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Physiology, The University of Western Australia, and The Western Australian Institute of Medical Research, Crawley, WA, 6009, Australia
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(Received 19 September 2003;
accepted after revision 18 November 2003;
first published online 21 November 2003)
Corresponding author L. C. Hool: Physiology M311, School of Biomedical and Chemical Sciences, The University of Western Australia, Stirling Highway, Crawley, WA 6009, Australia. Email: lhool{at}cyllene.uwa.edu.au
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Introduction |
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Since the discovery of O2-sensitive K+ channels in the carotid body a number of other cell types have been shown to possess ion channels that respond to changes in oxygen tension. Hypoxia-sensitive K+ channels have been found in pulmonary and systemic myocytes, central neurones, chromaffin cells, smooth muscle and neuroepithelial cells (Lopez-Barneo et al. 2001). Hypoxic pulmonary vasoconstriction is a fast response that reduces blood flow through poorly ventilated alveoli in order to match perfusion to ventilation. This involves a reduction in macroscopic voltage-dependent K+ channels in resistance vessel pulmonary myocytes (Post et al. 1992; Yuan et al. 1993; Osipenko et al. 1997).
However, not all ion channels respond to hypoxia alike. For example, while hypoxia inhibits K+ channels in pulmonary myocytes (Yuan et al. 1993), renal arteries dilate in response to low oxygen as a result of an increase in K+ conductance (Michelakis et al. 2002). The varied responses by ion channels to changes in O2 tension have made it difficult to assign a universal O2-sensing component to the channel. Nevertheless, it has been proposed that the ion channel is itself the oxygen sensor because modulation by hypoxia is rapid and occurs in excised membrane patches where cytosolic variables such as second messengers, ATP and Ca2+ are absent. However, hypoxia also alters cellular redox state by altering cellular production of reactive oxygen species (Ferrari, 1995, 1996; Kroll & Czyzyk-Krzeska, 1998; Hanson & Leibold, 1998; Duranteau et al. 1998; Hool & Arthur, 2002), which can induce changes in protein function (Khan & Wilson, 1995; Wolin, 2000). In addition, alterations in ion channel redox state have been shown to influence ion channel function (Chiamvimonvat et al. 1995; Lacampagne et al. 1995; Campbell et al. 1996; Fearon et al. 1999, 2000; Hool, 2000; Liu & Gutterman, 2002).
Despite considerable characterization of the effects of hypoxia on K+ channels in non-cardiac tissue, no studies have been performed to date that characterize the effects of acute hypoxia on the cardiac delayed rectifier K+ channel. The channel, which is responsible for repolarization in cardiac myocytes is composed of two currents. The slower component of the channel (IKs) is characterized by a delayed onset of activation that can be blocked by chromanol and is encoded by the genes KVLQT1 and KCNE1 (Sanguinetti et al. 1996; Barhanin et al. 1996; McDonald et al. 1997). Mutations in KVLQT1 result in Long QT Syndrome 1 (LQT1) whereby a lengthening of the QT interval leads to increased risk of torsades de pointes and sudden cardiac death (Keating & Sanguinetti, 2001). The rapid current (IKr) is sensitive to block by lanthanum and benzenesulphonamide antiarrhythmics. It activates very rapidly and typically exhibits prominent rectification (Sanguinetti & Jurkiewicz, 1990a). The human ether-a-go-go-related gene (HERG) encodes IKr and mutations in this gene cause Long QT Syndrome 2 (LQT2) (Sanguinetti et al. 1995). Patients with LQT1 typically develop arrhythmias under emotional stress when the adrenergic system is activated (Schwartz et al. 2001).
