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Perioperative Medicine  |   March 2015
Intravenous Anesthetic Propofol Inhibits Multiple Human Cardiac Potassium Channels
Author Notes
  • From the Department of Anesthesiology, Union Hospital (L.Y.), Department of Pharmacology (H.L.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; and Department of Physiology (L.Y., H.L., G.-R.L.) and Department of Medicine (H.-Y.S., G.-R.L.), Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong, China.
  • Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are available in both the HTML and PDF versions of this article. Links to the digital files are provided in the HTML text of this article on the Journal’s Web site (www.anesthesiology.org).
    Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are available in both the HTML and PDF versions of this article. Links to the digital files are provided in the HTML text of this article on the Journal’s Web site (www.anesthesiology.org).×
  • Submitted for publication November 16, 2013. Accepted for publication September 22, 2014.
    Submitted for publication November 16, 2013. Accepted for publication September 22, 2014.×
  • Address correspondence to Dr. Liu: Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan, China. china.liuh@gmail.com. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
Article Information
Perioperative Medicine / Basic Science / Cardiovascular Anesthesia / Pharmacology / Respiratory System
Perioperative Medicine   |   March 2015
Intravenous Anesthetic Propofol Inhibits Multiple Human Cardiac Potassium Channels
Anesthesiology 03 2015, Vol.122, 571-584. doi:10.1097/ALN.0000000000000495
Anesthesiology 03 2015, Vol.122, 571-584. doi:10.1097/ALN.0000000000000495
Abstract

Background:: Propofol is widely used clinically for the induction and maintenance of anesthesia. Clinical case reports have shown that propofol has an antiatrial tachycardia/fibrillation effect; however, the related ionic mechanisms are not fully understood. The current study investigates the effects of propofol on human cardiac potassium channels.

Methods:: The whole cell patch voltage clamp technique was used to record transient outward potassium current (Ito) and ultrarapidly activating delayed rectifier potassium current (IKur) in human atrial myocytes and hKv1.5, human ether-à-go-go-related gene (hERG), and hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells. Current clamp mode was used to record action potentials in human atrial myocytes.

Results:: In human atrial myocytes, propofol inhibited Ito in a concentration-dependent manner (IC50 = 33.5 ± 2.0 μM for peak current, n = 6) by blocking open channels without affecting the voltage-dependent kinetics or the recovery time constant; propofol decreased IKur (IC50 = 35.3 ± 1.9 μM, n = 6) in human atrial myocytes and inhibited hKv1.5 current expressed in HEK 293 cells by preferentially binding to the open channels. Action potential duration at 90% repolarization was slightly prolonged by 30 μM propofol in human atrial myocytes. In addition, propofol also suppressed hERG and hKCNQ1/hKCNE1 channels expressed in HEK 293 cells.

Conclusion:: Propofol inhibits multiple human cardiac potassium channels, including human atrial Ito and IKur, as well as hKv1.5, hERG, and hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells, and slightly prolongs human atrial action potential duration, which may contribute to the antiatrial tachycardia/fibrillation effects observed in patients who receive propofol.

Abstract

Propofol blocks several important potassium currents including IKur in human atrial myocytes or stably expressing cell lines and prolongs the action potential in human atria. The findings have particular relevance to the understanding of the potential antitachycardia and antiatrial fibrillation effects of propofol on our patients.

Supplemental Digital Content is available in the text.

What We Already Know about This Topic
  • Propofol has potential proarrhythmic and antiarrhythmic effects on our patients

  • The mechanisms involved with the effects of propofol on heart rhythms are not fully understood, and there is currently no information on the effects of propofol on the important ultra rapid delayed rectifier potassium current (IKur) or action potential in human atria

What This Article Tells Us That Is New
  • Propofol blocks several important potassium currents including IKur in human atrial myocytes or stably expressing cell lines and prolongs the action potential in human atria

  • The findings have particular relevance to the understanding of the potential antitachycardia and antiatrial fibrillation effects of propofol on our patients

PROPOFOL is an intravenously administered anesthetic that is commonly used clinically to induce and/or maintain anesthesia. This anesthetic has multiple advantages, for example, quick onset and rapid recovery, with minimal side effects. In addition, propofol has been reported to have both proarrhythmic and antiarrhythmic effects.1  It has been reported that a slowed atrioventricular conduction was frequently observed in children with paroxysmal supraventricular tachycardia undergoing radiofrequency catheter ablation,2  and a completed atrioventricular blockade was found in an elderly patient undergoing knee replacement arthroplasty.3  Sinus arrest,4  severe bradycardia, atrioventricular blockade,5  polymorphic ventricular tachycardia,6,7  and some particular arrhythmias similar to Brugada syndrome8  have been reported in patients receiving propofol. Among the arrhythmias induced by propofol, bradycardia (<50 beats/min) was the most frequent (4.8% of patients with propofol).9  Experimental studies demonstrated that blockade of cardiac L-type calcium current (ICa.L),10–12  sodium current (INa),13,14  and/or pacemaker current15,16  likely contributes to the proarrhythmia observed in patients receiving propofol.
