Differential sensitivity of inward rectifier K+ channels to metabolic inhibitors

Inhibition of inward rectifier K(+) channels under ischemic conditions may contribute to electrophysiological consequences of ischemia such as cardiac arrhythmia. Ischemia causes metabolic inhibition, and the use of metabolic inhibitors is one experimental method of simulating ischemia. The effects of metabolic inhibitors on the activity of inward rectifier K(+) channels K(ir)2.1, K(ir)2.2, and K(ir)2.3 were studied by heterologous expression in Xenopus oocytes and two-electrode voltage clamp. 10 microm carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited K(ir)2.2 and K(ir)2.3 currents but was without effect on K(ir)2.1 currents. The rate of decline of current in FCCP was faster for K(ir)2.3 than for K(ir)2.2. K(ir)2.3 was inhibited by 3 mm sodium azide (NaN(3)), whereas K(ir)2.1 and K(ir)2.2 were not. K(ir)2.2 was inhibited by 10 mm NaN(3). All three of these inward rectifiers were inhibited by lowering the pH of the solution perfusing inside-out membrane patches. K(ir)2.3 was most sensitive to pH (pK = 6.9), whereas K(ir)2.1 was least sensitive (pK = 5.9). For K(ir)2.2 the pK was 6.2. These results demonstrate the differential sensitivity of these inward rectifiers to metabolic inhibition and internal pH. The electrophysiological response of a particular cell type to ischemia may depend on the relative expression levels of different inward rectifier genes.     

Hyaluronan export by the ABC transporter MRP5 and its modulation by intracellular cGMP

Hyaluronan must be exported from its site of synthesis, the inner side of plasma membrane, to the extracellular matrix. Here, we identified the multidrug-associated protein MRP5 as the principle hyaluronan exporter from fibroblasts. The expression of the MRP5 (ABC-C5) transporter was silenced in fibroblasts using RNA interference, and a dose-dependent inhibition of hyaluronan export was observed. Hyaluronan oligosaccharides introduced into the cytosol competed with the export of endogenously labeled hyaluronan and the MRP5 substrate fluorescein. Because cGMP is a physiological substrate of MRP5, the intracellular concentrations of cGMP were modulated by the drugs 3-isobutyl-1-methylxanthin, propentofyllin, L-NAME, zaprinast, and bromo-cGMP, and the effects on hyaluronan export were analyzed. Increasing the cGMP levels inhibited hyaluronan export and decreasing it afforded higher concentrations of zaprinast to inhibit the export. Thus, cGMP may be a physiological regulator of hyaluronan export at the level of the export MRP5.     

Inwardly rectifying K(+) channels in esophageal smooth muscle

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.     

Hyaluronan enters keratinocytes by a novel endocytic route for catabolism.

Hyaluronan synthesized in the epidermis has an exceptionally short half-life, indicative of its catabolism by epidermal keratinocytes. An intracellular pool of endogenously synthesized hyaluronan, from 1 to 20 fg/cell, inversely related to cell density, was observed in cultured rat epidermal keratinocytes. More than 80% of the intracellular hyaluronan was small (<90 kDa). Approximately 25% of newly synthesized hyaluronan was endocytosed by the keratinocytes and had a half-life of 2-3 h. A biotinylated aggrecan G(1) domain/link protein probe demonstrated hyaluronan in small vesicles of approximately 100 nm diameter close to the plasma membrane, and in large vesicles and multivesicular bodies up to 1300 nm diameter around the nucleus. Hyaluronan did not co-localize with markers of lysosomes. However, inhibition of lysosomal acidification with NH(4)Cl or chloroquine, or treating the cells with the hyaluronidase inhibitor apigenin increased intracellular hyaluronan staining, suggesting that it resided in prelysosomal endosomes. Competitive displacement of hyaluronan from surface receptors using hyaluronan decasaccharides, resulted in a rapid disappearance of this endosomal hyaluronan (t(12) approximately 5 min), indicating its transitory nature. The ultrastructure of the hyaluronan-containing vesicles, co-localization with marker proteins for different vesicle types, and application of specific uptake inhibitors demonstrated that the formation of hyaluronan-containing vesicles did not involve clathrin-coated pits or caveolae. Treatment of rat epidermal keratinocytes with the OX50 monoclonal antibody against the hyaluronan receptor CD44 increased endosomal hyaluronan. However, no CD44-hyaluronan co-localization was observed intracellularly unless endosomal trafficking was retarded by monensin, or cultivation at 20 degrees C, suggesting CD44 recycling. Rat epidermal keratinocytes thus internalize a large proportion of their newly synthesized hyaluronan into non-clathrin-coated endosomes in a receptor mediated way, and rapidly transport it to slower degradation in the endosomal/lysosomal system.     