The aim of this study was to examine the effect of acute hypoxia on both the slow and rapid components of the native delayed rectifier K+ channel in the absence and presence of ß-adrenergic receptor stimulation in isolated guinea-pig ventricular myocytes. We have shown previously that hypoxia can modulate the function of the cardiac L-type Ca2+ channel (Hool, 2000, 2001; Hool & Arthur, 2002). The results of this study indicate that hypoxia also regulates the function of cardiac IKs. Basal IKs is inhibited by hypoxia and the inhibition may involve redox modification of the channel protein because the thiol-specific reducing agent DTT mimicked the effect. In addition, hypoxia increases the sensitivity of IKs to ß-adrenergic receptor stimulation that appears to involve a classical isoform of PKC. This effect can also be mimicked by DTT. However the site of thiol group modification may differ from that involved in basal inhibition of the current by hypoxia alone because the membrane-impermeant thiol-specific oxidizing compound DTNB attenuated the inhibition of basal IKs by hypoxia without altering the increase in sensitivity to Iso. In contrast, hypoxia and ß-adrenergic receptor stimulation do not appear to alter IKr. These findings provide insight into the regulation of the two components of the delayed rectifier K+ channel by ß-adrenergic receptor stimulation during hypoxia.
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Methods |
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For all studies Tricolour guinea-pigs (Cavea porcellis) weighing between 200 and 250 g were used. A total number of 110 animals were anaesthetized with intraperitoneal injection of pentobarbitone sodium (240 mg kg-1) prior to excision of the heart, as approved by The Animal Ethics Committee of The University of Western Australia in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NH & MRC, 6th Edition, 1997). Hearts were initially perfused retrogradely via the aorta with a Krebs Henseleit Buffer (KHB) which contained (mM): NaCl 120, KCl 4.8, CaCl2 1.5, MgSO4 2.2, NaH2PO4 1.2, NaHCO3 25, and glucose 11 (pH maintained at 7.35). The pefusate was then switched to Ca2+-free KHB for 5 min after which Collagenase B (Boehringer Mannheim) was added. After 30–45 min of digestion, the ventricles were cut down and minced in a solution containing (mM): potassium glutamate 110, KCl 25, KH2PO4 10, MgSO4 2, taurine 20, creatine 5, EGTA 0.5, Hepes 5 and glucose 20 (pH adjusted to 7.4 with KOH). The minced tissue was then gently triturated to free myocytes for use within 8 h of isolation.
Data acquisition
The whole-cell configuration of the patch-clamp technique was used to record currents. Microelectrodes with tip diameters of 3–5 µm and resistances of 0.5–1.5 M
contained (mM): potassium glutamate 115, Hepes 10, EGTA 10, KCl 20, MgATP 5, Tris-GTP 0.1, phosphocreatine 10, CaCl2 1 (pH adjusted to 7.05 at 37°C with KOH). Macroscopic currents were recorded using an Axopatch 200B voltage-clamp amplifier (Axon Instruments) and an IBM compatible computer with a Digidata 1200 interface and pCLAMP software (Axon Instruments). A Ag–AgCl electrode (Clark Electrodes, Clark Electromedical Instruments) was used to ground the bath. Currents were measured in extracellular modified Tyrode solution that contained (mM): NaCl 140, KCl 5.4, CaCl2 2.5, MgCl2 0.5, Hepes 5.5, glucose 11, glibenclamide 0.01 (pH adjusted to 7.4 with NaOH). The solution was delivered to cells through a fast flow apparatus that allowed rapid changes (< 1 s) in extracellular solutions delivered to the myocyte (Hool, 2000). The solution was made hypoxic by bubbling with 100% nitrogen in a glass reservoir and delivered to the cells via a combination of stainless steel and Polyethylene tubing as previously described (Hool, 2000). All hypoxia experiments were performed at 
17 mmHg oxygen tension as determined by an oxygen-sensitive probe placed in front of the outlet of the fast flow apparatus delivering hypoxic solution at the point where currents are recorded (Hool, 2000). All experiments were performed at 36°C.