However, propofol has been reported to suppress ventricular tachycardia17,18  and improve QTc dispersion19,20  and has been found to be effective in the conversion of supraventricular tachycardia21,22  or atrial fibrillation23  to sinus rhythm. Nonetheless, the ionic mechanism underlying the antiatrial tachycardia or antiatrial fibrillation effects of propofol is not fully understood. The ultrarapid delayed rectifier potassium current IKur (encoded by hKv1.5) is present in the atria, but not in the ventricles of the human heart,24  and is therefore believed to be a target for antiatrial fibrillation therapy.25  No information is available in the literature regarding the effect of propofol on human atrial IKur and action potential. We hypothesized that propofol would inhibit multiple human cardiac potassium channels including IKur. The current study was therefore designed to determine the potential effect of propofol on IKur, transient outward potassium current (Ito), and action potential in human atrial myocytes using the whole cell patch clamp technique. We also investigated the effects of propofol on hKv1.5, human ether-à-go-go-related gene (hERG or KCNH2, coding for the α subunit of the rapid delayed rectifier potassium current IKr) channels, and hKCNQ1/hKCNE1 (coding for the slow delayed rectifier potassium current IKs) channels stably expressed in HEK 293 cells. Our results demonstrated that propofol inhibited multiple human cardiac ion channels including Ito, IKur, hERG, and hKCNQ1/hKCNE1 channels, and slightly prolonged human atrial action potential duration.
Materials and Methods
Human Atrial Myocytes Preparation
Atrial myocytes were isolated from right atrial appendage specimens obtained from 15 patients (46 to 71 yr old) undergoing coronary artery bypass. The procedure for obtaining the tissue was approved (reference No. UW-10–174) by the institutional review board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (Hong Kong, China) and a written consent was obtained from each patient. All atrial specimens were grossly normal at the time of cardiac surgery, and all patients were free of supraventricular tachyarrhythmias and symptomatic congestive heart failure. The patients were administered angiotensin-converting enzyme inhibitors, β-blockers, or calcium channel blockers before surgery. After excision, the samples were quickly immersed in an oxygenated calcium-free cardioplegic solution and immediately transported to the laboratory. Atrial myocytes were enzymatically dissociated with the procedure described previously.25,26  The isolated myocytes were kept in a high potassium medium26,27  at room temperature for at least 1 h before experimental recording. No randomization or blind methods were used in the present electrophysiological study.
Cell Culture
The HEK 293 cell line stably expressing hKv1.5 (KCNA5, coding IKur),28  hKCNQ1/hKCNE1 (IKs),29  or hERG (IKr)30  was maintained in Dulbecco modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 400 μg/ml G418 (Sigma-Aldrich, St. Louis, MO) for the cell line expressing hKv1.5 and hERG, or 100 μg/ml hygromycin (Invitrogen) for the cell line expressing hKCNQ1/hKCNE1. Cells used for electrophysiology were seeded on a glass cover slip.
Whole Cell Patch Clamp Recordings
Membrane currents were recorded with the whole cell patch clamp technique as described previously.26,28,31  In brief, a small aliquot of the solution containing the isolated myocytes was placed in an open perfusion chamber (1 ml) mounted on the stage of an inverted microscope (IX50; Olympus, Tokyo, Japan). Myocytes were allowed to adhere to the bottom of the chamber for 10 to 20 min and then superfused at 2 to 3 ml/min with Tyrode solution. For current recording in cell lines, the glass coverslips with HEK 293 cells expressing different ion channels were transferred into the cell chamber and superfused with Tyrode solution.
Borosilicate glass electrodes (1.2-mm outside diameter) were pulled with a Brown-Flaming puller (model P-97; Sutter Instrument Co., Novato, CA) and had tip resistances of 2 to 3 MΩ when filled with pipette solution. Membrane currents were recorded in voltage clamp mode, and action potentials were recorded in current clamp mode using an EPC-9 amplifier and Pulse software (HEKA, Lambrecht, Germany). After a giga-ohm seal was obtained, the cell membrane was ruptured by gentle suction to establish whole cell configuration. The cell membrane capacitance (Cm) was directly measured using the lock-in module of the Pulse software and used for normalizing the current in individual cells. The series resistance (3 to 5 MΩ) was compensated by 50 to 80% to minimize voltage errors. Electrical signals were recorded with a sampling rate of 5 kHz and filtered at 2 kHz.
Pulse Protocols and Current Measurements
Voltage-dependent Ito traces were recorded using a protocol with 300-ms voltage steps in 10-mV increments between −40 and +60 mV from a holding potential of −50 mV at 0.2 Hz. The current was measured from the peak to “‘quasi”-steady-state level. IKur was determined using a 100-ms prepulse to +40 mV (to inactivate Ito) followed by a 10-ms interval before 200-ms test potentials between −40 to +60 mV from a holding potential of −50 mV, then to −30 mV (pulse interval of 10 s). The current was measured from zero current level to the current at the end of the voltage step. Voltage-dependent hKv1.5 current was elicited by 5-s voltage pulses to potentials between −40 mV and +60 mV from a holding potential of −80 mV (pulse interval, 20 s). The current amplitude was measured at the end of the 5-s depolarizing pulse. Voltage-dependent hERG current was recorded with 3-s voltage steps from −80 mV to potentials between −60 to +60 mV, then to −50 mV every 15 s. The step current was measured as the difference between zero current and the level at the end of voltage step. The tail current was measured at its peak. Voltage-dependent hKCNQ1/hKCNE1 current traces were recorded with 3-s voltage steps from −80 mV to potentials between −40 to +60 mV, then to −50 mV (pulse interval of 15 s). The voltage step-activated current was measured from the beginning of the significant activation of time-dependent current to the level at the end of the depolarization step.