Localization of voltage-gated K(+) channels in squid giant axons

We have localized the classical voltage-gated K(+) channel within squid giant axons by immunocytochemistry using the Kv1 antibody of Rosenthal et al. (1996). Widely dispersed patches of intense immunofluorescence were observed in the axonal membrane. Punctate immunofluorescence was also observed in the axoplasm and was localized to approximately 25-50-microm-wide column down the length of the nerve (axon diameter approximately 500 microm). Immunoelectronmicroscopy of the axoplasm revealed a K(+) channel containing vesicles, 30-50 nm in diameter, within this column. These and other vesicles of similar size were isolated from axoplasm using a novel combination of high-speed ultracentrifugation and controlled-pore size, glass bead separation column techniques. Approximately 1% of all isolated vesicles were labeled by K(+) channel immunogold reacted antibody. Incorporation of isolated vesicle fractions within an artificial lipid bilayer revealed K(+) channel electrical activity similar to that recorded directly from the axonal membrane by Llano et al. (1988). These K(+) channel-containing vesicles may be involved in cycling of K(+) channel protein into the axonal membrane. We have also isolated an axoplasmic fraction containing approximately 150-nm-diameter vesicles that may transport K(+) channels back to the cell body.     

Interaction of the K(+)-channel KAT1 with the coat protein complex II coat component Sec24 depends on a di-acidic endoplasmic reticulum export motif

The correct functioning of ion channels depends not only on the control of their activity but also on the regulation of the number of channels in the membrane. For example, it has been proposed that the density of the plant K(+)-channel KAT1 may be adjusted by controlling its export from its site of synthesis, the endoplasmic reticulum (ER). Efficient transport of the channel to the plasma membrane was found to depend on a di-acidic ER export signal in the C-terminus of the protein. Studies in yeast and mammals indicate that di-acidic ER export motifs are essential for enrichment of proteins into ER-derived coat protein complex II (COPII) vesicles and are recognized by Sec24 a component of the COPII coat. To investigate whether similar mechanisms also exist in plants we have analysed the interaction of KAT1 with Sec24 in vivo using fluorescence resonance energy transfer (FRET) measurements in Vicia faba guard cells. These measurements revealed a FRET signal between KAT1 and Sec24 fused to the cyan fluorescent protein and the yellow fluorescent protein, respectively, indicating an interaction between KAT1 and Sec24. The FRET signal only occurred in the perinuclear region of the ER and was dependent on the di-acidic ER export motif of KAT1. Together, the results point to a highly conserved mechanism for ER export of KAT1 whereby the channel is recruited into COPII vesicles via binding of the di-acidic motif to Sec24.     

Potassium channels regulate tone in rat pulmonary veins

Intrapulmonary veins (PVs) contribute to pulmonary vascular resistance, but the mechanisms controlling PV tone are poorly understood. Although smooth muscle cell (SMC) K(+) channels regulate tone in most vascular beds, their role in PV tone is unknown. We show that voltage-gated (K(V)) and inward rectifier (K(ir)) K(+) channels control resting PV tone in the rat. PVs have a coaxial structure, with layers of cardiomyocytes (CMs) arrayed externally around a subendothelial layer of typical SMCs, thus forming spinchterlike structures. PVCMs have both an inward current, inhibited by low-dose Ba(2+), and an outward current, inhibited by 4-aminopyridine. In contrast, PVSMCs lack inward currents, and their outward current is inhibited by tetraethylammonium (5 mM) and 4-aminopyridine. Several K(V), K(ir), and large-conductance Ca(2+)-sensitive K(+) channels are present in PVs. Immunohistochemistry showed that K(ir) channels are present in PVCMs and PV endothelial cells but not in PVSMCs. We conclude that K(+) channels are present and functionally important in rat PVs. PVCMs form sphincters rich in K(ir) channels, which may modulate venous return both physiologically and in disease states including pulmonary edema.     

Activating of ATP-dependent K+ channels comprised of K(ir) 6.2 and SUR 2B by PGE2 through EP2 receptor in cultured interstitial cells of Cajal from murine small intestine.

