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Departments of *Anesthesiology,
Physiology, and
Pharmacology & Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin
Address correspondence and reprint requests to Wai-Meng Kwok, PhD, Department of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Address e-mail to wmkwok{at}mcw.edu
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Abstract |
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IMPLICATIONS: The effects of a volatile anesthetic, isoflurane, were determined on a cardiac potassium channel current, IKs, a major ionic component underlying the cardiac action potential. The result shows that IKs is significantly inhibited by isoflurane. This may contribute to anesthetic-induced changes in the electrocardiogram, particularly the prolongation of the QT interval.
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Introduction |
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The IK is composed of two different current types. Recent studies have clearly identified IK as two distinct channel entities: the slowly activating delayed-rectifier potassium channel current, IKs, and the rapidly activating type, IKr. Dysfunctions of IKs and IKr have been genetically linked to the LQTS, and 90% of the identified LQTS mutations found to date are related to these channels (4). It is interesting to note, however, that the acquired LQTS caused by various drugs, such as dofetilide and sotalol (5), is mostly induced by inhibition of IKr because of its unique channel structure causing the drugs to be trapped in the pore region, causing channel dysfunction (6). Although the total IK (combination of IKs and IKr) is highly sensitive to anesthetics, IKr seems less sensitive to isoflurane, whereas even a large concentration of isoflurane attenuated IKr to a small degree (7). Thus, the inhibition of IKr alone cannot explain the observed significant IK attenuation by volatile anesthetics (2,3,8).
Because the structures of volatile anesthetics differ from those drugs that cause acquired LQTS, we hypothesized that IKs has an important role during anesthetic-induced AP prolongation, unlike the common drug-induced AP prolongation caused by IKr inhibition. However, the anesthetic effect on IKs alone has not been characterized. In this study, the effect of isoflurane on IKs was investigated at both room (22°C) and physiological (36°C) temperatures.
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Methods |
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All chemicals were purchased from Sigma Chemical Co. unless otherwise noted. To isolate for IKs, the following reagents were used in the external solution (in mM): N-methyl-D-glucamine (for sodium ion replacement) 132, CaCl2 1, MgCl2 2, KCl 1 (to reduce the inward-rectifier potassium current contribution), HEPES 10, lanthanum chloride 0.05 (to block IKr), and cadmium chloride 0.2 (to block the L-type calcium current) (pH adjusted to 7.4 with HCl). The pipette solution contained the following (in mM): K-glutamate 60, KCl 50, HEPES 10, MgCl2 1, EGTA 11, CaCl2 1, and K2-adenosine triphosphate 5 (pH adjusted to 7.4 with KOH). This ratio of EGTA to CaCl2 resulted in an estimated free intracellular Ca2+ of 
10 nM (11).
Isoflurane was dispersed in the external potassium solution by sonication, kept in glass-syringe reservoirs to ensure a constant concentration, and delivered to the recording chamber by using syringe pumps. To verify anesthetic concentrations, 1 mL of the superfusate containing isoflurane was collected in a metal-capped 2-mL glass vial at the end of each experiment. Samples were then analyzed by gas chromatography as reported previously (9). Anesthetic concentrations are expressed as aqueous concentrations, which are relatively insensitive to temperature.
A drop of cells suspended in the modified Tyrode solution was placed in a flow-through chamber mounted on the stage of an inverted microscope. Only rod-shaped cells with clear borders and striations were selected for the experiments performed within 12 h after isolation. Experiments were conducted at 22°C–25°C or 35°C–37°C by using a recording chamber connected to a temperature-controller unit. Patch pipettes were pulled from borosilicate glass capillaries with a micropipette puller and heat-polished by using a microforge. The resistances of pipettes, when filled with the internal solution and immersed in the modified Tyrode solution, were 2–4 M
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Standard whole-cell configuration of the patch-clamp technique was used to monitor IKs. Gigaohm seal and rupture of the membrane were achieved in the modified Tyrode solution, followed by superfusion of the chamber with the external potassium solution. Negative pressure was maintained on these electrodes to minimize cell dialysis and related current rundown. Currents were recorded with a List EPC-7 amplifier interfaced to a computer via an Axon Instruments 1200A Digidata board, sampled at 10 kHz, and low-pass filtered at a cutoff frequency of 3 kHz. Series resistance compensation was adjusted to give the fastest possible capacity transient without feedback instability in the voltage clamp circuit, and the maximum voltage error was calculated as <5 mV.
