Cellular localization of mitochondria contributes to Kv channel-mediated regulation of cellular excitability in pulmonary but not mesenteric circulation (original) (raw)
Related papers
2008
Mitochondria are proposed to be a major oxygen sensor in hypoxic pulmonary vasoconstriction (HPV), a unique response of the pulmonary circulation to low oxygen tension. Mitochondrial factors including reactive oxygen species, cytochrome c, ATP, and magnesium are potent modulators of voltage-gated K ϩ (Kv) channels in the plasmalemmal membrane of pulmonary arterial (PA) smooth muscle cells (PASMCs). Mitochondria have also been found close to the plasmalemmal membrane in rabbit main PA smooth muscle sections. Therefore, we hypothesized that differences in mitochondria localization in rat PASMCs and systemic mesenteric arterial smooth muscle cells (MASMCs) may contribute to the divergent oxygen sensitivity in the two different circulations. Cellular localization of mitochondria was compared with immunofluorescent labeling, and differences in functional coupling between mitochondria and Kv channels was evaluated with the patch-clamp technique and specific mitochondrial inhibitors antimycin A (acting at complex III of the mitochondrial electron transport chain) and oligomycin A (which inhibits the ATP synthase). It was found that mitochondria were located significantly closer to the plasmalemmal membrane in PASMCs compared with MASMCs. Consistent with these findings, the effects of the mitochondrial inhibitors on Kv current (IKv) were significantly more potent in PASMCs than in MASMCs. The cytoskeletal disruptor cytochalasin B (10 M) also altered mitochondrial distribution in PASMCs and significantly attenuated the effect of antimycin A on the voltage-dependent parameters of IKv. These findings suggest a greater structural and functional coupling between mitochondria and Kv channels specifically in PASMCs, which could contribute to the regulation of PA excitability in HPV. pulmonary artery; vascular smooth muscle cells; mesenteric artery; K ϩ channel activation; K ϩ channel inactivation; confocal imaging; patch-clamp technique
Mitochondria Dependent Regulation of Kv Currents in Rat Pulmonary Artery Smooth Muscle Cells
Voltage-gated K + (Kv) channels are important in the regulation of pulmonary vascular function having both physiological and pathophysiological implications. The pulmonary vasculature is essential for reoxygenation of the blood, supplying oxygen for cellular respiration. Mitochondria have been proposed as the major oxygen sensing organelles in the pulmonary vasculature. Using electrophysiological techniques and immunofluorescence an interaction of the mitochondria with Kv channels was investigated. Inhibitors, blocking the mitochondrial electron transport chain at different complexes were shown to have a dual effect on Kv currents in freshly isolated rat pulmonary arterial smooth muscle cells (PASMCs). These dual effects comprised of an enhancement of Kv current in a negative potential range (manifested as a 5-14 mV shift in the Kv activation to more negative membrane voltages) with a decrease in current amplitude at positive potentials. Such effects were most prominent as a result of inhibition of Complex III by antimycin A. Investigation of the mechanism of antimycin A-mediated effects on I Kv revealed the presence of a mitochondrial-mediated Mg 2+ and ATP dependent regulation of Kv channels in PASMCs, which exists in addition to that currently proposed to be caused by changes in intracellular reactive oxygen species.
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
Voltage activation of heart inner mitochondrial membrane channels
Journal of Bioenergetics and Biomembranes, 1992
The patch clamp records obtained from mitoplast membranes prepared in the presence of a calcium chelator generally lack channel activity. However, multiconductance channel (MCC) activity can be induced by membrane potentials above + 60 mV [Kinnally et al., Biochem. Biophys. Res. Commun. 176, 1183-1188]. Once activated, the MCC activity persists at all voltages. The present report characterizes the activation by voltage of multiconductance channels of rat heart inner mitochondrial membranes using patch-clamping. In some membrane patches, the size of single current transitions progressively increases with time upon application of voltage. The inhibitor cyclosporin has also been found to decrease channel conductance in steps. The results suggest that voltage-induced effects which are inhibited by cyclosporin Aare likely to involve either an increase in effective pore diameter or the assembly of low-conductance units. In activated patches, we have found at high membrane potentials (e.g., 130 mV) changes in conductance as high as 5 nS occurring in large steps (up to 2.7 nS). These were generally preceded by a smaller transition. Similar results were obtained less frequently at lower voltages. These results can be explained on the assumption that once assembled the channels may act in unison.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008
Both ATP-regulated (mitoK ATP ) and large conductance calcium-activated (mitoBK Ca ) potassium channels have been proposed to regulate mitochondrial K + influx and matrix volume and to mediate cardiac ischaemic preconditioning (IP). However, the specificity of the pharmacological agents used in these studies and the mechanisms underlying their effects on IP remain controversial. Here we used increasing concentrations of K + -ionophore (valinomycin) to stimulate respiration by rat liver and heart mitochondria in the presence of the K + /H + exchanger nigericin. This allowed rates of valinomycin-induced K + influx to be determined whilst parallel measurements of light scattering (A 520 ) and matrix volume ( 3 H 2 O and [ 14 C]sucrose) enabled rates of K + influx to be correlated with increases in matrix volume. Light scattering readily detected an increase in K + influx of b 5 nmol K + min − 1 per mg protein corresponding to b 2% mitochondrial matrix volume increase. In agreement with earlier data no light-scattering changes were observed in response to any mitoK ATP channel openers or blockers. However, the mitoBK Ca opener NS1619 (10-50 µM) did decrease light scattering slightly, but this was also seen in K + -free medium and was accompanied by uncoupling. Contrary to prediction, the mitoBK Ca blocker paxilline (10-50 µM) decreased rather than increased light scattering, and it also slightly uncoupled respiration. Our data argue against the presence of significant activities of either the mitoK ATP or the mitoBK Ca channel in rat liver and heart mitochondria and provide further evidence that preconditioning induced by pharmacological openers of these channels is more likely to involve alternative mechanisms.