Once the whole-cell configuration was achieved, the holding potential was set at –80 mV. Na+ channels and T-type Ca2+ channels were inactivated by applying a 50 ms prepulse to –40 mV immediately before each test pulse. L-type Ca2+ channels were blocked by adding 2 µM nisoldipine to all external solutions. When studying the slow component of the delayed rectifier K+ channel (IKs), the time course of changes in K+ conductance was monitored by applying a 3 s test pulse to +50 mV once every 10 s. At the completion of the 3 s test pulse the cell was stepped to –40 mV. IKs was defined as the time-dependent current elicited during the step to +50 mV. When studying the rapid component of the delayed rectifier K+ channel (IKr), 10 µM chromanol 293B (Tocris) was added to the bath to block IKs, and IKr was defined as the lanthanum-sensitive tail currents elicited upon return to –40 mV after test pulses to various voltages from –40 to +40 mV (200 ms duration, 20 mV increments).
Results are reported as means ±S.E.M. with n indicating the number of cells studied. Statistical comparisons of responses between unpaired data were made using the Student's t test or between groups of cells using one-way ANOVA and Tukey's post hoc test (GraphPad Prism 3.02).
Synthesis of PKC peptides
The PKCß peptide was synthesized from ßC2-4 (SLNPEWNET; amino acids 218–226) (Ron et al. 1995) and PKC
peptide from V1-2 (EAVSLKPT; amino acids 285–292) (Dorn et al. 1999). Both peptides were synthesized and purified at the Protein Facility, Biochemistry, The University of Western Australia. All peptides were > 86% pure.
Solutions
Most drugs were prepared as stock solutions in water so that the desired final concentration was achieved by 1: 1000 dilution with the control extracellular solution. Glibenclamide (Research Biochemicals International) was initially dissolved in dimethyl sulphoxide and nisoldipine (a gift from Bayer Australia Ltd) was dissolved in polyethylene glycol before being diluted 1: 1000 in extracellular solution. All other drugs were obtained from Sigma Chemical Co. and dissolved in water as stock. To minimize the possibility of oxidative degradation, ascorbic acid is commonly added to solutions containing the ß-adrenergic receptor agonist isoproterenol (Iso). However, to prevent any direct effect of the antioxidant on channel function, ascorbic acid was omitted from all solutions and solutions containing Iso were prepared fresh and changed every 60–90 min
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Results |
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The effect of lowering oxygen tension on basal IKs channel activity was examined. IKs was initially recorded in cells superfused with Tyrode solution at room oxygen tension (PO2 of 150 mmHg) and then in cells exposed to Tyrode solution made hypoxic to a PO2 of 17 mmHg. In 29 cells, hypoxia caused a 21.9 ± 1.8% decrease in basal current that could be reversed upon re-exposure of the cells to control solution at room oxygen tension (Fig. 1A). This represented a decrease in current density from 3.89 ± 0.5 to 3.03 ± 0.32 pA pF-1 (P < 0.05; Student's paired t test).
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), derived from single exponential fits to the IKs tail currents, increased from 165 ± 6.0 to 296 ± 8.2 ms during hypoxia (P < 0.001; Student's paired t test). We examined whether the inhibition of basal IKs during hypoxia involved the reduction of thiol groups on the channel protein or an alternative protein that may alter channel function. If this were true, then exposure of cells to a thiol-specific oxidizing compound should attenuate the effect of hypoxia. Cells were exposed firstly to hypoxia followed by hypoxia in the presence of the relatively membrane-impermeant thiol-specific oxidizing agent DTNB. Figure 2A illustrates the protocol in a typical experiment. In six cells, hypoxia caused a 25.0 ± 2.2% decrease in basal current. Subsequent application of 200 µM DTNB significantly attenuated the inhibition associated with hypoxia by 74.8 ± 1.3% (P < 0.01; Fig. 2C). When cells were exposed first to DTNB and subsequently to hypoxia and DTNB the effect of hypoxia was also attenuated. In five cells, consistent with previous results (Coetzee et al. 1995; Chiamvimonvat et al. 1995; Lacampagne et al. 1995; Han et al. 1996; Szabo et al. 1997; Hool, 2000; Tang et al. 2001), exposure of cells to 200 µM DTNB alone initially caused a small rapid decrease in basal current of 9.8 ± 5.0%. However, when the cells