The interstitial cells of Cajal (ICC) are pacemaker cells in gastrointestinal tract and generate an electrical rhythm in gastrointestinal muscles. We investigated the possibility that PGE(2) might affect the electrical properties of cultured ICC by activating ATP-dependent K(+) channels and, the EP receptor subtypes and the subunits of ATP-dependent K(+) channels involved in these activities were identified. In addition, the regulation of intracellular Ca(2+) ([Ca(2+)](i)) mobilization may be involved the action of PGE(2) on ICC. Treatments of ICC with PGE(2) inhibited electrical pacemaker activities in the same manner as pinacidil, an ATP-dependent K(+) channel opener and PGE(2) had only a dose-dependent effect. Using RT-PCR technique, we found that ATP-dependent K(+) channels exist in ICC and that these are composed of K(ir) 6.2 and SUR 2B subunits. To characterize the specific membrane EP receptor subtypes in ICC, EP receptor agonists and RT-PCR were used: Butaprost (an EP(2) receptor agonist) showed the actions on pacemaker currents in the same manner as PGE(2). However sulprostone (a mixed EP(1) and EP(3) agonist) had no effects. In addition, RT-PCR results indicated the presence of the EP(2) receptor in ICC. To investigate cAMP involvement in the effects of PGE(2) on ICCs, SQ-22536 (an inhibitor of adenylate cyclase) and cAMP assays were used. SQ-22536 did not affect the effect of PGE(2) on pacemaker currents, and PGE(2) did not stimulate cAMP production. Also, we found PGE(2) inhibited the spontaneous [Ca(2+)](i) oscillations in cultured ICC. These observations indicate that PGE(2) alters pacemaker currents by activating the ATP-dependent K(+) channels comprised of K(ir) 6.2-SUR 2B in ICC and this action of PGE(2) are through EP(2) receptor subtype and also the activation of ATP-dependent K(+) channels involves intracellular Ca(2+) mobilization.     

Basolateral potassium channels of rabbit colon epithelium: role in sodium absorption and chloride secretion

In order to assess the role of different classes of K(+) channels in recirculation of K(+) across the basolateral membrane of rabbit distal colon epithelium, the effects of various K(+) channel inhibitors were tested on the activity of single K(+) channels from the basolateral membrane, on macroscopic basolateral K(+) conductance, and on the rate of Na(+) absorption and Cl(-) secretion. In single-channel measurements using the lipid bilayer reconstitution system, high-conductance (236 pS), Ca(2+)-activated K(+) (BK(Ca)) channels were most frequently detected; the second most abundant channel was a low-conductance K(+) channel (31 pS) that exhibited channel rundown. In addition to Ba(2+) and charybdotoxin (ChTX), the BK(Ca) channels were inhibited by quinidine, verapamil and tetraethylammonium (TEA), the latter only when present on the side of the channel from which K(+) flow originates. Macroscopic basolateral K(+) conductance, determined in amphotericin-permeabilised epithelia, was also markedly reduced by quinidine and verapamil, TEA inhibited only from the lumen side, and serosal ChTX was without effect. The chromanol 293B and the sulphonylurea tolbutamide did not affect BK(Ca) channels and had no or only a small inhibitory effect on macroscopic basolateral K(+) conductance. Transepithelial Na(+) absorption was partly inhibited by Ba(2+), quinidine and verapamil, suggesting that BK(Ca) channels are involved in basolateral recirculation of K(+) during Na(+) absorption in rabbit colon. The BK(Ca) channel inhibitors TEA and ChTX did not reduce Na(+) absorption, probably because TEA does not enter intact cells and ChTX is 'knocked off' its extracellular binding site by K(+) outflow from the cell interior. Transepithelial Cl(-) secretion was inhibited completely by Ba(2+) and 293B, partly by quinidine but not by the other K(+) channel blockers, indicating that the small (<3 pS) K(V)LQT1 channels are responsible for basolateral K(+) exit during Cl(-) secretion. Hence different types of K(+) channels mediate basolateral K(+) exit during transepithelial Na(+) and Cl(-) transport.     

