Overexpression of human KCNA5 increases IK V and enhances apoptosis

Apoptotic cell shrinkage, an early hallmark of apoptosis, is regulated by K⁺ efflux and K⁺ channel activity. Inhibited apoptosis and downregulated K⁺ channels in pulmonary artery smooth muscle cells (PASMC) have been implicated in development of pulmonary vascular medial hypertrophy and pulmonary hypertension. The objective of this study was to test the hypothesis that overexpression of KCNA5, which encodes a delayed-rectifier voltage-gated K⁺ (Kv) channel, increases K⁺ currents and enhances apoptosis. Transient transfection of KCNA5 caused 25- to 34-fold increase in KCNA5 channel protein level and 24- to 29-fold increase in Kv channel current (I_K(V)) at +60 mV in COS-7 and rat PASMC, respectively. In KCNA5-transfected COS-7 cells, staurosporine (ST)-mediated increases in caspase-3 activity and the percentage of cells undergoing apoptosis were both enhanced, whereas basal apoptosis (without ST stimulation) was unchanged compared with cells transfected with an empty vector. In rat PASMC, however, transfection of KCNA5 alone caused marked increase in basal apoptosis, in addition to enhancing ST-mediated apoptosis. Furthermore, ST-induced apoptotic cell shrinkage was significantly accelerated in COS-7 cells and rat PASMC transfected with KCNA5, and blockade of KCNA5 channels with 4-aminopyridine (4-AP) reduced K⁺ currents through KCNA5 channels and inhibited ST-induced apoptosis in KCNA5-transfected COS-7 cells. Overexpression of the human KCNA5 gene increases K⁺ currents (i.e., K⁺ efflux or loss), accelerates apoptotic volume decrease (AVD), increases caspase-3 activity, and induces apoptosis. Induction of apoptosis in PASMC by KCNA5 gene transfer may serve as an important strategy for preventing the progression of pulmonary vascular wall thickening and for treating patients with idiopathic pulmonary arterial hypertension (IPAH). potassium ion channel; pulmonary hypertension     

Plasma membrane aquaporin activity can affect the rate of apoptosis but is inhibited after apoptotic volume decrease

Apoptosis is characterized by a conserved series of morphological events beginning with the apoptotic volume decrease (AVD). This study investigated a role for aquaporins (AQPs) during the AVD. Inhibition of AQPs blocked the AVD in ovarian granulosa cells undergoing growth factor withdrawal and blocked downstream apoptotic events such as cell shrinkage, changes in the mitochondrial membrane potential, DNA degradation, and caspase-3 activation. The effects of AQP inhibition on the AVD and DNA degradation were consistent in thymocytes and with two additional apoptotic signals, thapsigargin and C₆-ceramide. Overexpression of AQP-1 in Chinese hamster ovary (CHO-AQP-1) cells enhanced their rate of apoptosis. The AVD is driven by loss of K⁺ from the cell, and we hypothesize that after the AVD, AQPs become inactive, which halts further water loss and allows K⁺ concentrations to decrease to levels necessary for apoptotic enzyme activation. Swelling assays on granulosa cells, thymocytes, and CHO-AQP-1 cells revealed that indeed, the shrunken (apoptotic) subpopulation has very low water permeability compared with the normal-sized (nonapoptotic) subpopulation. In thymocytes, AQP-1 is present and was shown to colocalize with the plasma membrane receptor tumor necrosis factor receptor-1 (TNF-R1) both before and after the AVD, which suggests that this protein is not proteolytically cleaved and remains on the cell membrane. Overall, these data indicate that AQP-mediated water loss is important for the AVD and downstream apoptotic events, that the water permeability of the plasma membrane can control the rate of apoptosis, and that inactivation after the AVD may help create the low K⁺ concentration that is essential in apoptotic cells. Furthermore, inactivation of AQPs after the AVD does not appear to be through degradation or removal from the cell membrane. water movement; major intrinsic protein; channel; enzyme     