were then exposed to hypoxia in the presence of DTNB, there was either no change in current magnitude or a small further decrease in current. In five cells there was an average decrease of 1.6 ± 0.9%, which was not statistically significantly different from the initial decrease in current produced by DTNB alone. To further examine a role for redox modification of thiol groups in the hypoxic response, cells were exposed to the thiol reducing agent dithiothreitol (DTT). In five cells, 1 mM DTT caused a 21.2 ± 5.4% decrease in current which was similar to the magnitude of inhibition caused by hypoxia alone (21.9 ± 1.8%, Fig. 1, n= 24; n.s.). Additionally, when cells were exposed to hypoxia first and then DTT in the presence of hypoxia, DTT did not cause any further decrease in current (23.8 ± 4.5% inhibition during hypoxia alone versus 22.8 ± 5.6% inhibition during hypoxia and DTT; n= 5, n.s.). Figure 2C summarizes the results. These data suggest that the inhibition of basal IKs by hypoxia involves the reduction of thiol groups. Since DTNB is membrane impermeant (Aizenman et al. 1989; Lei et al. 1992), the results with DTNB suggest the effect of hypoxia may involve thiol groups on the extracellular side of the channel protein or a nearby effector protein.
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It is well recognized that activation of ß-adrenergic receptors leads to an increase in magnitude of IKs via a protein kinase A-dependent phosphorylation of the channel (Walsh et al. 1989; Walsh & Kass, 1991). The effect of hypoxia on the sensitivity of IKs to the ß-adrenergic receptor agonist Iso was examined next. In the absence of hypoxia, exposure of myocytes to 1 nM Iso produced a subthreshold response and the current was maximally stimulated in the presence of 1 µM Iso (Fig. 3A). The K0.5 for activation of the current in the absence of hypoxia was 18.3 ± 3.9 nM (Fig. 4A). As before, when cells were exposed to hypoxia alone basal IKs was inhibited (Fig. 3B). This time 0.1 nM Iso produced a threshold response and IKs was near-maximally activated at 10 nM Iso (Fig. 3B). The K0.5 for activation of the current in the presence of hypoxia was significantly decreased to 1.88 ± 0.43 nM (Fig. 4A; P < 0.05). In addition, hypoxia did not alter the response to 1 µM Iso, a maximally stimulating concentration of the agonist (5.3 ± 0.66 pA pF-1, n= 11 during hypoxia versus 6.01 ± 1.14 pA pF-1n= 8 during normoxia; n.s.).
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The altered sensitivity of IKs to isoproterenol involves the redox modification of thiol groups
We hypothesized that redox modification of critical thiol groups was involved in the increase in sensitivity of IKs to Iso. The effect of hypoxia on basal IKs can be mimicked by the thiol reducing agent DTT (Fig. 2). We examined the effect of DTT on the sensitivity of IKs to Iso. Cells were exposed to DTT alone followed by DTT in the presence of 1 nM and 3 nM Iso. These responses were then normalized to a maximally stimulating concentration of Iso (1 µM) in the same cell. As before, 1 mM DTT caused a 21.1 ± 5.4% decrease in basal IKs. However, in the continued presence of DTT, 1 nM and 3 nM Iso produced currents that were 13.8 ± 3.9 and 62.5 ± 5.9% of the current elicited by 1 µM Iso within the same cell (n= 6; Fig. 5A). These currents were significantly larger (P < 0.001) than currents elicited by the same concentrations of Iso in room oxygen tension (Fig. 4A). To further determine whether the altered sensitivity of IKs to Iso involved redox modification of thiol groups, cells were exposed first to hypoxia and then to 200 µM DTNB and Iso in the continued presence of hypoxia. In six cells, hypoxia caused a 25.0 ± 2.2% decrease in basal IKs. The addition of DTNB attenuated the effect of hypoxia by 74.8 ± 1.3%. However, subsequent exposure to 3 and 10 nM Iso elicited currents that were 84.1 ± 7.5 and 100% of the current elicited by 1 µM Iso in the same cell (n= 6; Fig. 5B). The magnitude of these currents was significantly larger (P < 0.001) than the currents elicited by the same concentrations of Iso in room oxygen (Figs 3A and 4A) and similar to currents elicited by the same concentrations of Iso during hypoxia in the absence of DTNB (Figs 3B and 4A). These data suggest that redox modification of thiol groups is involved in the increase in sensitivity of IKs to Iso. Since DTNB is membrane impermeant, the site of the mechanism may differ from the site involved in the inhibition of basal IKs by hypoxia.