Analysis of Na+,K+-ATPase motion and incorporation into the plasma membrane in response to G protein-coupled receptor signals in living cells

Dopamine (DA) increases Na(+),K(+)-ATPase activity in lung alveolar epithelial cells. This effect is associated with an increase in Na(+),K(+)-ATPase molecules within the plasma membrane (). Analysis of Na(+),K(+)-ATPase motion was performed in real-time in alveolar cells stably expressing Na(+),K(+)-ATPase molecules carrying a fluorescent tag (green fluorescent protein) in the alpha-subunit. The data demonstrate a distinct (random walk) pattern of basal movement of Na(+),K(+)-ATPase-containing vesicles in nontreated cells. DA increased the directional movement (by 3.5 fold) of the vesicles and an increase in their velocity (by 25%) that consequently promoted the incorporation of vesicles into the plasma membrane. The movement of Na(+),K(+)-ATPase-containing vesicles and incorporation into the plasma membrane were microtubule dependent, and disruption of this network perturbed vesicle motion toward the plasma membrane and prevented the increase in the Na(+),K(+)-ATPase activity induced by DA. Thus, recruitment of new Na(+),K(+)-ATPase molecules into the plasma membrane appears to be a major mechanism by which dopamine increases total cell Na(+),K(+)-ATPase activity.     

Participation of a K(+) channel modulated directly by cGMP in the speract-induced signaling cascade of strongylocentrotus purpuratus sea urchin sperm

Speract, a decapeptide from Strongylocentrotus purpuratus sea urchin eggs, transiently stimulates a membrane guanylyl cyclase and activates a K(+)-selective channel that hyperpolarizes sperm. However, previous studies of sperm and of sperm membrane vesicles reached conflicting conclusions about the mechanisms that open these channels. We find that speract hyperpolarizes and increases the cGMP content of flagellar vesicles. We confirm previous findings that intravesicular GTPgammaS and GTP enhance this hyperpolarization, but not GDPbetaS. The G protein activators AlF(-)(4) and mastoparan also are ineffective. Thus, it is unlikely that a G protein participates in the speract response. In contrast, hyperpolarization responses to speract are increased by 3-isobutyl-1-methylxanthine, which preferentially inhibits cGMP-selective phosphodiesterases of sperm, and the 8Br-cGMP derivative hyperpolarizes vesicles in the absence of speract. The responses to speract and to 8Br-cGMP have similar ionic selectivities (K(+) > Rb(+) > > Li(+) > Na(+)) and sensitivities to the channel blockers 4-aminopiridine and 3, 4-dichlorobenzamil, indicating that they likely result from opening of the same K(+) channel. Inhibitors that preferentially inhibit cAMP-selective phosphodiesterases do not alter responses to speract, and permeant cAMP analogs do not hyperpolarize vesicles. In addition, inhibitors of protein kinases and phosphatases fail to alter vesicle hyperpolarization by speract. The increase in vesicular cGMP content produced by speract therefore may directly mediate opening of the channel that hyperpolarizes sperm membrane vesicles. Similar mechanisms presumably operate in intact sperm.     

Contribution of K(ir)2 potassium channels to ATP-induced cell death in brain capillary endothelial cells and reconstructed HEK293 cell model

Cellular turnover of brain capillary endothelial cells (BCECs) by the balance of cell proliferation and death is essential for maintaining the homeostasis of the blood-brain barrier. Stimulation of metabotropic ATP receptors (P2Y) transiently increased intracellular Ca2(+) concentration ([Ca2(+)](i)) in t-BBEC 117, a cell line derived from bovine BCECs. The [Ca2(+)](i) rise induced membrane hyperpolarization via the activation of apamin-sensitive small-conductance Ca2(+)-activated K(+) channels (SK2) and enhanced cell proliferation in t-BBEC 117. Here, we found anomalous membrane hyperpolarization lasting for over 10 min in response to ATP in ～15% of t-BBEC 117, in which inward rectifier K(+) channel (K(ir)2.1) was extensively expressed. Once anomalous hyperpolarization was triggered by ATP, it was removed by Ba2(+) but not by apamin. Prolonged exposure to ATPγS increased the relative population of t-BBEC 117, in which the expression of K(ir)2.1 mRNAs was significantly higher and Ba2(+)-sensitive anomalous hyperpolarization was observed. The cultivation of t-BBEC 117 in serum-free medium also increased this population and reduced the cell number. The reduction of cell number was enhanced by the addition of ATPγS and the enhancement was antagonized by Ba2(+). In the human embryonic kidney 293 cell model, where SK2 and K(ir)2.1 were heterologously coexpressed, [Ca2(+)](i) rise by P2Y stimulation triggered anomalous hyperpolarization and cell death. In conclusion, P2Y stimulation in BCECs enhances cell proliferation by SK2 activation in the majority of cells but also triggers cell death in a certain population showing a substantial expression of K(ir)2.1. This dual action of P2Y stimulation may effectively facilitate BCEC turnover.     