Contribution of KCNQ1 to the regulatory volume decrease in the human mammary epithelial cell line MCF-7

Using the human mammary epithelial cell line MCF-7, we have investigated volume-activated changes in response to hyposmotic stress. Switching MCF-7 cells from an isosmotic to a hyposmotic solution resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). This RVD response was inhibited by the nonselective K⁺ channel inhibitors Ba²⁺, quinine, and tetraethylammonium chloride, implicating K⁺ channel activity in this volume-regulatory mechanism. Additional studies using chromonol 293B and XE991 as inhibitors of the KCNQ1 K⁺ channel, and also a dominant-negative NH₂-terminal truncated KCNQ1 isoform, showed complete abolition of the RVD response, suggesting that KCNQ1 plays an important role in regulation of cell volume in MCF-7 cells. We additionally confirmed that KCNQ1 mRNA and protein is expressed in MCF-7 cells, and that, when these cells are cultured as a polarized monolayer, KCNQ1 is located exclusively at the apical membrane. Whole cell patch-clamp recordings from MCF-7 cells revealed a small 293B-sensitive current under hyposmotic, but not isosmotic conditions, while recordings from mammalian cells heterologously expressing KCNQ1 alone or KCNQ1 with the accessory subunit KCNE3 reveal a volume-sensitive K⁺ current, inhibited by 293B. These data suggest that KCNQ1 may play important physiological roles in the mammary epithelium, regulating cell volume and potentially mediating transepithelial K⁺ secretion. potassium channel; volume regulation; mammary gland     

Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents

Cell shrinkageis an early prerequisite in programmed cell death, and cytoplasmicK⁺ is a dominant cation that controls intracellular ionhomeostasis and cell volume. Blockade of K⁺ channelsinhibits apoptotic cell shrinkage and attenuates apoptosis. We examined whether apoptotic repressor with caspase recruitment domain (ARC), an antiapoptotic protein, inhibits cardiomyocyte apoptosis by reducing K⁺ efflux throughvoltage-gated K⁺ (Kv) channels. In heart-derived H9c2cells, whole cell Kv currents (I_K(V)) wereisolated by using Ca²⁺-free extracellular (bath) solutionand including 5 mM ATP and 10 mM EGTA in the intracellular (pipette)solution. Extracellular application of 5 mM 4-aminopyridine (4-AP), ablocker of Kv channels, reversibly reduced I_K(V)by 50-60% in H9c2 cells. The remaining currents during 4-APtreatment may be generated by K⁺ efflux through4-AP-insensitive K⁺ channels. Overexpression of ARC inheart-derived H9c2 cells significantly decreasedI_K(V), whereas treatment with staurosporine, apotent apoptosis inducer, enhanced I_K(V)in wild-type cells. The staurosporine-induced increase inI_K(V) was significantly suppressed and thestaurosporine-mediated apoptosis was markedly inhibited incells overexpressing ARC compared with cells transfected with thecontrol neomycin vector. These results suggest that theantiapoptotic effect of ARC is, in part, due to inhibition of Kvchannels in cardiomyocytes.

     

Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling

Apoptotic cell death in mammalian models is frequently associated with cell shrinkage. Inhibition of apoptotic volume decrease (AVD) is cytoprotective, suggesting that cell shrinkage is an important early event in apoptosis. In salmonid hepatoma and gill cells staurosporine induced apoptosis, as assessed by activation of effector caspases, nuclear condensation, and a decrease of mitochondrial membrane potential (MMP), and these changes were accompanied by cell shrinkage. The Cl⁻ transport inhibitor DIDS and the K⁺ channel inhibitor quinidine prevented AVD, but only DIDS inhibited apoptosis. Other Cl⁻ flux inhibitors, as well as a pan-caspase inhibitor, did not prevent cell shrinkage, but still prevented caspase activation. Furthermore, regulatory volume decrease (RVD) under hypotonic conditions was not facilitated, but diminished in apoptotic cells. Since all transport inhibitors used blocked RVD, but only DIDS and quinidine inhibited AVD, the ion transporters involved in both processes are apparently not identical. In addition, our data indicate that inhibition of Cl⁻ fluxes rather than blocking cell shrinkage or K⁺ fluxes is important for preventing apoptosis. In line with this, inhibition of MAP kinases reduced RVD and not AVD, but still diminished caspase activation. Finally, we observed that MAP kinases were activated upon staurosporine treatment and that at least activation of ERK was prevented when AVD was inhibited.     