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To examine whether the effect of hypoxia is occurring at the level of the ß-adrenergic receptor, experiments were performed to examine the effect of hypoxia on IKs activated by histamine. Histamine activates IKs through the same cAMP-dependent pathway as ß-adrenergic receptor agonists but by binding H2-histaminergic receptors. We exposed cells to 30 nM histamine followed by 3 µM histamine, a maximally stimulating concentration of the agonist, and compared the response within the same cell. In the absence of hypoxia, 30 nM histamine activated a current that was 20.2 ± 9.3% of the current activated by 3 µM histamine within the same cell (n= 5, Fig. 6A). When a separate group of cells were exposed to hypoxia, basal IKs was inhibited 19.6 ± 2.8%. In the continued presence of hypoxia, 30 nM histamine now activated a current that was 95.5 ± 3.3% of the current elicited by 3 µM histamine within the same cell (n= 6, P < 0.01, Fig. 6B). To determine whether hypoxia acts a point distal to the ß-adrenergic receptor, additional experiments were performed to examine the effect of hypoxia on the response of the channel to forskolin. Forskolin increases IKs by the same cAMP-dependent pathway as ß-adrenergic receptor agonists but by direct activation of cAMP itself. In the absence of hypoxia, 50 nM forskolin activated a current that was 7.6 ± 4.5% of the current elicited by a maximally stimulating concentration of forskolin in the same cell (10 µM, n= 5). In the presence of hypoxia, as before basal IKs was inhibited 19.1 ± 2.0%. However, in the continued presence of hypoxia, 50 nM forskolin activated a current that was 52.1 ± 11.5% of the current elicited by 10 µM forskolin (n= 7, P < 0.05). These data indicate that hypoxia increases the sensitivity of IKs to Iso at a point downstream from the ß-adrenergic receptor.
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The sensitivity of the cardiac L-type Ca2+ channel to Iso is increased in the presence of hypoxia and this increase can be attenuated when PKCß peptide is placed in the pipette solution (Hool, 2000). In addition, recent work has implicated PKCßII and PKC in the regulation of human cardiac delayed rectifier K+ channels (Xiao et al. 2003). The C2-4 peptide derived from PKCß can block the classical PKC isoforms by preventing the translocation and binding of the isoform to its receptor for activated C kinase (Ron et al. 1995). The classical PKC isoforms include PKC
, PKCßI, PKCßII and PKC
and are calcium sensitive. Since the results of the current study suggest that the effect of hypoxia is mediated downstream from the ß-adrenergic receptor, we hypothesized an involvement of PKC. To determine which isoforms of PKC may be involved, we synthesized ßC2-4 peptide and V1-2 peptide (which prevents binding of the calcium-insensitive PKC isoform) and added 100 nM each peptide inhibitor to the pipette solution in separate sets of experiments. First, cells were dialysed with the PKCß peptide and then exposed to hypoxia followed by 3 and 10 nM Iso. The responses to 3 and 10 nM Iso were then compared with the current elicited by 1 µM Iso in the same cell. Figure 7A illustrates the protocol in a typical experiment. In the presence of the PKCß peptide, hypoxia caused a 20.3 ± 2.4% reduction in basal IKs. However, 3 and 10 nM Iso activated currents that were 23.3 ± 4.2 and 59.8 ± 7.3% of the current elicited by 1 µM Iso (n= 5). The magnitude of these currents was significantly less (P < 0.05) than currents activated by the same concentrations of Iso during hypoxia in the absence of PKCß peptide (Fig. 4A). To determine a possible role for PKC and any non-specific effect of the peptide inhibitors on IKs, cells were dialysed with 100 nM PKC peptide and exposed to increasing concentrations of Iso in the presence of hypoxia. Contrary to the effects of PKCß peptide inhibitor, dialysis of cells with PKC peptide did not alter the increase in sensitivity of IKs to Iso during hypoxia. In the presence of PKC peptide, hypoxia caused a 21.3 ± 2.4% decrease in basal IKs. In the continued presence of the peptide inhibitor and hypoxia, 3 and 10 nM Iso activated currents that were 79.4 ± 9.7 and 88.6 ± 9.7% of the current elicited by 1 µM Iso (n= 5; Fig. 7B). The magnitude of these currents was not significantly different from currents activated by the same concentrations of Iso in the presence of hypoxia and in the absence of peptide inhibitor (Figs 3B and 4A). These results suggest that the mechanism by which hypoxia increases the sensitivity of IKs to Iso involves a classical isoform of PKC.
