Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells (original) (raw)
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American Journal of Physiology-cell Physiology, 2007
in mouse pulmonary artery smooth muscle cells channels + Functional characterization of voltage-gated K You might find this additional info useful... 61 articles, 43 of which you can access for free at: This article cites http://ajpcell.physiology.org/content/293/3/C928.full#ref-list-1 5 other HighWire-hosted articles: This article has been cited by http://ajpcell.physiology.org/content/293/3/C928#cited-by including high resolution figures, can be found at: Updated information and services http://ajpcell.physiology.org/content/293/3/C928.full found at: can be American Journal of Physiology -Cell Physiology about Additional material and information http://www.the-aps.org/publications/ajpcell This information is current as of October 24, 2012. 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/.
Ca2+-activated K+ channels in human red cells. Comparison of single-channel currents with ion fluxes
Biophysical Journal, 1984
Exposure of the inner surface of intact red cells or red cell ghosts to Ca2" evokes unitary currents that can be measured in cell-attached and cell-free membrane patches. The currents are preferentially carried by K+ (PK/ PNa 17) and show rectification. Increasing the Ca2" concentration from 0 to 5 IuM increases the probability of the open state of the channels parallel to the change of K+ permeability as observed in suspensions of red cell ghosts. Prolonged incubation of red cell ghosts in the absence of external K+ prevents the Ca2" from increasing K+ permeability. Similarly, the probability to find Ca2"-activated unitary currents in membrane patches is drastically reduced. These observations suggest that the Ca2"-induced changes of K+ permeability observed in red cell suspensions are causally related to the appearance of the unitary K+ currents. Attempts to determine the number of K+ channels per cell were made by comparing fluxes measured in suspensions of red cells with the unitary currents in membrane patches as determined under comparable ionic conditions. At 100 mM KC1 in the external medium, where no net movements of K+ occur, the time course of equilibration of 86Rb+ does not follow a single exponential. This indicates a heterogeneity of the response to Ca2" of the cells in the population. The data are compatible with the assumption that 25% of the cells respond with Pk = 33.2 x 10-'4cm3/s and 75% with Pk = 3.
The Journal of General Physiology, 1994
An electrochemical gating model is presented to account for the effects described in the companion paper by M. R. Silver, M. S. Shapiro, and T. E. DeCoursey (1994. Journal of General Physiology, 103:519-548) of Rb+ and Rb+/K+ mixtures on the kinetics and voltage dependence of an inwardly rectifying (IR) K+ channel. The model proposes that both Rb+ and K+ act as allosteric modulators of an intrinsically voltage dependent isomerization between open and closed states. Occupancy of binding sites on the outside of the channel promotes channel opening and stabilizes the open state. Rb+ binds to separate sites within the pore and plugs IR channels. Occupancy of the pore by Rb+ can modify the rates of isomerization and the affinity of the allosteric sites for activator ions. The model also incorporates the proposed triple-barreled nature of the IR channel (Matsuda, H., 1988. Journal of Physiology. 397:237-258.) by proposing that plugging of the channel is a cooperative process involving a s...
An ATP-sensitive potassium conductance in rabbit arterial endothelial cells
The Journal of physiology, 1995
1. Whole-cell patch clamp recording was used to study an ATP-sensitive, sulphonylurea-inhibitable potassium (K+) conductance in freshly dissociated endothelial cells from rabbit arteries. 2. The ATP-sensitive K+ conductance was activated by micromolar concentrations of the K+ channel opener, levcromakalim, and by metabolic inhibition of endothelial cells using dinitrophenol and iodoacetic acid. The current-voltage (I-V) relationship obtained in isotonic K+ solutions was linear between -150 and -50 mV and had a slope conductance of approximately 1 nS. 3. The permeability of the ATP-sensitive K+ conductance determined from reversal potential measurements exhibited the following ionic selectivity sequence: Rb+ > K+ > Cs+ > Na+ > NH4+ > Li+. 4. Membrane currents activated by either levcromakalim or metabolic inhibition were inhibited by the sulphonylurea drugs, glibenclamide and tolbutamide, with half-maximal inhibitory concentrations of 43 nM and 224 microM and Hill coef...
