Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes
1997; Springer Nature; Volume: 16; Issue: 22 Linguagem: Inglês
10.1093/emboj/16.22.6615
ISSN1460-2075
Autores Tópico(s)Cardiac Ischemia and Reperfusion
ResumoArticle15 November 1997free access Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes Amanda J. Patel Amanda J. Patel Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Search for more papers by this author Michel Lazdunski Corresponding Author Michel Lazdunski Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Search for more papers by this author Eric Honoré Eric Honoré Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Search for more papers by this author Amanda J. Patel Amanda J. Patel Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Search for more papers by this author Michel Lazdunski Corresponding Author Michel Lazdunski Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Search for more papers by this author Eric Honoré Eric Honoré Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Search for more papers by this author Author Information Amanda J. Patel1, Michel Lazdunski 1 and Eric Honoré1 1Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:6615-6625https://doi.org/10.1093/emboj/16.22.6615 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The molecular structure of oxygen-sensitive delayed-rectifier K+ channels which are involved in hypoxic pulmonary artery (PA) vasoconstriction has yet to be elucidated. To address this problem, we identified the Shab K+ channel Kv2.1 and a novel Shab-like subunit Kv9.3, in rat PA myocytes. Kv9.3 encodes an electrically silent subunit which associates with Kv2.1 and modulates its biophysical properties. The Kv2.1/9.3 heteromultimer, unlike Kv2.1, opens in the voltage range of the resting membrane potential of PA myocytes. Moreover, we demonstrate that the activity of Kv2.1/Kv9.3 is tightly controlled by internal ATP and is reversibly inhibited by hypoxia. In conclusion, we propose that metabolic regulation of the Kv2.1/Kv9.3 heteromultimer may play an important role in hypoxic PA vasoconstriction and in the possible development of PA hypertension. Introduction Oxygen is an essential requirement for cell survival. However, the organism has the ability to adapt rapidly to hypoxia. Ion channels, in particular K+ channels, play a key role in adaptive hypoxic mechanisms and are thought to be involved in the sensing of oxygen (Lopez-Barneo, 1994, 1996; Kozlowski, 1995; Weir and Archer, 1995; Lopez-Barneo et al., 1997). One such adaptation is the hypoxia-induced vasoconstriction of resistance pulmonary artery (PA) smooth muscle which leads to a redistribution of the non-oxygenated blood towards better ventilated regions of the lung (for review, see Kozlowski, 1995; Weir and Archer, 1995). In the fetus, hypoxic pulmonary vasoconstriction (HPV) diverts blood through the ductus arteriosus and is essential for fetal survival. Although HPV fulfils an essential physiological function, it also contributes to the development of pulmonary hypertension in patients with chronic obstructive lung diseases (e.g. chronic bronchitis, emphysema) and people living at high altitudes (Barnes and Liu, 1995; Weir and Archer, 1995). It has been demonstrated that hypoxic vasoconstriction of resistance PA smooth muscle cells is mainly mediated by the closing of voltage-dependent K+ channels leading to cell depolarization, calcium influx and myocyte contraction (Post et al., 1992; Archer et al., 1993; Yuan et al., 1993a; Cornfield et al., 1994; Osipenko et al., 1997). Chronic hypoxia has also been associated with reduced delayed-rectifier K+ current in rat pulmonary artery smooth muscle cells (Smirnov et al., 1994). Recently, a novel oxygen-sensitive non-inactivating K+ current active at resting membrane potential (IK(N)) has been described in rabbit PA myocytes (Evans et al., 1996; Osipenko et al., 1997). The molecular identity of these oxygen-sensitive K+ channels and the nature of the oxygen sensor(s) remain unknown. The present work reports the molecular cloning, characterization and regulation by hypoxia of a novel voltage- and ATP-dependent K+ channel subunit called Kv9.3, isolated from rat PA smooth muscle cells. The molecular identification of Kv9.3 could be of tremendous importance in the understanding of pulmonary hypertension and in the design of novel therapeutic strategies. Results Delayed-rectifier K+ channels in rat pulmonary artery myocytes In order to record