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To determine whether hypoxia or Iso modulate IKr, first IKs was recorded. Cells were then exposed to 10 µM chromanol 293B a highly specific blocker of IKs (Busch et al. 1996). In 24 cells, chromanol 293B inhibited IKs 93.2 ± 3.4%. The cells were then stepped from –40 to +40 mV (200 ms duration) in 20 mV increments and IKr, defined as the lanthanum-sensitive tail current, was elicited upon return to –40 mV (Sanguinetti & Jurkiewicz, 1990b; Sanguinetti et al. 1995). Lanthanum chloride (10 µM) completely inhibited IKr. The effect of Iso on IKr in the absence of hypoxia was examined first. Exposure of cells to concentrations of Iso up to 1 µM did not alter IKr, even when measurements were repeated after 2 min direct exposure to the agonist in nine cells. These results are consistent with previous reports in guinea-pig ventricular myocytes (Sanguinetti et al. 1991, 1995). In addition, exposure of seven cells to hypoxia alone or in the presence of Iso did not alter the magnitude of IKr at any potential.
An absence of an effect of hypoxia on IKr may suggest that the channel protein is unable to be modified by redox agents. To test this, cells were exposed to 1 mM DTT and 200 µM DTNB and IKr was recorded. Similar to the effect of hypoxia, DTT and DTNB did not appear to alter IKr at any test potential (n= 8). Measurements were made after up to 2 min direct exposure to each drug. Although 1 mM DTT may not be sufficient to reduce all thiol groups in all proteins in the native channel, the data presented here are consistent with data reported in HERG channels exposed to 5 mM DTT for 10–15 min (Dun et al. 1999; Fan et al. 1999). These results suggest that Iso, hypoxia and thiol-specific reducing or oxidizing compounds appear to be without effect on the rapid component of native IK.
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Discussion |
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In contrast, the results of this study indicate that hypoxia does not appear to affect the rapid component of the delayed rectifier K+ channel. Current amplitudes of native guinea-pig IKr channels are small and we concede that a small effect of hypoxia cannot be totally excluded. Although there is not general agreement, functional cardiac IKr channels may require the expression of a minK and HERG complex (McDonald et al. 1997) or a minK-related peptide (MiRP1) and HERG complex (Pond & Nerbonne, 2001; Weerapura et al. 2002). One possible explanation for the absence of an effect of hypoxia on IKr could be that channel function is unable to be modulated by redox modification of thiol groups. Consistent with this, we could not record any effect of DTT or DTNB on IKr. In further support of this argument, DTT, hydrogen peroxide, [2-(trimethylammonium)ethyl]methanethiosulphonate (MTSET) and 2-sulphonatoethylmethanethiosulphonate (MTSES) are without effect on HERG channels expressed in Xenopus oocytes (Fan et al. 1999; Dun et al. 1999).