Activation of K+ channels induces apoptosis in vascular smooth muscle cells

Intracellular K⁺ playsan important role in controlling the cytoplasmic ion homeostasis formaintaining cell volume and inhibiting apoptotic enzymes in thecytosol and nucleus. Cytoplasmic K⁺ concentration is mainlyregulated by K⁺ uptake viaNa⁺-K⁺-ATPase and K⁺ efflux throughK⁺ channels in the plasma membrane. Carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), a protonophorethat dissipates the H⁺ gradient across the inner membraneof mitochondria, induces apoptosis in many cell types. In ratand human pulmonary artery smooth muscle cells (PASMC), FCCP opened thelarge-conductance, voltage- and Ca²⁺-sensitiveK⁺ (maxi-K) channels, increased K⁺ currentsthrough maxi-K channels [I_K(Ca)], and inducedapoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM)decreased I_K(Ca) by blocking the sarcolemmalmaxi-K channels and inhibited the FCCP-induced apoptosis inPASMC cultured in media containing serum and growth factors.Furthermore, inhibition of K⁺ efflux by raisingextracellular K⁺ concentration from 5 to 40 mM alsoattenuated PASMC apoptosis induced by FCCP and theK⁺ ionophore valinomycin. These results suggest thatFCCP-mediated apoptosis in PASMC is partially due to anincrease of maxi-K channel activity. The resultant K⁺ lossthrough opened maxi-K channels may serve as a trigger for cellshrinkage and caspase activation, which are major characteristics ofapoptosis in pulmonary vascular smooth muscle cells.

     

TREK-2 (K2P10.1) and TRESK (K2P18.1) are major background K+ channels in dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons express mRNAs for many two-pore domain K⁺ (K_2P) channels that behave as background K⁺ channels. To identify functional background K⁺ channels in DRG neurons, we examined the properties of single-channel openings from cell-attached and inside-out patches from the cell bodies of DRG neurons. We found seven types of K⁺ channels, with single-channel conductance ranging from 14 to 120 pS in 150 mM KCl bath solution. Four of these K⁺ channels showed biophysical and pharmacological properties similar to TRESK (14 pS), TREK-1 (112 pS), TREK-2 (50 pS), and TRAAK (73 pS), which are members of the K_2P channel family. The molecular identity of the three other K⁺ channels could not be determined, as they showed low channel activity and were observed infrequently. Of the four K_2P channels, the TRESK-like (14 pS) K⁺ channel was most active at 24°C. At 37°C, the 50-pS (TREK-2 like) channel was the most active and contributed the most (69%) to the resting K⁺ current, followed by the TRESK-like 14-pS (16%), TREK-1-like 112-pS (12%), and TRAAK-like 73-pS (3%) channels. In DRG neurons, mRNAs of all four K_2P channels, as well as those of TASK-1 and TASK-3, were expressed, as judged by RT-PCR analysis. Our results show that TREKs and TRESK together contribute >95% of the background K⁺ conductance of DRG neurons at 37°C. As TREKs and TRESK are targets of modulation by receptor agonists, they are likely to play an active role in the regulation of excitability in DRG neurons. two-pore domain K⁺ channel; conductance; excitability     

Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes

Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated K_V1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K_2P) channels in the RVD of human CD4⁺ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K_2P5.1 and K_2P18.1 as the most important K_2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K_2P5.1 and K_2P18.1 on the RVD was comparable to that of K_V1.3. K_2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K_2P channels.     