selectively the activity of voltage-dependent K+ channels in PA smooth muscle, cells were voltage-clamped in the whole-cell configuration in the absence of external and internal calcium (10 mM EGTA) and in the presence of 5 mM internal ATP to avoid contamination with both large conductance calcium-dependent K+ channels and KATP channels (Yuan et al., 1996). Figure 1A and B compare the I–V relationships of the currents recorded in freshly dissociated and cultured PA cells. The characteristics of the outward currents observed in freshly dissociated cells were identical to those in primary (I) culture. However, when the cells were trypsinized and plated in secondary (II) culture, the outward K+ currents were absent (Figure 1B). Similar data were obtained with passages III to VIII (not shown). During maintained depolarization, the outward K+ current declined with an exponential time course (τ = 271 ± 48 ms at + 50 mV, n = 18). Steady-state activation and inactivation parameters of currents recorded in primary cultured PA cells are presented in Figure 1C. The activation voltage threshold was −50 mV, a value close to the resting membrane potential (−54 ± 4 mV, n = 6). About 15% of the channels failed to inactivate at +20 mV (Figure 1C). The activation and inactivation steady-state curves overlapped, revealing a window K+ current. The pharmacological properties of the PA myocyte K+ channels are illustrated in Figure 1D. Charybdotoxin (CTX; 20 nM), dendrotoxin (DTX; 200 nM) and mast cell degranulating peptide (MCD; 300 nM), potent blockers of both Kv1.2 and Kv1.3, inhibited the outward current by ∼20%, with the effects of DTX and CTX being non-additive. In agreement with previous reports (Post et al., 1995; Yuan, 1995; Archer et al., 1996), we observed that outward K+ currents in PA smooth muscle cells are relatively resistant to tetraethylammonium (TEA) but sensitive to 4-aminopyridine (4-AP). Finally, the appetite-suppressant drug dex-fenfluramine (DFF) which has been shown to induce PA vasoconstriction (Weir et al., 1996) inhibited the outward K+ current by 60% at a concentration of 1 mM. The sensitivity of resistance PA myocytes K+ channels to hypoxia is illustrated in Figure 1E. The hypoxic inhibition of K+ currents in the presence and in the absence of 200 nM DTX was compared (Figure 1F). Hypoxic inhibitions (25 ± 4% and 17 ± 2% in control and in the presence of DTX, respectively) were not statistically different (P 10% inhibition). Figure 9 shows the reversible hypoxic inhibition of Kv2.1 and Kv2.1/Kv9.3. The mean hypoxic inhibitions of the responsive cells were 34 ± 5.5% and 28 ± 2.5% for Kv2.1 and Kv2.1/Kv9.3, respectively. Kv1.2 and Kv1.3 did not display any hypoxic sensitivity in COS cells (n = 11; data not shown). The I–V curves illustrated in Figure 9D show that the effects of hypoxia were independent of the voltage. Figure 9.Hypoxic inhibition of Kv2.1 and Kv2.1/Kv9.3 in COS cells. (A) Reversible inhibition of Kv2.1 by a 5-min hypoxic period. (B) Reversible hypoxic inhibition of Kv2.1/Kv9.3. Timing of hypoxia is indicated in panel (C) by numbers. The holding potential was −60 mV and the test pulse was +30 mV. (C) Time course of Kv2.1/Kv9.3 current inhibition by hypoxia. (D) I–V curves of Kv2.1/Kv9.3 showing the reversible inhibition by 5 min of hypoxia. The holding potential was −80 mV and the cell was depolarized with a voltage ramp of 350 ms in duration to 100 mV. Download figure Download PowerPoint Discussion Molecular identification of K+ channel subunits in rat PA smooth muscle cells The aim of the present work was to identify the oxygen-sensitive voltage-dependent delayed-rectifier K+ channels in rat pulmonary artery smooth muscle cells. Using a PCR approach, we isolated two Shaker subunits Kv1.2 and Kv1.3 (Baumann et al., 1988; Stuhmer et al., 1989; Beckh and Pongs, 1990; Pongs, 1992), one Shab subunit Kv2.1 (Frech et al., 1989) and a novel Shab-related subunit called Kv9.3. The presence of Kv1.2 and Kv1.3 was confirmed at the pharmacological level in PA cells using CTX (blocker of both Kv1.2 and Kv1.3) and DTX and MCD peptide (blockers of Kv1.2). This analysis revealed that the DTX-sensitive channels underly ∼20% of the total K+ channel current in PA cells and do not play a significant role in the oxygen-sensitive K+ channel complexes. Previous reports have clearly demonstrated that hypoxic pulmonary artery vasoconstriction is unaffected by TEA and CTX, blockers of both Kv1.2 and Kv1.3 (Post et