The FASEB Journal, 2001
Hypoxic pulmonary vasoconstriction (HPV) is initiated by the inhibition of several 4aminopyridine (4-AP)-sensitive, voltage-gated, K+ channels (Kv). Several O 2 -sensitive candidate channels (Kv1.2, Kv1.5, Kv2.1, and Kv3.1b) have been proposed, based on similarities between their characteristics in expression systems and the properties of the O 2 -sensitive K+ current (I K ) in pulmonary artery smooth muscle cells (PASMCs). We used gene targeting to delete Kv1.5 in mice by creating a SWAP mouse that is functionally a Kv1.5 knockout. We hypothesized that SWAP mice would display impaired HPV. The Kv1.5 α-subunits present in the endothelium and PASMCs of wild-type mice were absent in the lungs of SWAP mice, whereas expression of other channels Kv (1.1, 1.2, 2.1, 3.1, 4.3), Kir 3.1, Kir 6.1, and BK Ca was unaltered. In isolated lungs and resistance PA rings, HPV was reduced significantly in SWAP versus wild-type mice. Consistent with this finding, PASMCs from SWAP PAs were slightly depolarized and lacked I Kv1.5 , a 4-AP and hypoxia-sensitive component of I K that activated between -50 mV and -30 mV. We conclude that a K+ channel containing Kv1.5α-subunits is an important effector of HPV in mice.
Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O 2 constriction. It occurs by an incompletely understood mechanism that is intrinsic to the DA smooth muscle cell (DASMC). We hypothesized that O 2 alters the function of an O 2 sensor (the mitochondrial electron transport chain, ETC) thereby increasing production of a diffusible redox-mediator (H 2 O 2 ), thus triggering an effector mechanism (inhibition of DASMC voltage-gated K ϩ channels, Kv). O 2 constriction was evaluated in 26 human DAs (12 female, aged 9Ϯ2 days) studied in their normal hypoxic state or after normoxic tissue culture. In fresh, hypoxic DAs, 4-aminopyridine (4-AP), a Kv inhibitor, and O 2 cause similar constriction and K ϩ current inhibition (I K ). Tissue culture for 72 hours, particularly in normoxia, causes ionic remodeling, characterized by decreased O 2 and 4-AP constriction in DA rings and reduced O 2 -and 4-AP-sensitive I K in DASMCs. Remodeled DAMSCs are depolarized and express less O 2 -sensitive channels (including Kv2.1, Kv1.5, Kv9.3, Kv4.3, and BK Ca ). Kv2.1 adenoviral gene-transfer significantly reverses ionic remodeling, partially restoring both the electrophysiological and tone responses to 4-AP and O 2 . In fresh DASMCs, ETC inhibitors (rotenone and antimycin) mimic hypoxia, increasing I K and reversing constriction to O 2 , but not phenylephrine. O 2 increases, whereas hypoxia and ETC inhibitors decrease H 2 O 2 production by altering mitochondrial membrane potential (⌬⌿m). H 2 O 2 , like O 2 , inhibits I K and depolarizes DASMCs. We conclude that O 2 controls human DA tone by modulating the function of the mitochondrial ETC thereby varying ⌬⌿m and the production of H 2 O 2 , which regulates DASMC Kv channel activity and DA tone. (Circ Res. 2002;91:478-486.)
Endothelial mitochondria as a possible target for potassium channel modulators
Pharmacological reports : PR, 2006
Variety of ion channels is present in plasma membrane of endothelial cells. These include the potassium channels such as Ca(2+)-activated K(+) channels, inwardly rectifying K(+) channels, voltage-dependent K(+) channels and also ATP-regulated K(+) channels. Due to an influence on the membrane potential they are important regulators of vascular tone by modulating endothelial calcium ions signaling and synthesis of vasodilating factors. Potassium channels in mitochondrial membranes of various tissues, similar to plasma membrane potassium channels, are described. Mitochondrial potassium channels such as ATP-regulated or large conductance Ca(2+)-activated and voltage gated channels are implicated in cytoprotective phenomenon in different tissues. In this paper we describe the pharmacological properties of mitochondrial potassium channels and discuss their role of as novel pharmacotherapeutic targets in endothelium.