Hypotonicity activates a lanthanide-sensitive pathway for K+ release in A6 epithelia

The nature of the pathway forK⁺ release activated duringregulatory volume decrease (RVD) in A6 epithelia was investigated bymeasuring cell thickness (T_c) asan index of cell volume and by probingK⁺ efflux with⁸⁶Rb as tracer forK⁺(R_Rb). Cell swelling was inducedby sudden reduction of basolateral osmolality (from 260 to 140 mosmol/kgH₂O). Experiments wereperformed in the absence of Na⁺transport. Apical R_Rb wasnegligible in iso- and hyposmotic conditions. On the other hand,osmotic shock increased basolateralR_Rb(R^bl_Rb) rapidly, reaching a maximum 7 minafter the peak in T_c. Quinine (0.5 mM) completely inhibited RVD and R^bl_Rb.Also verapamil (0.2 mM) impeded volume recovery considerably; lidocaine(0.2 mM) did not exert a noticeable effect. TheK⁺ channel blockerBa²⁺ (30 mM) delayed RVD but couldnot prevent complete volume recovery. Cs⁺ inhibited RVD noticeably atconcentrations <40 mM. With large Cs⁺ concentrations (>40 mM), theinitial osmometric swelling was followed by a gradual increase ofT_c, suggesting activation of Cs⁺ influx. Chronic exposure ofthe basolateral surface to 0.5 mM La³⁺ orGd³⁺ completely abolished RVD andR^bl_Rb. Acute administration oflanthanides at the time of osmolality decrease did not affect theinitial phase of RVD and reduced R^bl_Rbonly slightly. Apical Gd³⁺ exertedan inhibitory effect on RVD and R^bl_Rb.The effect of Gd³⁺ shouldtherefore be localized at an intracellular site. The role ofCa²⁺ entry could be excluded byfailure of extracellular Ca²⁺removal to inhibit volume recovery. In contrast to lanthanides, chronically and acutely administeredMg²⁺ (0.5 mM) inhibited RVD andR^bl_Rb by ~50%. These data suggest thatK⁺ excretion during RVD occursthrough a rather poorly selective pathway that does not seem to bedirectly activated by membrane stretch.

     

Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl^– channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V_Post-RVD < V_baseline). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl^– channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd²⁺ (V_Post-RVD > 1.4*V_baseline). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K⁺-Cl^– cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15°C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15°C, the channel blockers NPPB + Cd²⁺ completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl^– channels vs. transporters to this process is 60–70% and 30–40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl^– channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a. voltage-gated chloride channel family; potassium-chloride cotransporters; peritumoral edema     

Significance of Ca2+ and K+ in Micrasterias Growth and Morphogenesis

Significance of Ca²⁺ and K⁺ for the complex morphogenesis ofMicrasterias, which takes place through multipolar tip growth,was investigated. Studies with different external Ca²⁺ concentrationsand Ca²⁺ channel inhibitors LaCl₃ and verapamil indicate thatCa²⁺ and Ca²⁺ channels are essential in the development, whiletreatments with different K⁺ concentrations and K⁺ channel inhibitorTEA demonstrate that potassium or K⁺ channels are not neededin the process, albeit the existence of K⁺ channels. K⁺ is notneeded even for the regulation of turgor pressure, which wasfound to decrease clearly during cell development. The plasmamembrane ATPase inhibitors diethylstilbesterol (DES) and Na-orthovanadatestop morphogenesis and indicate the importance of ion pumpsin the developmental process. Both supraoptimal, external K⁺and Ca²⁺ cause abundant Ca²⁺ precipitate formation in chloroplasts,which shows that chloroplasts are important in regulation ofcytoplasmic Ca²⁺ metabolism and that K⁺ activates the uptakeof Ca²⁺ through Ca²⁺ channels. (Received June 13, 1995; Accepted September 13, 1996)     