IKs was elicited during 2-s depolarizing test pulses to +80 mV from a holding potential of -40 mV (in 20-mV increments). The IKs deactivating tail currents were monitored during the return to the -40 mV holding potential after the depolarizing test pulses. Data acquisition and analyses were performed with the pClamp software. Additional data and statistical analyses were performed with ORIGIN software (Version 6.0; OriginLab Inc., Northampton, MA).
Because a unique feature of IKs is its noninactivating characteristic with nonsteady-state activation kinetics, all analyses were performed on the IKs tail currents. Results are compared by using the value on return to -40 mV after a test pulse to +60 mV. The effect of isoflurane on IKs is reported as a percentage change of the tail current amplitude in the presence of isoflurane compared with the control conditions. The deactivating tail current was fitted with a standard double-exponential function (12): equation
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where I is the current amplitude; 
f and s are the fast and slow time constants, respectively; and A1, A2, and C are constants. The peak IKs tail-current amplitude was determined by extrapolation of the fitted curves to the end of the depolarizing test pulse (13).
The voltage dependence of IKs activation was determined by constructing an activation curve based on tail-current amplitude and normalized in reference to the tail-current amplitude recorded after a test pulse to +80 mV (I80). The IKs activation curves were fitted to a Boltzmann equation of the following form: equation
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where Vt is the test-pulse potential, V1/2 is the half-activation voltage, and k is the slope factor.
IKr was elicited during 250-ms depolarizing test pulses to +60 mV from a holding potential of -40 mV (in 20-mV increments), similar to the protocol that was previously reported (14). Chromanol 293B (30 µM; Tocris, Ellisville, MO) was used to block IKs. The effect of isoflurane on IKr tail-current amplitude was determined as described previously.
Data are presented as means ± SEM. Paired and unpaired Student’s t-tests were used to compare means between control and anesthetic treatments and between two groups. For comparisons among three different anesthetic-concentration groups, a one-way analysis of variance withposthocpair wise correction (Fisher’s protected least significant difference test) was used. In all comparisons, a value of P< 0.05 was considered statistically significant.
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Results |
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f and s, to accommodate the fast and slow components. Isoflurane did not affect the deactivation kinetics, and both time constants (f and s) remained unchanged (Table 1). The effect of isoflurane (0.3 mM) on IKr at 36°C was also investigated. On the basis of the tail-current amplitude, isoflurane was found to attenuate IKr by 21% ± 2% (n = 6). This inhibition was significantly less than that on IKs (P < 0.05; Fig. 3).
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Compared with the IKs wave form at 36°C, the current activation and deactivation kinetics were slower at 22°C (Fig. 4A). Similar to the effect observed at 36°C, 0.6 mM isoflurane reversibly inhibited IKs at room temperature, and this inhibition was concentration dependent. However, the magnitude of inhibition was temperature dependent, where the effect of isoflurane on IKs at 22°C was significantly more than that at 36°C (Fig. 2). This effect was evident at the clinically relevant concentrations of 0.3 and 0.6 mM but not at the large concentration of 1.1 mM.
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The IKs deactivation kinetics were markedly slower compared with those at 36°C, where both f and s were significantly greater (P < 0.05) at 22°C. Thus, as expected, the open- to closed-state transitions were slowed at room temperature. Isoflurane accelerated the IKs deactivation at 22°C, but not at 36°C. The slow time constant for deactivation, s, was significantly decreased by isoflurane at 22°C, resulting in an acceleration of IKs deactivation (Table 1). Although there was a tendency for f to decrease in the presence of isoflurane, it was not significantly affected by the anesthetic.
Because the effect of isoflurane was diminished at 36°C, a temperature-dependent modulation of IKs by an intracellular second-messenger pathway may be involved. PKC modulates IKs in a temperature-dependent manner (17). At or near physiological temperatures, IKs is enhanced by PKC. However, at room temperature, PKC does not modulate IKs. Thus, the diminished effect of isoflurane on IKs at 36°C may be due to an enhancement of IKs by basal PKC. This hypothesis was tested by investigating the effect of PKC activation by phorbol 12-myristate 13-acetate (PMA) on IKs.
PKC activation by PMA (0.2 µM) had no significant effect on IKs at 22°C, in agreement with previously published reports (17). The current amplitude, voltage dependence of activation (V1/2, 38.9 ± 2.2 mV; k, 14.6 ± 1.1 mV), and deactivation kinetics (f, 420 ± 20 ms; s, 1545 ± 113 ms) were not affected by PMA. The effect of isoflurane (0.6 mM) in the presence of PMA was not significantly different from that without PKC activation. Consequently, as expected, PKC did not modulate isoflurane’s action on IKs at room temperature.