急性低温/再复温对大鼠心室肌膜电位和钾电流的影响

本文旨在研究急性低温/再复温对大鼠心室肌膜电位和钾电流的影响.膜电位和膜电流分别在全细胞膜片钳的电压钳和电流钳模式下记录.当细胞外灌流液从25℃降低到4℃后,一过性外向电流(transient outward current, Ito)完全消失,膜电位为 60mV时的稳态外向K 电流(sustained outward K current, Iss)和膜电位为-120mV时的内向整流K 电流(inward rectifier K current, IK1)分别降低(48.5±14.1)%和(35.7±18.2)%,同时,膜电位绝对值降低.当细胞外灌流液从4℃再升高到36℃后,膜电位出现一过性超级化,然后恢复到静息电位水平;在58个细胞中,有36个细胞伴随复温出现ATP-敏感性K (ATP-sensitive K , KATP)通道的激活.再复温引起的上述变化可以被Na /K -ATP酶抑制剂哇巴因(100μmol/L)所抑制.再复温引起的KATP通道激活也能被蛋白激酶A抑制剂H-89(100μmol/L)所抑制.在细胞膜电位被钳制在0mV时,当细胞外灌流液温度从25℃降低到4℃后,细胞的体积没有发生明显改变,但当再复温引起KATP通道激活后,细胞很快发生皱缩,同时细胞内部出现许多折光较强的斑点.上述结果表明急性低温/再复温对大鼠心室肌膜电位和K 电流有明显影响,并提示KATP通道激活可能与心肌低温/再复温损伤有关.     

Ba2+-sensitive K+ channels in luminal-membrane vesicles from pars convoluta of rabbit proximal tubule

This paper describes properties of 86Rb+ fluxes through a novel K+ channel in luminal-membrane vesicles isolated from pars convoluta of rabbit proximal tubule. The uptake of 86Rb+ into potassium salt loaded vesicles was specifically inhibited by Ba2+. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using these membrane vesicles loaded with anions of different membrane permeability and was as follows: gluconate greater than SO4(2-) greater than Cl-. Furthermore, the vesicles containing the channels show a cation selectivity with the order K+ greater than Rb+ greater than Li+ greater than Na+ = choline+.     

Augmented K(+) currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis

The balance between apoptosis and proliferation in pulmonary artery smooth muscle cells (PASMCs) is important in maintaining normal pulmonary vascular structure. Activity of voltage-gated K(+) (K(V)) channels has been demonstrated to regulate cell apoptosis and proliferation. Treatment of PASMCs with staurosporine (ST) induced apoptosis in PASMCs, augmented K(V) current [I(K(V))], and induced mitochondrial membrane depolarization. High K(+) (40 mM) negligibly affected the ST-induced mitochondrial membrane depolarization but inhibited the ST-induced I(K(V)) increase and apoptosis. Blockade of K(V) channels with 4-aminopyridine diminished I(K(V)) and markedly decreased the ST-mediated apoptosis. Furthermore, the ST-induced apoptosis was preceded by the increase in I(K(V)). These results indicate that ST induces PASMC apoptosis by activation of plasmalemmal K(V) channels and mitochondrial membrane depolarization. The increased I(K(V)) would result in an apoptotic volume decrease due to a loss of cytosolic K(+) and induce apoptosis. The mitochondrial membrane depolarization would cause cytochrome c release, activate the cytosolic caspases, and induce apoptosis. Inhibition of K(V) channels would thus attenuate PASMC apoptosis.     