Cellular mechanisms involved in CO(2) and acid signaling in chemosensitive neurons

An increase in CO₂/H⁺ is a major stimulus for increased ventilation and is sensed by specialized brain stem neurons called central chemosensitive neurons. These neurons appear to be spread among numerous brain stem regions, and neurons from different regions have different levels of chemosensitivity. Early studies implicated changes of pH as playing a role in chemosensitive signaling, most likely by inhibiting a K⁺ channel, depolarizing chemosensitive neurons, and thereby increasing their firing rate. Considerable progress has been made over the past decade in understanding the cellular mechanisms of chemosensitive signaling using reduced preparations. Recent evidence has pointed to an important role of changes of intracellular pH in the response of central chemosensitive neurons to increased CO₂/H⁺ levels. The signaling mechanisms for chemosensitivity may also involve changes of extracellular pH, intracellular Ca²⁺, gap junctions, oxidative stress, glial cells, bicarbonate, CO₂, and neurotransmitters. The normal target for these signals is generally believed to be a K⁺ channel, although it is likely that many K⁺ channels as well as Ca²⁺ channels are involved as targets of chemosensitive signals. The results of studies of cellular signaling in central chemosensitive neurons are compared with results in other CO₂- and/or H⁺-sensitive cells, including peripheral chemoreceptors (carotid body glomus cells), invertebrate central chemoreceptors, avian intrapulmonary chemoreceptors, acid-sensitive taste receptor cells on the tongue, and pain-sensitive nociceptors. A multiple factors model is proposed for central chemosensitive neurons in which multiple signals that affect multiple ion channel targets result in the final neuronal response to changes in CO₂/H⁺. hypercapnia; brain stem; ventilation; peripheral chemoreceptor; glia; gap junction; glomus; channel; calcium; potassium; carbonic anhydrase; taste receptor; nociception     

System-specific O2 sensitivity of the tandem pore domain K+ channel TASK-1

Hypoxic inhibition of TASK-1, a tandem pore domain background K⁺ channel, provides a critical link between reduced O₂ levels and physiological responses in various cell types. Here, we examined the expression and O₂ sensitivity of TASK-1 in immortalized adrenomedullary chromaffin (MAH) cells. In physiological (asymmetrical) K⁺ solutions, 3 µM anandamide or 300 µM Zn²⁺ inhibited a strongly pH-sensitive current. Under symmetrical K⁺ conditions, the anandamide- and Zn²⁺-sensitive K⁺ currents were voltage independent. These data demonstrate the functional expression of TASK-1, and cellular expression of this channel was confirmed by RT-PCR and Western blotting. At concentrations that selectively inhibit TASK-1, anandamide and Zn²⁺ were without effect on the magnitude of the O₂-sensitive current or the hypoxic depolarization. Thus TASK-1 does not contribute to O₂ sensing in MAH cells, demonstrating the failure of a known O₂-sensitive K⁺ channel to respond to hypoxia in an O₂-sensing cell. These data demonstrate that, ultimately, the sensitivity of a particular K⁺ channel to hypoxia is determined by the cell, and we propose that this is achieved by coupling distinct hypoxia signaling systems to individual channels. Importantly, these data also reiterate the indirect O₂ sensitivity of TASK-1, which appears to require the presence of an intracellular mediator. hypoxia; background K⁺ channels; TASK-1; MAH cells     

Swelling-activated cation-selective channels in A6 epithelia are permeable to large cations