To obtain IC50 values, the fractional blocks obtained at different drug concentrations were fitted with the Hill equation: E = Emax/[1 + (IC50/C)b], where E is the inhibition of currents in percentage at concentration C, Emax is the maximum inhibition, IC50 is the concentration for 50% inhibition of maximum effect, and b is the Hill coefficient. The activation or inactivation conductance variables of Ito, hKv1.5, hERG, or hKCNQ1/hKCNE1 were determined with normalized currents. Current activation and inactivation were fitted by the Boltzmann distribution: y = 1/{1 + exp[(VmV0.5)/S], where Vm is the membrane potential, V0.5 is the midpoint, and S is the slope factor. The relation of 1/τblock against the concentration is described by the linear function: 1/τblock = k[D] + l, where 1/τblock is the time constant of development of block, and k and l are the apparent rate constants for association and dissociation of the drug.
Solutions and Chemicals
Tyrode solution contained 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.33 mM NaH2PO4, 5 mM HEPES, and 10 mM glucose; pH was adjusted to 7.4 with NaOH. The pipette solution for current recording contained 20 mM KCl, 110 mM K-aspartate, 1 mM MgCl2, 10 mM HEPES, 5 mM EGTA, 0.1 mM guanosine-5'-triphosphate, 5 mM Na2-phosphocreatine, and 5 mM adenosine triphosphate; pH was adjusted to 7.2 with KOH. The pipette solution for action potential recording contained 20 mM KCl, 110 mM K-aspartate, 1 mM MgCl2, 10 mM HEPES, 0.05 mM EGTA, 0.1 mM GTP, 5 mM Na2-phosphocreatine, and 5 mM adenosine triphosphate; pH was adjusted to 7.2 with KOH. For Ito and IKur recording, BaCl2 (200 μM) and CdCl2 (200 μM) were added to the bath solution to block IK1 and ICa.L. Atropine (1.0 μM) was used to minimize possible IK,ACh contamination during the current recording. Diphenyl phosphine oxide-1 (DPO-1; 2 μM) was added to inhibit IKur for determining Ito.27  A stock solution of propofol (Sigma-Aldrich) at 200 mM was made in dimethyl sulfoxide.
Statistical Analysis
A group size of number of 5 or more was determined based on previous experience.25,26  Nonlinear curve fitting was performed using PulseFit (HEKA) and Sigmaplot (SPSS, Chicago, IL). Paired and/or unpaired Student two-tailed t test was used as appropriate to evaluate the statistical significance of differences between two group means. Two-way ANOVA followed by the Newman–Keuls test was used for multiple comparisons, which is referred to as “ANOVA” in the results, unless otherwise noted. Data are presented as mean ± SEM. Values of P less than 0.05 were considered to indicate statistical significance.
Results
Inhibition of Cardiac Ito by Propofol in Human Atrial Myocytes
It is well established that both Ito and IKur are present in human atrial myocytes.24,26,27,32  Voltage-dependent membrane currents were elicited in a representative human atrial myocyte with a voltage protocol as shown in the inset in the absence and the presence of propofol (fig. 1A). Propofol at 50 μM inhibited both Ito peak and the sustained IKur. To accurately evaluate the propofol effect on Ito, the selective IKur/Kv1.5 blocker DPO-1 was used to separate Ito as in a previous study.27 Figure 1B shows the voltage-dependent Ito traces during control, in the presence of 2 μM DPO-1, co-presence of DPO-1 and 30 μM propofol, and washout of propofol. DPO-1 (2 μM) induced a slight reduction of peak current amplitude and almost a full inhibition of IKur. Propofol at 30 μM remarkably suppressed Ito, and the effect was partially reversed by washout. Figure 1C displays the time course of Ito recorded with a protocol as shown in the inset in the absence or presence of 30 μM propofol in a typical experiment with DPO-1 treatment. Ito was gradually decreased by 30 μM propofol, and the inhibition reached a steady-state level within 4 min and was partially reversed upon washout. Experiments to determine the blocking properties of Ito by propofol were conducted in the presence of 2 μM DPO-1.
Fig. 1.
Inhibition of Ito by propofol. (A) Membrane currents were recorded with a voltage protocol shown in the inset in a representative human atrial myocyte in the absence (control) and the presence of 50 μM propofol. (B) Membrane currents recorded in another myocyte in the absence and the presence of 2 μM diphenyl phosphine oxide-1 (DPO-1; to inhibit IKur), DPO-1 plus 30 μM propofol, and washout of propofol. (C) Time course of Ito recorded in a representative cell pretreated with 2 μM DPO-1. Propofol at 30 μM gradually inhibited Ito, and the inhibition was reversed by washout.
Inhibition of Ito by propofol. (A) Membrane currents were recorded with a voltage protocol shown in the inset in a representative human atrial myocyte in the absence (control) and the presence of 50 μM propofol. (B) Membrane currents recorded in another myocyte in the absence and the presence of 2 μM diphenyl phosphine oxide-1 (DPO-1; to inhibit IKur), DPO-1 plus 30 μM propofol, and washout of propofol. (C) Time course of Ito recorded in a representative cell pretreated with 2 μM DPO-1. Propofol at 30 μM gradually inhibited Ito, and the inhibition was reversed by washout.
Fig. 1.