Pfl�gers Archiv European Journal of Physiology, 1994
A novel class of Ca2+-activated K + channel, also activated by Mg-ATR exists in the main pulmonary artery of the rat. In view of the sensitivity of these "Kca,ATp" channels to such charged intermediates it is possible that they may be involved in regulating cellular responses to hypoxia. However, their electrophysiologicat profile is at present unknown. We have therefore characterised the sensitivity of KCa,ATe channels to voltage, intracellular Ca 2+ ([Ca2+]i) and Mg-ATR They have a conductance of 245 pS in symmetrical K + and are approximately 20 times more selective for K § ions than Na + ions, with a K + permeability (PrO of 4.6• s -1. Ca 2+ ions applied to the intracellular membrane surface of KCa,ATP channels causes a marked enhancement of their activity. This activation is probably the result of simultaneous binding of at least two Ca 2+ ions, determined using Hill analysis, to the channel or some closely associated protein. This results in a shift of the voltage activation threshold to more hyperpolarized membrane potentials. The activation of KCa.AT~, channels by Mg-ATP has an ECso of approximately 50 laM. Although the ECso is unaffected by [Ca2+]~, channel activation by Mg-ATP is enhanced by increasing [Ca2+]~. One possible interpretation of these data is that Mg-ATP increases the sensitivity of Kca,A~ channels to Ca 2+. It is therefore possible that under hypoxic conditions, where lower levels of Mg-ATP may be encountered, the sensitivity of Kca,ATe channels to Ca 2+ and therefore voltage is reduced. This would tend to induce a depolarising influence, which would favour the influx of Ca 2+ through voltage-activated Ca 2 § channels, ultimately leading to increased vascular tone.
Journal of Cellular Physiology, 1993
The transmembrane potential (V,,,) of vascular endothelial cells (ECj is an important property that may be involved in intra-and intercellular signal transduction for various vascular functions. In this study, V,,, of intact aortic and vena caval EC from hamsters were measured using conventional microelectrodes. Vascular strips with the luminal surface upwards were suffused in a tissue chamber with Krebs solution in physiological conditions. The resting V, of aortic and vend caval EC was found to be -40 ? 1 mV (n = 55) and -43 5 1 mV (n = 15j, respectively. The V, recordings were confirmed to have originated from EC by scanning and transmission electron microscopy combined with the comparison of electrical recordings between normal and endothelium-denuded aortic strips. The input resistance varied from 10-240 MdZ, which implied the presence of electrical coupling between vascular EC. Elevating the K + level in the suffusate from 4.7 mM to 50 and 100 mM depolarized aortic EC by 19% and 29% and vena caval EC by 18% and 29%, respectively. These low percentages indicated a relatively small contribution of [ K f ] to the resting V,, of vascular EC. A positive correlation (r > 0.69) between the resting V, and the magnitude of depolarization by the high [K+], may be related to the involvement of voltage-dependent K' channels. The hyperpolarization caused by lowering both "at], and [CIp], suggested the disengagement of some electrogenic transport systems in the membrane, such as a Naf-Kf-CIp cotransporter. The transference number (t , , , ), as an index of membrane conductance for specific ions, was calculated for K' (1 5-20%), Na+ (1 6%), and CI-(9-15%), demonstrating that both Naf and CI-as well as K+ contribute (b 1993 WILEY-LISS, INC c) 1993 Wiley-Liis, Inc Klepper, 19901, membrane electrical conductivity (O'Donnell and Vargas, 1986), and electrical coupling between endothelial cells (Daut et al., 1988; Beny and Gribi, 1989), have been documented, but most of these studies were completed on cells in culture.