Effects of basolateral monovalent cation replacements(Na⁺ byLi⁺,K⁺,Cs⁺, methylammonium, andguanidinium) on permeability to⁸⁶Rb of volume-sensitive cationchannels (VSCC) in the basolateral membrane and on regulatory volumedecrease (RVD), elicited by a hyposmotic shock, were studied in A6epithelia in the absence of apicalNa⁺ uptake. A complete and quickRVD occurred only when the cells were perfused withNa⁺ orLi⁺ saline. With both cations,hypotonicity increased basolateral ⁸⁶Rb release(R^bl_Rb), which reached a maximum after 15 min and declined back to control level. When the major cation wasK⁺,Cs⁺, methylammonium, orguanidinium, the RVD was abolished. Methylammonium induced a biphasictime course of cell thickness(T_c), with an initial decline ofT_c followed by a gradual increase.With K⁺,Cs⁺, or guanidinium,T_c increased monotonously afterthe rapid initial rise evoked by the hypotonic challenge. In thepresence of K⁺,Cs⁺, or methylammonium,R^bl_Rb remained high during most of thehypotonic period, whereas with guanidinium blockage of R^bl_Rb was initiated after 6 min ofhypotonicity, suggesting an intracellular location of the site ofaction. With all cations, 0.5 mM basolateralGd³⁺ completely blocked RVD andfully abolished the R^bl_Rb increaseinduced by the hypotonic shock. The lanthanide also blocked theadditional volume increase induced byCs⁺,K⁺, guanidinium, ormethylammonium. When pH was lowered from 7.4 to 6.0, RVD andR^bl_Rb were markedly inhibited. This studydemonstrates that the VSCCs in the basolateral membrane of A6 cells arepermeable to K⁺,Rb⁺,Cs⁺, methylammonium, andguanidinium, whereas a marked inhibitory effect is exerted byGd³⁺, protons, and possiblyintracellular guanidinium.

     

ATP-sensitive potassium channels mediate hyperosmotic stimulation of NKCC in slow-twitch muscle

In mildly hyperosmotic medium, activation of the Na⁺-K⁺-2Cl^- cotransporter (NKCC) counteracts skeletal muscle cell water loss, and compounds that stimulate protein kinase A (PKA) activity inhibit the activation of the NKCC. The aim of this study was to determine the mechanism for PKA inhibition of NKCC activity in resting skeletal muscle. Incubation of rat slow-twitch soleus and fast-twitch plantaris muscles in isosmotic medium with the PKA inhibitors H-89 and KT-5720 caused activation of the NKCC only in the soleus muscle. NKCC activation caused by PKA inhibition was insensitive to MEK MAPK inhibitors and to insulin but was abolished by the PKA stimulators isoproterenol and forskolin. Furthermore, pinacidil [an ATP-sensitive potassium (K_ATP) channel opener] or inhibition of glycolysis increased NKCC activity in the soleus muscle but not in the plantaris muscle. Preincubation of the soleus muscle with glibenclamide (a K_ATP channel inhibitor) prevented the NKCC activation by hyperosmolarity, PKA inhibition, pinacidil, and glycolysis inhibitors. In contrast, glibenclamide stimulated NKCC activity in the plantaris muscle. In cells stably transfected with the Kir6.2 subunit of the of K_ATP channel, inhibition of glycolysis activated potassium current and NKCC activity. We conclude that activation of K_ATP channels in slow-twitch muscle is necessary for activation of the NKCC and cell volume restoration in hyperosmotic conditions. protein kinase A; glibenclamide; glycolysis; Na⁺-K⁺-2Cl^- cotransporter; Kir6.2     

Two types of voltage-dependent potassium channels in outer hair cells from the guinea pig cochlea

