedipine-insensitive but isradipine-sensitive steady state voltage dependent R-type Ca²⁺ channels present on rabbit VSMCs and these channels are directly coupled to PTX and CTX sensitive G protein(s).     

Single-channel analysis of the potassium permeability in HeLa cancer cells: Evidence for a calcium-activated potassium channel of small unitary conductance

Summary Cell-attached and inside-out patch-clamp experiments (O.P. Hamill et al.,Pfluegers Arch. 391: 85–100, 1981) were undertaken in order to characterize the molecular mechanisms responsible for the calcium-dependent potassium permeability observed in HeLa cancer cells. Our result essentially indicate that the HeLa cell external membrane contains potassium channels whose activity can be triggered within an internal calcium concentration range of 0.1 to 1 m. This particular channel was found to behave as an inward rectifier in symmetrical 200mm KCl with a conductance of 50 and 10 pS at large negative and large positive membrane potentials, respectively.I/V curves were also measured in 10, 20, 75, 200 and 300mm KCl and the data interpreted in terms of a one-site-two-barrier model. The channel activity appeared to be nearly voltage independent within the voltage range –100 to +100mV, an increase ofP _o, the open channel probability, being observed at large negative potentials only. In addition, the results obtained from inside-out experiments on the relationship betweenP _o and the cytoplasmic freecalcium concentration have led to conclude that four calcium ions are probably required in order to open the channel. In this regard it was found that an increase of the internal free-calcium level affects more the number of channel openings per second than the actual channel mean lifetime. Finally, it is concluded following a time interval distribution analysis, that this particular channel has at least three closed states and two open states.     

Estradiol rapidly induces the translocation and activation of the intermediate conductance calcium activated potassium channel in human eccrine sweat gland cells

Background and aims

Steroid hormones target K⁺ channels as a means of regulating electrolyte and fluid transport. In this study, ion transporter targets of Estradiol (E2) were investigated in the human eccrine sweat gland cell line NCL-SG3.

Results

Whole cell patch-clamp studies revealed E2 (10 nM) rapidly activates a whole cell K⁺ conductance, which is abolished by clotrimazole (30 μM), an inhibitor of the intermediate conductance calcium activated K⁺ channel (IK_Ca). The estrogen receptor (ER) antagonist ICI 182, 780 had no effect on this E2 activated K⁺ conductance, suggesting an estrogen receptor independent mechanism of activation. Confocal microscopy studies revealed under basal conditions that the IK_Ca channel is located within the cell cytoplasm and in the presence of E2, rapidly translocates to both the apical and basolateral membrane. In the presence of E2, tyrosine phosphorylation of calmodulin, which is known to regulate trafficking of the IK_Ca channel, is increased, and treatment of cells with the calmodulin inhibitor trifluoperazine (TFP) prevents the E2-induced translocation.

Conclusions

Estradiol rapidly regulates a K⁺ conductance through the IK_Ca channel in an estrogen receptor independent manner. E2 stimulates the translocation of IK_Ca to the cell membrane in a calmodulin dependent manner, representing a novel paradigm of estrogen action in sweat gland epithelial cells.     

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK-OV3 ovarian cancer cells and OVCAR-3 ovarian cancer cells were responsive to LPE. LPE-stimulated intracellular calcium concentration ([Ca(2+)](i)) increase was inhibited by U-73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca(2+)](i) increase by LPE, indicating the involvement of PTX-sensitive G-proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE-stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B-driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells.     

Muscarinic receptor stimulation induces TASK1 channel endocytosis through a PKC-Pyk2-Src pathway in PC12 cells

Muscarinic receptor stimulation or protein kinase C (PKC) activation in rat adrenal medullary and PC12 cells rapidly induces tyrosine phosphorylation of TWIK-related-acid-sensitive K⁺ 1 (TASK1) channels with the subsequent clathrin-dependent endocytosis. Our previous study suggested that the muscarinic signal is transmitted to the non-receptor tyrosine kinase Src through PKC and Pyk2. Although PKC activation is known to stimulate Pyk2 in certain types of cells, its molecular mechanism remains unclear. In this study, proximity ligation assay (PLA) and other molecular biological approaches were used to elucidate the details of this muscarinic signaling in PC12 cells. When green fluorescent protein (GFP)-TASK1 was expressed, the majority of GFP-TASK1 was located at the cell periphery. However, the simultaneous expression of GFP-TASK1 and PKCα, but not PKCδ, led to GFP-TASK1 internalization. Muscarinic receptor stimulation resulted in transient co-localization of Pyk2 and Src at the cell periphery, and expression of kinase dead (KD) Pyk2 and Src, but not Pyk2 and KD Src, resulted in GFP-TASK1 internalization. PLA analysis revealed that in response to muscarine, PKCα activates Pyk2 through phosphorylating its serine residues. These results indicate that muscarinic receptor stimulation induces TASK1 channel endocytosis sequentially through PKCα, Pyk2, and Src, and PKCα activates Pyk2 through phosphorylation.     

Calcitriol inhibits Ether-à go-go potassium channel expression and cell proliferation in human breast cancer cells

Antiproliferative actions of calcitriol have been shown to occur in many cell types; however, little is known regarding the molecular basis of this process in breast carcinoma. Ether-à-go-go (Eag1) potassium channels promote oncogenesis and are implicated in breast cancer cell proliferation. Since calcitriol displays antineoplastic effects while Eag1 promotes tumorigenesis, and both factors antagonically regulate cell cycle progression, we investigated a possible regulatory effect of calcitriol upon Eag1 as a mean to uncover new molecular events involved in the antiproliferative activity of this hormone in human breast tumor-derived cells. RT real-time PCR and immunocytochemistry showed that calcitriol suppressed Eag1 expression by a vitamin D receptor (VDR)-dependent mechanism. This effect was accompanied by inhibition of cell proliferation, which was potentiated by astemizole, a nonspecific Eag1 inhibitor. Immunohistochemistry and Western blot demonstrated that Eag1 and VDR abundance was higher in invasive-ductal carcinoma than in fibroadenoma, and immunoreactivity of both proteins was located in ductal epithelial cells. Our results provide evidence of a novel mechanism involved in the antiproliferative effects of calcitriol and highlight VDR as a cancer therapeutic target for breast cancer treatment and prevention.