Journal of Membrane Biology, 1998
The pharmacological profile of a voltage-independent Ca 2+-activated potassium channel of intermediate conductance (IK(Ca 2+)) present in bovine aortic endothelial cells (BAEC) was investigated in a series of inside-out and outside-out patch-clamp experiments. Channel inhibition was observed in response to external application of ChTX with a half inhibition concentration of 3.3 ± 0.3 nM (n ס 4). This channel was insensitive to IbTX, but channel block was detected following external application of MgTX and StK leading to the rank order toxin potency ChTX > StK > MgTX >>IbTX. A reduction of the channel unitary current amplitude was also measured in the presence of external TEA, with half reduction occurring at 23 ± 3 mM TEA (n ס 3). The effect of TEA was voltage insensitive, an indication that TEA may bind to a site located on external side of the pore region of this channel. Similarly, the addition of d-TC to the external medium caused a reduction of the channel unitary current amplitude with half reduction at 4.4 ± 0.3 mM (n ס 4). In contrast, application of d-TC to the bathing medium in inside-out experiments led to the appearance of long silent periods, typical of a slow blocking process. Finally, the IK(Ca 2+) in BAEC was found to be inhibited by NS1619, an activator of the Ca 2+-activated potassium channel of large conductance (Maxi K(Ca 2+)), with a half inhibition value of 11 ± 0.8 M (n ס 4). These results provide evidence for a pharmacological profile distinct from that reported for the Maxi K(Ca 2+) channel, with some features attributed to the voltage-gated K V 1.2 potassium channel.
2006
Objectives: Inhibition of voltage-gated K+ (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs) contributes to the development of hypoxic pulmonary vasoconstriction (HPV). Mitochondria have been proposed as the major oxygen sensing organelles in PASMCs. Although a role for mitochondrial-dependent cellular redox state changes that modulate Kv channels has been proposed, the precise mechanism of the interaction between Kv channels and mitochondria remains unclear. To understand these mechanisms the effect of various mitochondrial inhibitors on Kv channel currents (IKv) were investigated in rat PASMCs and PAs. Comparisons were drawn to the mesenteric circulation. Methods: Patch-clamp technique under different intracellular conditions and in the presence o f a variety o f pharmacological tools. Three key parameters of Ikv were O I assessed; activation, inactivation and Ikv block. Additionally, Mg and Ca fluorescent measurements were performed and whole vessel contractility was assessed using a Mulvany-Halpem myograph. Results: The mitochondrial uncoupler CCCP, and mitochondrial electron transport chain (mETC) inhibitors, rotenone, myxothiazol, antimycin and cyanide, induced similar significant changes in all three Ikv parameters. Antimycin-induced effects, as the most pronounced were studied in detail. It was found that these effects 1) cannot be entirely explained by changes in cellular redox state, 2) are mimicked by the ATP synthase inhibitor oligomycin, 3) were significantly inhibited by cell dialysis with 5 mM Na2ATP, EDTA or with 5 (instead of 0.5) mM MgCl2, whereas intracellular MgATP partially reversed the effect, 4) both CCCP and antimycin caused a significant increase in intracellular Mg2+ (Mg2+i) and 5) hypoxia caused an increase in Mg2+i and a leftward shift in Ikv activation, mimicking the effect of mitochondrial inhibitors. Additionally, the involvement of the Na+-Mg2+ (NME), alongside the Na+-Ca2+ (NCE) and Na+-H+ (NHE) exchangers on 7kv activation and block was evaluated using various extracellular and intracellular conditions. 1) Na+0 removal (to block the exchangers) caused i) a leftward in 7kv activation, ii) a significant increase in the slope of the dependency and iii) a decrease in the maximal whole-cell conductance. 