Cell-attached and cell-free configurations of the patch-clamptechnique were used to investigate the conductive properties andregulation of the major K⁺channels in the basolateral membrane of outer hair cells freshly isolated from the guinea pig cochlea. There were two majorvoltage-dependent K⁺ channels. ACa²⁺-activatedK⁺ channel with a high conductance(220 pS,P_K/P_Na = 8) was found in almost 20% of the patches. The inside-out activityof the channel was increased by depolarizations above 0 mV andincreasing the intracellular Ca²⁺concentration. External ATP or adenosine did not alter thecell-attached activity of the channel. The open probability of theexcised channel remained stable for several minutes without rundown andwas not altered by the catalytic subunit of protein kinase A (PKA)applied internally. The most frequentK⁺ channel had a low conductanceand a small outward rectification in symmetricalK⁺ conditions (10 pS for inwardcurrents and 20 pS for outward currents, P_K/P_Na = 28). It was found significantly more frequently in cell-attached andinside-out patches when the pipette contained 100 µM acetylcholine. It was not sensitive to internalCa²⁺, was inhibited by4-aminopyridine, was activated by depolarization above 30 mV,and exhibited a rundown after excision. It also had a slow inactivationon ensemble-averaged sweeps in response to depolarizing pulses. Thecell-attached activity of the channel was increased when adenosine wassuperfused outside the pipette. This effect also occurred with permeantanalogs of cAMP and internally applied catalytic subunit of PKA. Bothchannels could control the cell membrane voltage of outer hair cells.

     

IK channels are involved in the regulatory volume decrease in human epithelial cells

Parallel activation ofCa²⁺-dependent K⁺ channels and volume-sensitiveCl channels is known to be responsible for KCl effluxduring regulatory volume decrease (RVD) in human epithelial Intestine407 cells. The present study was performed to identify theK⁺ channel type. RT-PCR demonstrated mRNA expression ofCa²⁺-activated, intermediate conductance K⁺(IK), but not small conductance K⁺ (SK1) or largeconductance K⁺ (BK) channels in this cell line. Whole cellrecordings showed that ionomycin or hypotonic stress activated inwardlyrectifying K⁺ currents that were reversibly blocked by IKchannel blockers [clotrimazole (CLT) and charybdotoxin] but not by SKand BK channel blockers (apamin and iberiotoxin). Inside-out recordingsrevealed the existence of CLT-sensitive single K⁺-channelactivity, which exhibited an intermediate unitary conductance (30 pS at100 mV). The channel was activated by cytosolic Ca²⁺ ininside-out patches and by a hypotonic challenge in cell-attached patches. The RVD was suppressed by CLT, but not by apamin oriberiotoxin. Thus we conclude that the IK channel is involved in theRVD process in these human epithelial cells.

     

Differential effects of volatile and intravenous anesthetics on the activity of human TASK-1

Volatile anesthetics have been shown to activate various two-pore (2P) domain K⁺ (K_2P) channels such as TASK-1 and TREK-1 (TWIK-related acid-sensitive K⁺ channel), and mice deficient in these channels are resistant to halothane-induced anesthesia. Here, we investigated whether K_2P channels were also potentially important targets of intravenous anesthetics. Whole cell patch-clamp techniques were used to determine the effects of the commonly used intravenous anesthetics etomidate and propofol on the acid-sensitive K⁺ current in rat ventricular myocytes (which strongly express TASK-1) and selected human K_2P channels expressed in Xenopus laevis oocytes. In myocytes, etomidate decreased both inward rectifier K⁺ (K_ir) current (I_K1) and acid-sensitive outward K⁺ current at positive potentials, suggesting that this drug may inhibit TASK channels. Indeed, in addition to inhibiting guinea pig Kir2.1 expressed in oocytes, etomidate inhibited human TASK-1 (and TASK-3) in a concentration-dependent fashion. Propofol had no effect on human TASK-1 (or TASK-3) expressed in oocytes. Moreover, we showed that, similar to the known effect of halothane, sevoflurane and the purified R-(–)- and S-(+)-enantiomers of isoflurane, without stereoselectivity, activated human TASK-1. We conclude that intravenous and volatile anesthetics have dissimilar effects on K_2P channels. Human TASK-1 (and TASK-3) are insensitive to propofol but are inhibited by supraclinical concentrations of etomidate. In contrast, stimulatory effects of sevoflurane and enantiomeric isoflurane on human TASK-1 can be observed at clinically relevant concentrations. volatile anesthetics; etomidate; propofol; ion channels     