Specific Electron Donor-Energized Transport of α-Aminoisobutyric Acid and K+ into Intact Cells of a Marine Pseudomonad

The transport of alpha-aminoisobutyric acid and K(+) into K(+)-depleted cells of a marine pseudomonad (ATCC 19855) was stimulated strongly by ethanol, reduced nicotinamide adenine dinucleotide (NADH), and ascorbate-reduced N, N, N', N'-tetramethyl-p-phenylenediamine. In the presence of the quinone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide, only ascorbate-reduced N, N, N', N'-tetramethyl-p-phenylenediamine was active. Primary and secondary, but not tertiary, alcohols from ethanol to n-amyl alcohol stimulated both alpha-aminoisobutyric acid and K(+) transport and were oxidized by the cells. Malate and succinate, which were oxidized rapidly by the cells, had little or no capacity to energize the transport of alpha-aminoisobutyric acid into K(+)-depleted cells but were partially effective in promoting K(+) uptake. Ethanol stimulated the transport of alpha-aminoisobutyric acid into K(+)-preloaded cells. The transport of both alpha-aminoisobutyric acid and K(+) was inhibited 20% by iodoacetate, 85% by N-ethylmaleimide, and 90 to 100% by both NaCN and p-chloromercuribenzoate. The addition of Na(3)Fe(CN)(6) permitted the ethanol-induced transport of alpha-aminoisobutyric acid into K(+)-preloaded cells in the presence of NaCN, but little or no uptake of alpha-aminoisobutyric acid or of K(+) into K(+)-depleted cells under the same conditions. The transport of alpha-aminoisobutyric acid into K(+)-depleted cells required both K(+) and an electron donor. The oxidation of NADH and ethanol by K(+)-depleted cells was stimulated strongly by K(+). Parallels between these studies and those with membrane vesicles show that results with membrane vesicles of the marine pseudomonad have physiological significance for the intact cells. The results support the conclusion that the energy for the active transport of both alpha-aminoisobutyric acid and K(+) into cells of this organism is provided by electron flow through a region of the respiratory chain lying between cytochrome c and O(2).     

Interactions between adenosine and K+ channel-related pathways in the coupling of somatosensory activation and pial arteriolar dilation

Multiple, perhaps interactive, mechanisms participate in the linkage between increased neural activity and cerebral vasodilation. In the present study, we assessed whether neural activation-related pial arteriolar dilation (PAD) involved interactions among adenosine (Ado) A(2) receptors (A(2)Rs), large-conductance Ca(2+)-operated K(+) (BK(Ca)) channels, and inward rectifier K(+) (K(ir)) channels. In rats with closed cranial windows, we monitored sciatic nerve stimulation (SNS)-induced PAD in the absence or presence of pharmacological blockade of A(2)Rs (ZM-241385), ecto-5'-nucleotidase (α,β-methylene-adenosine diphosphate), BK(Ca) channels (paxilline), and K(ir) channels (BaCl(2)). Individually, these interventions led to 53-66% reductions in SNS-induced PADs. Combined applications of these blockers led to little or no further repression of SNS-induced PADs, suggesting interactions among A(2)Rs and K(+) channels. In the absence of SNS, BaCl(2) blockade of K(ir) channels produced 52-80% reductions in Ado and NS-1619 (BK(Ca) channel activator)-induced PADs. In contrast, paxilline blockade of BK(Ca) channels was without effect on dilations elicited by KCl (K(ir) channel activator) and Ado suffusions, indicating that Ado- and NS-1619-associated PADs involved K(ir) channels. In addition, targeted ablation of the superficial glia limitans was associated with a selective 60-80% loss of NS-1619 responses, suggesting that the BK(Ca) channel participation (and paxilline sensitivity) derived largely from channels within the glia limitans. Additionally, blockade of either PKA or adenylyl cyclase caused markedly attenuated pial arteriolar responses to SNS and, in the absence of SNS, responses to Ado, KCl, and NS-1619. These findings suggested a key, possibly permissive, role for A(2)R-linked cAMP generation and PKA-induced K(+) channel phosphorylation in somatosensory activation-evoked PAD.     

A Ba2+-resistant, acid-sensitive K+ conductance in Na+-absorbing H441 human airway epithelial cells

By analysis of whole cell membrane currents in Na(+)-absorbing H441 human airway epithelial cells, we have identified a K(+) conductance (G(K)) resistant to Ba(2+) but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocaine, and quinidine inhibit basolateral membrane K(+) current (I(Bl)) whereas Ba(2+) has only a weak inhibitory effect. I(Bl) was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in I(Bl), acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive K(+) channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current (I(SC)), and basolateral bupivacaine, lidocaine, quinidine, and Ba(2+) inhibited I(SC) at concentrations similar to those needed to inhibit I(Bl), suggesting that the K(+) channels underlying I(Bl) are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain K(+) (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie G(K) in unstimulated cells and so maintain the driving force for Na(+) absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology.     