The effect of isoproterenol in the absence and presence of hypoxia on the two components of IK was also examined. Similar to results previously reported from this laboratory on L-type Ca2+ channels (Hool, 2000, 2001; Hool & Arthur, 2002), hypoxia decreased the K0.5 for activation of IKs by Iso (Figs 3 and 4). This effect could be mimicked by DTT but was unaffected by DTNB (Fig. 5), suggesting that the mechanism involves the reduction of thiol groups and the redox site for the increase in sensitivity to Iso may be different from the site involved in basal inhibition of IKs during hypoxia. Consistent with this, and effects of hypoxia on the L-type Ca2+ channel (Hool, 2000), hypoxia increased the sensitivity of IKs to histamine (Fig. 6) and forskolin. In addition, the increase in sensitivity to Iso could be attenuated when PKCß peptide was placed in the pipette solution but not in the presence of PKC peptide, while the inhibition of basal channel activity by hypoxia was unchanged (Fig. 7). An involvement of PKC, particularly the ß isoform, in hypoxic responses has been well described before (Takeishi et al. 1999; Bowling et al. 1999; Simpson, 1999). We have previously proposed that PKC may be facilitating PKA-dependent phosphorylation of the L-type Ca2+ channel and the redox modification of thiol groups either on the channel protein or an intermediate protein mediates this response (Hool, 2000, 2001; Hool & Arthur, 2002). The results of this study suggest that hypoxia may regulate the activity of IKs in a similar manner.
In contrast, IKr was unable to be modulated by hypoxia or Iso. Although the HERG channel contains a segment homologous to a cyclic nucleotide binding domain (Warmke & Ganetzky, 1994) and at least four putative phosphorylation sites for protein kinase A (Cui et al. 2000), agonists that increase protein kinase A or cAMP activity have been reported to have no effect (Sanguinetti et al. 1991, 1995) or an inhibition of IKr at high concentrations of isoproterenol in guinea-pig ventricular myocytes (1–10 µM) (Karle et al. 2002). Since the conditions during hypoxia were similar when recording IKs and IKr, this would suggest that the channel protein complex responsible for IKr is differentially regulated by hypoxia. We cannot rule out the possibility that hypoxia is altering the activity of cAMP, protein kinase A, a phosphatase or another intermediate protein. But it is also reasonable to propose that hypoxia and Iso are unable to modulate IKr activity because the channel proteins are not able to be redox modified in the same way that IKs is modified.
Mutations in KVLQT1, which codes for the subunit of IKs, cause LQT1 and mutations in KCNE1, which codes for the ß subunit of IKs, cause LQT5 (Splawski et al. 1997). cAMP-dependent protein kinase A phosphorylation of the KCNQ1 N terminus requires coexpression of KCNQ1–KCNE1 channels for functional responses (Kurokawa et al. 2003). Carriers of mutations in KVLQT1 are at greatest risk of experiencing fatal cardiac arrhythmia during elevated sympathetic nervous system activity (Schwartz et al. 2001) and treatment with ß-adrenergic receptor blocking drugs is effective in these patients (Moss et al. 2000). Mutations in HERG, which codes for IKr, cause LQT2. Both components of IK play a significant role in cardiac repolarization. It has been proposed that an important function of IKs is to counteract the excessive prolongation of the action potential duration induced by ß-adrenergic enhancement of the L-type Ca2+ channel (Han et al. 2001). Enhancement of the L-type Ca2+ channel promotes arrhythmogenic afterdepolarizations (Marban et al. 1986; January & Riddle, 1989). Results from this study and previous work (Hool, 2000; Hool & Arthur, 2002) indicate that hypoxia increases the sensitivity of IKs and the L-type Ca2+ channel without affecting IKr. These data provide some insight into relative risk of arrhythmia in patients with LQT syndrome. Patients who have a mutation in the genes KVLQT1 or KCNE1 may well be at greater risk of developing life-threatening arrhythmias during hypoxia and emotional stress than patients with a mutation in HERG alone.
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