Inhibition of Ito by propofol. (A) Membrane currents were recorded with a voltage protocol shown in the inset in a representative human atrial myocyte in the absence (control) and the presence of 50 μM propofol. (B) Membrane currents recorded in another myocyte in the absence and the presence of 2 μM diphenyl phosphine oxide-1 (DPO-1; to inhibit IKur), DPO-1 plus 30 μM propofol, and washout of propofol. (C) Time course of Ito recorded in a representative cell pretreated with 2 μM DPO-1. Propofol at 30 μM gradually inhibited Ito, and the inhibition was reversed by washout.
×
The effect of propofol on current–voltage (IV) relations of Ito was determined at 3, 10, 30, and 100 μM (fig. 2A). Propofol inhibited Ito in a concentration-dependent manner. Propofol at 10 to 100 μM suppressed Ito at test potentials of 0 mV to +60 mV (n = 6, P < 0.05 or P < 0.01 vs. control, ANOVA). No significant change in voltage dependence was observed at any concentration of propofol (fig. 2B). The concentration–response relation for inhibiting the peak current of Ito by propofol was evaluated at +50 mV and fitted with a Hill equation (fig. 2C). The IC50 for inhibiting peak current of Ito by propofol was 33.5 ± 2.0 μM (Hill coefficient 1.8 ± 0.2, n = 6). Propofol decreased the peak current and increased the inactivation of Ito, which implies open-channel blockade.33,34  Therefore, the inhibition was also estimated from the integral of the total current charge crossing the membrane at +50 mV, and propofol inhibited the Ito charge with an IC50 of 20.9 ± 5.0 μM (Hill coefficient 2.1 ± 0.4, n = 6; fig. 2C).
Fig. 2.
Concentration-dependent effects of propofol on Ito. (A) Current–voltage relations of Ito density in cells pretreated with 2 μM diphenyl phosphine oxide-1 (control) and in the co-presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited Ito at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (B) Mean percent inhibition of Ito from 0 to + 60 mV by propofol at 3 to 100 μM. (C) Concentration–response relation for reducing total Ito charge (n = 6) and peak current (n = 6) at + 50 mV. The data were fitted with a Hill equation.
Concentration-dependent effects of propofol on Ito. (A) Current–voltage relations of Ito density in cells pretreated with 2 μM diphenyl phosphine oxide-1 (control) and in the co-presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited Ito at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (B) Mean percent inhibition of Ito from 0 to + 60 mV by propofol at 3 to 100 μM. (C) Concentration–response relation for reducing total Ito charge (n = 6) and peak current (n = 6) at + 50 mV. The data were fitted with a Hill equation.
Fig. 2.
Concentration-dependent effects of propofol on Ito. (A) Current–voltage relations of Ito density in cells pretreated with 2 μM diphenyl phosphine oxide-1 (control) and in the co-presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited Ito at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (B) Mean percent inhibition of Ito from 0 to + 60 mV by propofol at 3 to 100 μM. (C) Concentration–response relation for reducing total Ito charge (n = 6) and peak current (n = 6) at + 50 mV. The data were fitted with a Hill equation.
×
To analyze the open-channel blocking property of Ito by propofol, the time to peak and the inactivation time constant were determined in human atrial myocytes in the absence and the presence of propofol. Figure 3A illustrates the expanded Ito traces recorded at +40 mV in the absence and the presence of 30 μM propofol. The time to peak of Ito was clearly reduced by propofol. The mean value of the time to peak of the current at 0 to +60 mV was significantly reduced by 30 μM propofol (fig. 3B, n = 6, P < 0.01 vs. control, ANOVA). Ito traces (+40 mV) were fitted by a mono-exponential function with the time constants shown before and after the application of 30 μM propofol (fig. 3C). The time constant of Ito inactivation was reduced by 30 μM propofol. The mean value of the time constant of Ito inactivation at 0 to +60 mV is shown in figure 3D. The time constant was significantly reduced by 10 or 30 μM propofol at all test potentials (n = 6, P < 0.05 or P < 0.01 vs. control, ANOVA).
Fig. 3.
Effects of propofol on time-dependent kinetics of Ito. (A) Expanded current traces of Ito at +40 mV before and after 30 μM propofol in a representative recording, showing the measurement of the time to peak of Ito. (B) Mean values of the time to the peak of Ito activation at 0 mV to +60 mV under control conditions and in the presence of 10 and 30 μM propofol (n = 6, P < 0.01, 30 μM vs. control, ANOVA). (C) Inactivation raw data (points) of Ito at +40 mV before and after 30 μM propofol fitted to a mono-exponential function (solid lines) with time constants shown. (D) Mean values of time constant of Ito inactivation at 0 mV to +60 mV before and after the application of 10 and 30 μM propofol (n = 6, P < 0.05 or P < 0.01 vs. control, ANOVA). (E) Development of Ito inhibition by propofol after channel activation. The solid lines represent that the mono-exponential function is fitted to the onset blocking data (points) by propofol at 10, 30, and 100 μM. (F) Rate constants for the block of Ito by propofol. 1/τblock is plotted against the concentration of propofol. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6).