Hypoxia reduces KCa channel activity by inducing Ca2+ spark uncoupling in cerebral artery smooth muscle cells

Arterial smooth muscle cell large-conductance Ca²⁺-activated potassium (K_Ca) channels have been implicated in modulating hypoxic dilation of systemic arteries, although this is controversial. K_Ca channel activity in arterial smooth muscle cells is controlled by localized intracellular Ca²⁺ transients, termed Ca²⁺ sparks, but hypoxic regulation of Ca²⁺ sparks and K_Ca channel activation by Ca²⁺ sparks has not been investigated. We report here that in voltage-clamped (–40 mV) cerebral artery smooth muscle cells, a reduction in dissolved O₂ partial pressure from 150 to 15 mmHg reversibly decreased Ca²⁺ spark-induced transient K_Ca current frequency and amplitude to 61% and 76% of control, respectively. In contrast, hypoxia did not alter Ca²⁺ spark frequency, amplitude, global intracellular Ca²⁺ concentration, or sarcoplasmic reticulum Ca²⁺ load. Hypoxia reduced transient K_Ca current frequency by decreasing the percentage of Ca²⁺ sparks that activated a transient K_Ca current from 89% to 63%. Hypoxia reduced transient K_Ca current amplitude by attenuating the amplitude relationship between Ca²⁺ sparks that remained coupled and the evoked transient K_Ca currents. Consistent with these data, in inside-out patches at –40 mV hypoxia reduced K_Ca channel apparent Ca²⁺ sensitivity and increased the K_d for Ca²⁺ from 17 to 32 µM, but did not alter single-channel amplitude. In summary, data indicate that hypoxia reduces K_Ca channel apparent Ca²⁺ sensitivity via a mechanism that is independent of cytosolic signaling messengers, and this leads to uncoupling of K_Ca channels from Ca²⁺ sparks. Transient K_Ca current inhibition due to uncoupling would oppose hypoxic cerebrovascular dilation. transient calcium-activated potassium current     

Modeling transmural heterogeneity of K(ATP) current in rabbit ventricular myocytes

To investigate the mechanisms regulating excitation-metabolic coupling in rabbit epicardial, midmyocardial, and endocardial ventricular myocytes we extended the LabHEART model (Puglisi JL and Bers DM. Am J Physiol Cell Physiol 281: C2049–C2060, 2001). We incorporated equations for Ca²⁺ and Mg²⁺ buffering by ATP and ADP, equations for nucleotide regulation of ATP-sensitive K⁺ channel and L-type Ca²⁺ channel, Na⁺-K⁺-ATPase, and sarcolemmal and sarcoplasmic Ca²⁺-ATPases, and equations describing the basic pathways (creatine and adenylate kinase reactions) known to communicate the flux changes generated by intracellular ATPases. Under normal conditions and during 20 min of ischemia, the three regions were characterized by different I_Na, I_to, I_Kr, I_Ks, and I_Kp channel properties. The results indicate that the ATP-sensitive K⁺ channel is activated by the smallest reduction in ATP in epicardial cells and largest in endocardial cells when cytosolic ADP, AMP, PCr, Cr, P_i, total Mg²⁺, Na⁺, K⁺, Ca²⁺, and pH diastolic levels are normal. The model predicts that only K_ATP ionophore (Kir6.2 subunit) and not the regulatory subunit (SUR2A) might differ from endocardium to epicardium. The analysis suggests that during ischemia, the inhomogeneous accumulation of the metabolites in the tissue sublayers may alter in a very irregular manner the K_ATP channel opening through metabolic interactions with the endogenous PI cascade (PIP₂, PIP) that in turn may cause differential action potential shortening among the ventricular myocyte subtypes. The model predictions are in qualitative agreement with experimental data measured under normal and ischemic conditions in rabbit ventricular myocytes. ATP-sensitive K⁺ channel; creatine and adenylate kinase reactions; phosphatidylinositol phosphates; heart; mathematical model