At 36°C, PKC activation by PMA enhanced IKs. At +60mV, IKs increased by 37.9% ± 9.9%. Although PMA enhanced the current amplitude, there was no significant effect of PMA on the activation curve (V1/2, 19.0 ± 0.6 mV; k, 13.6 ± 0.6 mV) or deactivation kinetics (f, 105 ± 13 ms; s, 389 ± 26 ms). Subsequent application of isoflurane (0.6 mM) attenuated IKs. Net inhibition of IKs by isoflurane under PKC activation was 53.0% ± 5.3%, which was significantly smaller than that without PKC stimulation (Fig. 5). This inhibition was calculated from the control (PMA-free, isoflurane-free) level. However, when the isoflurane effect was calculated from the maximal PMA effect, isoflurane inhibited the PMA-enhanced current by 63.8% ± 2.7%. This was not significantly different from the isoflurane effect on IKs in the absence of PKC activation. Thus, PKC did not modulate the isoflurane effect on IKs. The structural analog of PMA (4
-phorbol 12,13-didecanoate) that does not activate PKC (20) failed to enhance IKs at physiological temperature. Thus, the observed changes by PMA were confirmed as a PKC-related action.
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Discussion |
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On the basis of our results, the inhibitory effect of isoflurane on IKs appears to be the greatest among the other cardiac sarcolemmal ion channels. It was previously reported that 1 mM isoflurane depressed the fast sodium channel by 11% (9) and that 0.8 mM isoflurane attenuated the L-type calcium channel by approximately 23% (16). Isoflurane at 0.51 mM inhibited the transient outward potassium channel current by 25% in human atrial myocytes (15). However, isoflurane had a small but significant voltage-dependent biphasic effect on the inward-rectifier potassium channel (10). Isoflurane can also activate rather than inhibit the adenosine triphosphate-sensitive potassium channel (21). These effects of volatile anesthetics on the various cardiac ion channels were all obtained from experiments conducted at room temperature. Thus, it appears that IKs is relatively more sensitive to inhibition by isoflurane.
Isoflurane had a temperature-dependent inhibition of IKs at clinically relevant concentrations of 0.3 and 0.6 mM, where the inhibition was more at 22°C than that at 36°C. At 1.1 mM, the temperature-dependent block was not observed. Although not tested, inhibition of IKs by isoflurane may have reached a maximal effect at this concentration. Larger concentrations were not used because they would have been outside the clinically relevant range. Temperature-dependent anesthetic inhibition has been shown on ligand-gated channels, such as the nicotinic acetylcholine receptor (19) and the 
-aminobutyric acid type A receptor channel (18). For ligand-gated channels, this temperature-dependent inhibition supports the protein hypothesis (22), whereby volatile anesthetics act by competitive binding to a specific receptor protein. When the same aqueous concentration of volatile anesthetic is used, the increased inhibitory effect at the lower temperature cannot be adequately explained by the lipid hypothesis (23), where the anesthetic concentration at the lipid bilayer is less at the lower temperature. Under this condition, a smaller inhibition of IKs by isoflurane would have been expected. However, our results showed increased inhibition at the lower temperature. On the other hand, the temperature-dependent inhibitory effect of isoflurane and the effect on s at 22°C would appear to support the protein hypothesis of anesthetic interaction.
During IKs deactivation, the channel makes the transition from the open to closed state. This temperature-dependent result suggests that at 22°C, the slower rate of transition from the open to closed states may allow the anesthetic to affect the conformational transitions between the channel states. Although isoflurane accelerated the IKs deactivation kinetics at 22°C, it had no effect on deactivation kinetics at 36°C. At the higher temperature, faster transitions between channel states may deter the anesthetic from interacting with the transition states. These results implicate an overestimation of the anesthetic effect on IKs when experiments are conducted at room temperature. Whether this temperature-dependent effect of volatile anesthetics on ion channels is a common underlying feature of anesthetic interaction with voltage-gated channel proteins or whether this is specific for this particular channel needs to be determined in future studies. Therefore, this emphasizes the importance of temperature-dependent effects of volatile anesthetics on ion channels.