Effects of propofol on time-dependent kinetics of Ito. (A) Expanded current traces of Ito at +40 mV before and after 30 μM propofol in a representative recording, showing the measurement of the time to peak of Ito. (B) Mean values of the time to the peak of Ito activation at 0 mV to +60 mV under control conditions and in the presence of 10 and 30 μM propofol (n = 6, P < 0.01, 30 μM vs. control, ANOVA). (C) Inactivation raw data (points) of Ito at +40 mV before and after 30 μM propofol fitted to a mono-exponential function (solid lines) with time constants shown. (D) Mean values of time constant of Ito inactivation at 0 mV to +60 mV before and after the application of 10 and 30 μM propofol (n = 6, P < 0.05 or P < 0.01 vs. control, ANOVA). (E) Development of Ito inhibition by propofol after channel activation. The solid lines represent that the mono-exponential function is fitted to the onset blocking data (points) by propofol at 10, 30, and 100 μM. (F) Rate constants for the block of Ito by propofol. 1/τblock is plotted against the concentration of propofol. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6).
Fig. 3.
Effects of propofol on time-dependent kinetics of Ito. (A) Expanded current traces of Ito at +40 mV before and after 30 μM propofol in a representative recording, showing the measurement of the time to peak of Ito. (B) Mean values of the time to the peak of Ito activation at 0 mV to +60 mV under control conditions and in the presence of 10 and 30 μM propofol (n = 6, P < 0.01, 30 μM vs. control, ANOVA). (C) Inactivation raw data (points) of Ito at +40 mV before and after 30 μM propofol fitted to a mono-exponential function (solid lines) with time constants shown. (D) Mean values of time constant of Ito inactivation at 0 mV to +60 mV before and after the application of 10 and 30 μM propofol (n = 6, P < 0.05 or P < 0.01 vs. control, ANOVA). (E) Development of Ito inhibition by propofol after channel activation. The solid lines represent that the mono-exponential function is fitted to the onset blocking data (points) by propofol at 10, 30, and 100 μM. (F) Rate constants for the block of Ito by propofol. 1/τblock is plotted against the concentration of propofol. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6).
×
The onset of the open-channel blockade was further analyzed with the method described by Slawsky and Castle35  using the current traces recorded at +50 mV with 10, 30, and 100 μM propofol. Acceleration of Ito inactivation by 10, 30, and 100 μM propofol suggested an open-channel blocking effect. The drug-sensitive current expressed as a proportion of the current in the absence of the drug [(IcontrolIpropofol)/Icontrol], where Icontrol and Ipropofol are the current in the absence and the presence of propofol. The drug-induced block was then plotted as a function of time. The blockade developed in a time-dependent manner with an exponential onset as shown by the curve fits in figure 3E. The rate of blocking development was enhanced as the concentration increased; the time constants averaged 26.3 ± 2.9 ms, 19.1 ± 1.1 ms, and 7.6 ± 0.6 ms, at 10, 30, and 100 μM propofol, respectively (n = 6). The 1/τblock was plotted as a function of propofol concentrations as shown in figure 3F. The straight line is a regression fit of the equation 1/τblock = k[D] + l, and the apparent rate constants for association (k) and dissociation (l) were (1.1 ± 0.2) × 106 M−1 s−1 and 25.2 ± 5.8 s−1, respectively. The apparent Kd (Kd = l/k) derived from this relation for Ito current block by propofol was 22.9 μM, which is close to the IC50 of 20.9 μM obtained from the concentration–response curve determined by the integral of current charge.
Figure 4A illustrates the current and protocol (1-s conditioning pulses from −100 to +30 mV at a holding potential of −80 mV, followed by a 300-ms test pulse to +50 mV after a 30-ms interval at −50 mV) used for determining the steady-state inactivation (availability, I/Imax) of Ito, while figure 4B shows the tail current recorded by a voltage protocol (8-ms voltage steps from −50 mV to potentials between −40 to +50 mV, then to −40 mV) for determining the steady-state activation (g/gmax) of Ito. The normalized variable of I/Imax or g/gmax was calculated and fitted with a Boltzmann function in individual cells to obtain the half potential (V0.5) of Ito availability or activation and slope factor. Figure 4C displays the mean values of I/Imax fitted by a Boltzmann distribution in the absence or the presence of 30 μM propofol. The V0.5 of Ito availability was −22.0 ± 1.3 mV in the control and −24.6 ± 2.1 mV in the presence of propofol (n = 8, P = 0.168, paired Student t test). The fractional blockade was plotted as a function of the voltage of the preceding pulse as described by Caballero et al.,36  demonstrating that the blockade remained unchanged at potentials between −100 mV (48.1 ± 3.0%) and −50 mV (48.3 ± 3.1%, n = 8, P > 0.05, ANOVA). At more positive potentials, it tended to increase as the amount of inactivated channels increased, reaching a maximum at −20 mV (57.7 ± 6.4%), but the difference was not statistically significant (n = 8, P > 0.05 vs. blockade at −100 mV, ANOVA).
Fig. 4.
Effects of propofol on voltage-dependent kinetics and recovery of Ito from inactivation. (A) Representative current traces recorded with a protocol (1-s conditioning pulses from −100 to +30 mV at a holding potential of –80 mV, followed by a 300-ms test pulse to +50 mV after a 30-ms interval at –50 mV) for determining steady-state inactivation (availability, I/Imax) in a human atrial cell in the presence of 2 μM diphenyl phosphine oxide-1. (B) Tail current of Ito recorded by the protocol (8-ms voltage steps from −50 mV to potentials between −40 to +50 mV, then to −40 mV) for assessing steady-state activation (g/gmax). (C) Mean variables of I/Imax of Ito were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (D) Mean variables of g/gmax of Ito were fitted with a Boltzmann distribution. (E) Mean values of Ito recovery from inactivation determined with a protocol (a 300-ms step to +50 mV from a holding potential of –80 mV with variable P1–P2 interval) in the absence and the presence of 30 μM propofol in six cells. The data were best fitted with a mono-exponential function. (F) Mean percentage values of use-dependent inhibition of Ito by 30 μM propofol at 0.5, 1, 2, and 3 Hz (n = 6).