Constitutive expression of a Mg2+-inhibited K+ current and a TRPM7-like current in human erythroleukemia cells

Whole cell patch-clamp experiments were undertaken to define the basal K(+) conductance(s) in human erythroleukemia cells and its contribution to the setting of resting membrane potential. Experiments revealed a non-voltage-activated, noninactivating K(+) current. The magnitude of the current recorded under whole cell conditions was inhibited by an increase in free intracellular Mg(2+) concentration. Activation or inactivation of the Mg(2+)-inhibited K(+) current (MIP) was paralleled by activation or inactivation of a Mg(2+)-inhibited TRPM7-like current displaying characteristics indistinguishable from those reported for molecularly identified TRPM7 current. The MIP and TRPM7 currents were inhibited by 5-lipoxygenase inhibitors. However, inhibition of the MIP current was temporally distinct from inhibition of TRPM7 current, allowing for isolation of the MIP current. Isolation of the MIP conductance revealed a current reversing near the K(+) equilibrium potential, indicative of a highly K(+)-selective conductance. Consistent with this finding, coactivation of the nonselective cation current TRPM7 and the MIP current following dialysis with nominally Mg(2+)-free pipette solution resulted in hyperpolarized whole cell reversal potentials, consistent with an important role for the MIP current in the setting of a negative resting membrane potential. The MIP and TRPM7-like conductances were constitutively expressed under in vivo conditions of intracellular Mg(2+), as judged by their initial detection and subsequent inactivation following dialysis with a pipette solution containing 5 mM free Mg(2+). The MIP current was blocked in a voltage-dependent fashion by extracellular Cs(+) and, to a lesser degree, by Ba(2+) and was blocked by extracellular La(3+) and 2-aminoethoxydiphenyl borate. MIP currents were unaffected by blockers of ATP-sensitive K(+) channels, human ether-à-go-go-related gene current, and intermediate-conductance Ca(2+)-activated K(+) channels. In addition, the MIP current displayed characteristics distinct from conventional inwardly rectifying K(+) channels. A similar current was detected in the leukemic cell line CHRF-288-11, consistent with this current being more generally expressed in cells of leukemic origin.     

A synthetic channel-forming peptide induces Cl(-) secretion: modulation by Ca(2+)-dependent K(+) channels

A synthetic Cl(-) channel-forming peptide, C-K4-M2GlyR, applied to the apical membrane of human epithelial cell monolayers induces transepithelial Cl(-) and fluid secretion. The sequence of the core peptide, M2GlyR, corresponds to the second membrane-spanning region of the glycine receptor, a domain thought to line the pore of the ligand-gated Cl(-) channel. Using a pharmacological approach, we show that the flux of Cl(-) through the artificial Cl(-) channel can be regulated by modulating basolateral K(+) efflux through Ca(2+)-dependent K(+) channels. Application of C-K4-M2GlyR to the apical surface of monolayers composed of human colonic cells of the T84 cell line generated a sustained increase in short-circuit current (I(SC)) and caused net fluid secretion. The current was inhibited by the application of clotrimazole, a non-specific inhibitor of K(+) channels, and charybdotoxin, a potent inhibitor of Ca(2+)-dependent K(+) channels. Direct activation of these channels with 1-ethyl-2-benzimidazolinone (1-EBIO) greatly amplified the Cl(-) secretory current induced by C-K4-M2GlyR. The effect of the combination of C-K4-M2GlyR and 1-EBIO on I(SC) was significantly greater than the sum of the individual effects of the two compounds and was independent of cAMP. Treatment with 1-EBIO also increased the magnitude of fluid secretion induced by the peptide. The cooperative action of C-K4-M2GlyR and 1-EBIO on I(SC) was attenuated by Cl(-) transport inhibitors, by removing Cl(-) from the bathing solution and by basolateral treatment with K(+) channel blockers. These results indicate that apical membrane insertion of Cl(-) channel-forming peptides such as C-K4-M2GlyR and direct activation of basolateral K(+) channels with benzimidazolones may coordinate the apical Cl(-) conductance and the basolateral K(+) conductance, thereby providing a pharmacological approach to modulating Cl(-) and fluid secretion by human epithelia deficient in cystic fibrosis transmembrane conductance regulator Cl(-) channels.