The two time constants that characterize IKs deactivation were differentially affected by isoflurane at 22°C, where s was decreased but f was unaffected. In addition, the effect on s was not isoflurane concentration-dependent. This is in contrast to the concentration-dependent effect on current amplitude. These results suggest that the mechanism underlying the effects of isoflurane on IKs may involve multiple points of interaction. The action of isoflurane on the open channel and on the deactivating channel may not be identical. In other words, the effects of isoflurane may be dependent on the various conformations of the IKs channel protein. This conformation-dependent effect may also explain the differential effects of isoflurane on s and f, which reflects different transition states. Further experiments will be necessary to confirm this hypothesis.
The IKs activation curves were not affected by isoflurane at clinically relevant concentrations of 0.3 and 0.6 mM at both 22°C and 36°C. The slope factor k was not changed at either temperature, suggesting that the voltage sensor of the channel was not affected by isoflurane. Only at a large concentration of 1.1 mM did isoflurane induce a small hyperpolarizing shift of the activation curves. Physiologically, this can result in increased conductance of the current at a given membrane potential, leading to an enhanced current flow. Because IKs inhibition, and not enhancement, was observed at the three concentrations of isoflurane tested, it is highly unlikely that the observed shift in the activation at 1.1 mM isoflurane contributed to this inhibition.
The observed shift in the activation curve may be due to the effect of isoflurane on the minK protein, the accessory subunit of IKs. Mutations in the minK protein are known to suppress IKs function and to cause congenital LQTS (24); minK is also reported to shift the IKs activation curve in the depolarizing direction (25). Thus, a diminished influence of minK on the IKs protein may lead to a hyperpolarizing shift in the activation curve. This may have resulted from the action of isoflurane on the minK subunit.
The temperature-dependent modulation of IKs by PKC was similar to that reported previously (17). In the PKC experiments, when the absolute effect of isoflurane was calculated from the level of maximum PMA effect, there was no significant difference in the inhibition of IKs compared with that in the absence of PMA. This suggests that the inhibitory effect of isoflurane on IKs was not affected by PKC activation. However, when the net effect of isoflurane was calculated from the control (PMA-free, isoflurane-free) conditions, the anesthetic effect on IKs in the presence of PMA was diminished. Consequently, the difference in IKs inhibition at 36°C versus 22°C may be due to basal PKC activity at 36°C, which can result in enhanced current amplitude. In this case, the overall effect of isoflurane on IKs would appear diminished.
IKs inhibition leads to delayed repolarization and results in AP prolongation. We have previously shown that isoflurane prolongs AP at a clinically relevant concentration (3). Because the QT interval in the electrocardiogram reflects AP duration, our results suggest that the observed IKs inhibition is one of the factors that contributes to anesthetic-induced QT prolongation.
The high sensitivity of IKs to isoflurane suggests that the mechanism underlying isoflurane-induced QT prolongation is different from that often caused by various drugs that mostly block IKr (6). In addition, most of the drug-induced QT prolongation is caused by the unique pore structure of the IKr channel where the drugs are trapped inside. This may not be the case for the smaller isoflurane molecule.
The AP and QT prolongation itself has been proposed as an important antiarrhythmic mechanism, and IKs and IKr have been the targets for many Class III antiarrhythmic drugs. Thus, isoflurane can be expected to have Class III action. However, IKs is also regulated by other physiological factors, such as autonomic responses and intracellular calcium. Whether IKs contributes to antiarrhythmic action by isoflurane still needs to be determined. Therefore, more extensive studies are needed to predict the direct clinical outcome from the observed IKs inhibition by isoflurane.
In summary, isoflurane had a concentration-dependent reversible inhibitory effect on IKs. This inhibition was temperature dependent, where IKs was more sensitive to isoflurane at room temperature. Because IKs, together with IKr, contributes to the maintenance of the plateau phase of the cardiac AP, isoflurane-induced depression of IKs may be one of the causes of AP prolongation and, thus, QT prolongation.
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Acknowledgments |
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Footnotes |
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This article has been cited by other articles:
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  W. Ouyang and H. C. Hemmings Jr. Depression by Isoflurane of the Action Potential and Underlying Voltage-Gated Ion Currents in Isolated Rat Neurohypophysial Nerve Terminals J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 801 - 808. [Abstract] [Full Text] [PDF]
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) and in the presence of 0.6 mM isoflurane (
). C, Voltage dependence of IKs activation in the presence and absence of 0.6 mM isoflurane is shown. Relative current was determined as described in Methods. The curves were obtained by Boltzmann fit to the data.