Effects of propofol on voltage-dependent kinetics and recovery of Ito from inactivation. (A) Representative current traces recorded with a protocol (1-s conditioning pulses from −100 to +30 mV at a holding potential of –80 mV, followed by a 300-ms test pulse to +50 mV after a 30-ms interval at –50 mV) for determining steady-state inactivation (availability, I/Imax) in a human atrial cell in the presence of 2 μM diphenyl phosphine oxide-1. (B) Tail current of Ito recorded by the protocol (8-ms voltage steps from −50 mV to potentials between −40 to +50 mV, then to −40 mV) for assessing steady-state activation (g/gmax). (C) Mean variables of I/Imax of Ito were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (D) Mean variables of g/gmax of Ito were fitted with a Boltzmann distribution. (E) Mean values of Ito recovery from inactivation determined with a protocol (a 300-ms step to +50 mV from a holding potential of –80 mV with variable P1–P2 interval) in the absence and the presence of 30 μM propofol in six cells. The data were best fitted with a mono-exponential function. (F) Mean percentage values of use-dependent inhibition of Ito by 30 μM propofol at 0.5, 1, 2, and 3 Hz (n = 6).
Fig. 4.
Effects of propofol on voltage-dependent kinetics and recovery of Ito from inactivation. (A) Representative current traces recorded with a protocol (1-s conditioning pulses from −100 to +30 mV at a holding potential of –80 mV, followed by a 300-ms test pulse to +50 mV after a 30-ms interval at –50 mV) for determining steady-state inactivation (availability, I/Imax) in a human atrial cell in the presence of 2 μM diphenyl phosphine oxide-1. (B) Tail current of Ito recorded by the protocol (8-ms voltage steps from −50 mV to potentials between −40 to +50 mV, then to −40 mV) for assessing steady-state activation (g/gmax). (C) Mean variables of I/Imax of Ito were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (D) Mean variables of g/gmax of Ito were fitted with a Boltzmann distribution. (E) Mean values of Ito recovery from inactivation determined with a protocol (a 300-ms step to +50 mV from a holding potential of –80 mV with variable P1–P2 interval) in the absence and the presence of 30 μM propofol in six cells. The data were best fitted with a mono-exponential function. (F) Mean percentage values of use-dependent inhibition of Ito by 30 μM propofol at 0.5, 1, 2, and 3 Hz (n = 6).
×
Figure 4D displays the mean values of g/gmax fitted by a Boltzmann distribution in the absence or the presence of 30 μM propofol. The V0.5 of Ito activation was 21.9 ± 1.5 mV in the control and 15.9 ± 1.6 mV in the presence of propofol (n = 7, P = 0.055, paired Student t test). No difference was observed for the slope factor of Ito availability or activation before and after the application of propofol.
Time-dependent recovery of Ito from inactivation was determined with a paired-pulse protocol as shown in figure 4E. The recovery curves were fitted by a mono-exponential function in the absence and the presence of 30 μM propofol in individual cells. The recovery time constant of Ito from inactivation was 55.7 ± 6.1 ms in the control and 50.3 ± 7.8 ms in the presence of propofol (n = 6, P = 0.253, paired Student t test). These results indicate that propofol inhibits human atrial Ito without affecting the voltage-dependent kinetics and recovery time constant of the channel.
The use-dependent and rate-dependent effects of Ito by propofol were determined using 20 repetitive 200-ms depolarizations from a holding potential of −50 mV to +50 mV at 0.5, 1, 2, and 3 Hz. There was no significant difference when decreasing Ito by 30 μM propofol at the rate of 0.5, 1, 2, and 3 Hz (fig. 4F), suggesting that the use-dependent or rate-dependent blocking effect is not involved in the Ito inhibition by propofol in human atrial myocytes.
Blockade of Human Atrial IKur and hKv1.5 by Propofol
Figure 5A shows the time course of IKur recorded in a representative cell with a voltage protocol: as shown in the inset, a 100-ms prepulse to +40 mV to inactivate Ito, followed by a 200-ms test pulse to +50 mV, then to −30 mV every 15 s.26,27  The current was slowly reduced by 30 μM propofol and reached a steady state in 10 min, and the inhibitory effect was partially reversed by washout. The voltage-dependent IKur (fig. 5B) was recorded in a representative cell with a voltage protocol shown in the inset. Propofol at 30 μM substantially inhibited both IKur and tail current, and the effect partially recovered on washout. At test potential of +50 mV, IKur was reduced by 57.4 ± 5.2% with 30 μM propofol (n = 7, P = 0.002 vs. control, paired Student t test).
Fig. 5.
Inhibition of IKur by propofol. (A) Time course of IKur recorded in a typical experiment in the absence and the presence of 30 μM propofol with original IKur traces at corresponding time points shown. (B) Voltage-dependent IKur traces recorded in a representative cell with the voltage protocol shown in the inset. IKur was reversibly inhibited by 30 μM propofol. (C) Current–voltage relations of IKur in the absence and the presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited IKur at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (D) Mean percentage value of IKur at 0 to +60 mV in the presence of 3 to 100 μM propofol. Significant voltage dependence was observed for the drug effect at 10 to 100 μM, and a stronger effect was observed at potentials positive to between +20 and +60 mV (P < 0.05 or P < 0.01 vs. 0 mV, ANOVA). (E) Concentration–response relation for inhibiting IKur (at +50 mV) by propofol fitted with a Hill equation (n = 6).