al., 1995; Yuan, 1995; Archer et al., 1996). Therefore, these and our present data strongly suggest that Kv1.2 and Kv1.3 do not play a major role as oxygen-sensitive K+ channels in PA myocytes. Delayed-rectifier K+ channels IK(V) and IK(A) have been identified in PA myocytes (Post et al., 1992; Yuan et al., 1993b; Smirnov et al., 1994; Yuan, 1995; Archer, 1996; Archer et al., 1996). IK(V) activates and inactivates rather slowly, whereas IK(A) is a transient current. Kv1.3 shares similarities with the native IK(A) channel recorded in PA myocytes (Honoré et al., 1992), while the existence of both Kv2.1 and Kv1.2 may account for IK(V). Since Kv2.1, Kv1.2 and Kv1.3 are all sensitive to 4-AP, they may account for the 4-AP-sensitive component of PA myocytes. Kv9.3 associates with Kv2.1 and alters its biophysical and pharmacological properties The novel Shab-related subunit, Kv9.3 encodes an electrically silent K+ channel subunit when expressed in Xenopus oocytes and COS cells. However, this silent channel causes important alterations in the biophysical properties of the Shab channel Kv2.1. In particular, Kv9.3 consistently increased Kv2.1 channel current amplitude and shifted steady-state activation towards negative values (−60 to −50 mV). Furthermore, we observed that Kv2.1/Kv9.3 expression induced a shift in the resting membrane potential of both Xenopus oocytes and transfected COS cells (from +4 to −51 mV). The ability of Kv9.3 to ‘drag’ the Kv2.1 activation voltage threshold into the range of PA myocytes RMP suggests that the Kv2.1/Kv9.3 channel complex may contribute to the setting of the RMP (−54 ± 4 mV) and, consequently, in the setting of the resting pulmonary arterial pressure. Kv9.3 also speeded up Kv2.1 activation and dramatically slowed down deactivation. The slowing down of the deactivation was particularly evident in the voltage range corresponding to the resting membrane potential (RMP) of PA myocytes (−50 mV). The steep voltage-dependency of Kv2.1/Kv9.3 suggests that besides its possible role in the establishment of the RMP, this channel may also play an essential role in the control of the PA myocyte action potential duration. Recently, a novel K+ current IK(N) that is active at resting membrane potential, has been described in rabbit PA smooth muscle cells (Evans et al., 1996; Osipenko et al., 1997). This current is voltage-gated with an activation threshold between −80 and −65 mV in K+-rich solution. IK(N) was recorded at all levels of the pulmonary artery tree and was found to be blocked by 4-AP with 50% inhibition at 10 mM (Evans et al., 1996). Most importantly, the close similarity between the biophysical and pharmacological characteristics of IK(N) and the IKv2.1/Kv9.3 suggests that the channels underlying these currents may be one and the same. The alterations in the macroscopic properties of Kv2.1 channel current were confirmed at the single-channel level. Kv9.3 increased the single-channel conductance of Kv2.1, from 8.5 to 14.5 pS. At the pharmacological level, the sensitivity to both 4-AP and TEA were reduced when Kv2.1 was co-expressed with Kv9.3. The alterations in the biophysical and pharmacological properties of Kv2.1 by Kv9.3 suggest that these two subunits associate to form a heteromultimer whose properties are different from that of the Kv2.1 homomultimer. Indeed, immunoprecipitation experiments demonstrated a direct interaction between Kv2.1 and Kv9.3. Kv9.3 belongs to a novel family of electrically silent K+ channels and is the first to be identified in vascular smooth muscle. Other members of this new family have previously been identified in brain (Drewe et al., 1992; Hugnot et al., 1996; Salinas et al., 1997a). These channel subunits do not express a K+ channel current by themselves, but induce profound changes in the properties of the Shab channels Kv2.1 and Kv2.2 (Hugnot et al., 1996; Post et al., 1996; Salinas et al., 1997a). Most interestingly, these silent subunits have the ability to create a diverse range of effects, since Kv8.1 acts as a dominant inhibitory subunit (Hugnot et al., 1996) while Kv9.3 behaves as a stimulatory one (the present report). The Kv2.1/Kv9.3 complex is modulated by internal ATP Our electrophysiological data demonstrate that internal ATP plays an essential role in the control of Kv2.1 and Kv2.1/Kv9.3 channel activity. In inside-out patches, removal of ATP reversibly inhibits channel activity. Th
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