2) Removal of IV o I external Mg , intracellular EDTA and 100 pM amiloride, a putative NME inhibitor, all significantly attenuated Na+0-dependent effects on 7kv. 3) The effects on 7kv activation were significantly attenuated by 3 pM KB-R7943 (a reverse mode inhibitor of the NCE) and extracellular alkalanisation by 0.6 pH unit (the conditions facilitating accumulation I I of intracellular Na), but not by elevated [Ca ]j, intracellular BAPTA, extracellular acidification or by the NHE inhibitor 5-(N-methyl-N-isobutyl) amiloride. Significant differences between pulmonary and mesenteric circulation were found, suggesting specificity of the observed mechanism to the pulmonary circulation. Conclusions: Collectively, these findings suggest the presence of a novel 04mitochondrial-mediated Mg i-dependent mechanism in the regulation of Kv channels in PASMCs, which could be involved in HPV. It also suggests that Kv channel activity 04at physiological membrane potentials in PASMCs chiefly depends on Mg i 04" concentration determined by the balance between the extracellular Mg influx and release and its extrusion by the NME, thus representing a novel regulatory mechanism for Kv channels in PASMCs. Despite some similarities the same overall mechanism
Oxygen Sensitivity of Cloned Voltage-Gated K+ Channels Expressed in the Pulmonary Vasculature
Circulation Research, 1999
Hypoxic pulmonary vasoconstriction is initiated by inhibiting one or more voltage-gated potassium (Kv) channel in the vascular smooth muscle cells (VSMCs) of the small pulmonary resistance vessels. Although progress has been made in identifying which Kv channel proteins are expressed in pulmonary arterial (PA) VSMCs, there are conflicting reports regarding which channels contribute to the native O 2-sensitive K ϩ current. In this study, we examined the effects of hypoxia on the Kv1.2, Kv1.5, Kv2.1, and Kv9.3 ␣ subunits expressed in mouse L cells using the whole-cell patch-clamp technique. Hypoxia (PO 2 ϭϷ30 mm Hg) reversibly inhibited Kv1.2 and Kv2.1 currents only at potentials more positive than 30 mV. In contrast, hypoxia did not alter Kv1.5 current. Currents generated by coexpression of Kv2.1 with Kv9.3 ␣ subunits were reversibly inhibited by hypoxia in the voltage range of the resting membrane potential (E M) of PA VSMCs (Ϸ28% at Ϫ40 mV). Coexpression of Kv1.2 and Kv1.5 ␣ subunits produced currents that displayed kinetic and pharmacological properties distinct from Kv1.2 and Kv1.5 channels expressed alone. Moreover, hypoxia reversibly inhibited Kv1.2/Kv1.5 current activated at physiologically relevant membrane potentials (Ϸ65% at Ϫ40 mV). These results indicate that (1) hypoxia reversibly inhibits Kv1.2 and Kv2.1 but not Kv1.5 homomeric channels, (2) Kv1.2 and 1.5 ␣ subunits can assemble to form an O 2-sensitive heteromeric channel, and (3) only Kv1.2/Kv1.5 and Kv2.1/Kv9.3 heteromeric channels are inhibited by hypoxia in the voltage range of the PA VSMC E M. Thus, these heteromeric channels are strong candidates for the K ϩ channel isoforms initiating hypoxic pulmonary vasoconstriction.
Electrogenic Na+/K+-transport in human endothelial cells
Pfl�gers Archiv European Journal of Physiology, 1993
Na+/K + pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca2+]i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/1 [Na +] did not induce significant changes of [Ca2+]i. Superfusing the cells with K+-free solutions also did not significantly affect [Ca2+]i. Reapplication of K + after superfusion of the cells with K+-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 ~tmol/1 dihydroouabain (DHO) and was therefore identified as a Na+/K + pump current. During block and reactivation of the Na+/K + pump no changes in [Ca2+]i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 ~tmol/1. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10-1000U/1) did not affect the pump currents. An increase of the intracellular Na + concentration ([Na+]i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na+]i occurred at about 60 mmol/1. The concentration for half-maximal activation by extracellular K + was 2.4 _+ 1.2 mmol/1, and 0.4 _+ 0.1 and 8.7 _+ 0.7 mmol]l for T1 + and NH4 + respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than -150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 gmol/1 guanosine 5"-O-(3-thiotriphosphate) (GTPTS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforatedpatch technique prevents wash out of a guanine nucleoti-Correspondence to: B. Nilius de-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.