Inhibition of IKur by propofol. (A) Time course of IKur recorded in a typical experiment in the absence and the presence of 30 μM propofol with original IKur traces at corresponding time points shown. (B) Voltage-dependent IKur traces recorded in a representative cell with the voltage protocol shown in the inset. IKur was reversibly inhibited by 30 μM propofol. (C) Current–voltage relations of IKur in the absence and the presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited IKur at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (D) Mean percentage value of IKur at 0 to +60 mV in the presence of 3 to 100 μM propofol. Significant voltage dependence was observed for the drug effect at 10 to 100 μM, and a stronger effect was observed at potentials positive to between +20 and +60 mV (P < 0.05 or P < 0.01 vs. 0 mV, ANOVA). (E) Concentration–response relation for inhibiting IKur (at +50 mV) by propofol fitted with a Hill equation (n = 6).
Fig. 5.
Inhibition of IKur by propofol. (A) Time course of IKur recorded in a typical experiment in the absence and the presence of 30 μM propofol with original IKur traces at corresponding time points shown. (B) Voltage-dependent IKur traces recorded in a representative cell with the voltage protocol shown in the inset. IKur was reversibly inhibited by 30 μM propofol. (C) Current–voltage relations of IKur in the absence and the presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited IKur at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (D) Mean percentage value of IKur at 0 to +60 mV in the presence of 3 to 100 μM propofol. Significant voltage dependence was observed for the drug effect at 10 to 100 μM, and a stronger effect was observed at potentials positive to between +20 and +60 mV (P < 0.05 or P < 0.01 vs. 0 mV, ANOVA). (E) Concentration–response relation for inhibiting IKur (at +50 mV) by propofol fitted with a Hill equation (n = 6).
×
IV relations of IKur are illustrated in figure 5C in the absence and the presence of 3, 10, 30, and 100 μM propofol in a total of six myocytes. Propofol inhibited IKur in a concentration-dependent manner. The inhibition fraction of the current was more at potentials positive to +20 mV than that at 0 mV with 10 to 100 μM propofol (fig. 5D, n = 6, P < 0.05 or P < 0.01 vs. 0 mV, ANOVA). Figure 5E shows the concentration–response relation of propofol, which was fitted by a Hill equation. The IC50 (at +50 mV) of propofol for inhibiting IKur was 35.3 ± 1.9 μM (Hill coefficient 1.1 ± 0.05, n = 6).
IKur/Kv1.5 channel inhibitors usually block the open state of the channel and induce an increased inactivation of the current, that is, blocking increase during depolarization.33,34,37,38  However, propofol inhibited IKur without showing the increased inactivation in human atrial myocytes (fig. 5), suggesting that the open-channel blockade may not be involved in the current inhibition. Nevertheless, IKur was recorded in human atrial myocytes using short-duration test pulses with a prepulse, which might cover the open-channel blocking property of propofol. We therefore determined the effect of propofol on hKv1.5 channels expressed in HEK 293 cells using longer-duration pulses.
Figure 6A illustrates hKv1.5 current elicited by 5-s voltage pulses between −40 mV and +60 mV from a holding potential of −80 mV with a pulse interval of 20 s. Propofol at 50 μM significantly inhibited hKv1.5 current, and the effect partially recovered on washout. Figure 6B shows the IV relations of hKv1.5 current measured at the end of the depolarization step before and after the application of 10, 30, 50, and 100 μM propofol. The current was inhibited by propofol in a concentration-dependent manner (n = 9, P < 0.05 or P < 0.01 vs. control at 0 mV to +60 mV, ANOVA). Concentration–response relation for inhibiting the peak current, the “quasi”-steady-state current measured at the end of the voltage pulse, and the current charge crossing the membrane by propofol at 10 to 300 μM were evaluated at +50 mV and fitted with a Hill equation (fig. 6C). The IC50 values were 132.2 ± 6.9 μM (Hill coefficient 1.4 ± 0.1, n = 7), 58.1 ± 2.7 μM (Hill coefficient: 1.7 ± 0.1, n = 7), and 67.0 ± 2.8 μM (Hill coefficient 1.6 ± 0.1, n = 7), respectively.
Fig. 6.
Inhibition of hKv1.5 current by propofol. (A) Voltage-dependent hKv1.5 currents were recorded with a voltage protocol shown in the inset in a representative HEK 293 cell stably expressing KCNA5 gene before (control) and after the application of 50 μM propofol, and washout of propofol. (B) Current–voltage relations of hKv1.5 current density in the absence and the presence of 10, 30, 50, and 100 μM propofol. Propofol significantly inhibited hKv1.5 current at concentrations from 10 to 30, 50, and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (C) Concentration–response relation for inhibiting total hKv1.5 current charge and “quasi”-steady-state current measured at the end of the voltage pulse and current peak (n = 7) at + 50 mV. The data were fitted with a Hill equation.
Inhibition of hKv1.5 current by propofol. (A) Voltage-dependent hKv1.5 currents were recorded with a voltage protocol shown in the inset in a representative HEK 293 cell stably expressing KCNA5 gene before (control) and after the application of 50 μM propofol, and washout of propofol. (B) Current–voltage relations of hKv1.5 current density in the absence and the presence of 10, 30, 50, and 100 μM propofol. Propofol significantly inhibited hKv1.5 current at concentrations from 10 to 30, 50, and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (C) Concentration–response relation for inhibiting total hKv1.5 current charge and “quasi”-steady-state current measured at the end of the voltage pulse and current peak (n = 7) at + 50 mV. The data were fitted with a Hill equation.
Fig. 6.
Inhibition of hKv1.5 current by propofol. (A) Voltage-dependent hKv1.5 currents were recorded with a voltage protocol shown in the inset in a representative HEK 293 cell stably expressing KCNA5 gene before (control) and after the application of 50 μM propofol, and washout of propofol. (B) Current–voltage relations of hKv1.5 current density in the absence and the presence of 10, 30, 50, and 100 μM propofol. Propofol significantly inhibited hKv1.5 current at concentrations from 10 to 30, 50, and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (C) Concentration–response relation for inhibiting total hKv1.5 current charge and “quasi”-steady-state current measured at the end of the voltage pulse and current peak (n = 7) at + 50 mV. The data were fitted with a Hill equation.
×
Although the IC50 values are similar for inhibiting the “quasi”-steady-state current measured at the end of the voltage pulse and the current charge, the IC50 for inhibiting the peak current is greater, suggesting that propofol enhanced the inactivation of hKv1.5 current elicited by 5-s pulses, and open-channel blockade is involved in the current inhibition. The open-channel blocking properties were analyzed as for Ito (fig. 3).36,39  The current traces (at +50 mV) were fitted by a mono-exponential function with time constants shown in the absence and the presence of 50 μM propofol (fig. 7A). The mean values of the time constant (fig. 7B) were significantly reduced by 50 μM propofol at potentials of +10 to +60 mV (n = 11, P < 0.05 or P < 0.01 vs. control, paired Student t test). Figure 7C illustrates the current traces at +50 mV in the absence and the presence of 30, 50, and 100 μM propofol. The drug-sensitive hKv1.5 current expressed as a proportion of the current in the absence of the drug [(IcontrolIpropofol)/Icontrol]. 1/τblock as a function of the propofol concentration for data obtained at 30, 50, and 100 μM is shown in figure 7D. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6). The apparent rate constants for association (k) and dissociation (l) were (1.5 ± 0.1) × 104 M−1 s−1 and 0.88 ± 0.09 s−1, respectively. The apparent Kd (Kd = l/k) derived from this relation for hKv1.5 blocking by propofol was 58.7 μM, which is close to the IC50 of 57.3 μM obtained from the concentration–response curve.
Fig. 7.
Effects of propofol on kinetics and recovery of hKv1.5 current from inactivation. (A) Inactivation data of hKv1.5 current at +50 mV before and after 50 μM propofol fitted to a mono-exponential function with time constants shown. (B) Mean values of time constant of hKv1.5 current inactivation at +10 mV to +60 mV before and after the application of 50 μM propofol (n = 11, *P < 0.05 or **P < 0.01 vs. control, paired Student t test). (C) Currents elicited by 5-s pulses to +50 mV in the absence and the presence of increasing concentrations of propofol. (D) 1/τblock as a function of the propofol concentration for data obtained at concentrations of 30, 50, and 100 μM. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6). (E) Mean variables of I/Imax of hKv1.5 current in the absence and the presence of 50 μM propofol were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (F) Mean values of hKv1.5 current recovery from inactivation determined with a protocol shown in the inset in the absence and the presence of 50 μM propofol (n = 8). The data were best fitted with a mono-exponential function.
Effects of propofol on kinetics and recovery of hKv1.5 current from inactivation. (A) Inactivation data of hKv1.5 current at +50 mV before and after 50 μM propofol fitted to a mono-exponential function with time constants shown. (B) Mean values of time constant of hKv1.5 current inactivation at +10 mV to +60 mV before and after the application of 50 μM propofol (n = 11, *P < 0.05 or **P < 0.01 vs. control, paired Student t test). (C) Currents elicited by 5-s pulses to +50 mV in the absence and the presence of increasing concentrations of propofol. (D) 1/τblock as a function of the propofol concentration for data obtained at concentrations of 30, 50, and 100 μM. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6). (E) Mean variables of I/Imax of hKv1.5 current in the absence and the presence of 50 μM propofol were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (F) Mean values of hKv1.5 current recovery from inactivation determined with a protocol shown in the inset in the absence and the presence of 50 μM propofol (n = 8). The data were best fitted with a mono-exponential function.
Fig. 7.
Effects of propofol on kinetics and recovery of hKv1.5 current from inactivation. (A) Inactivation data of hKv1.5 current at +50 mV before and after 50 μM propofol fitted to a mono-exponential function with time constants shown. (B) Mean values of time constant of hKv1.5 current inactivation at +10 mV to +60 mV before and after the application of 50 μM propofol (n = 11, *P < 0.05 or **P < 0.01 vs. control, paired Student t test). (C) Currents elicited by 5-s pulses to +50 mV in the absence and the presence of increasing concentrations of propofol. (D) 1/τblock as a function of the propofol concentration for data obtained at concentrations of 30, 50, and 100 μM. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6). (E) Mean variables of I/Imax of hKv1.5 current in the absence and the presence of 50 μM propofol were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (F) Mean values of hKv1.5 current recovery from inactivation determined with a protocol shown in the inset in the absence and the presence of 50 μM propofol (n = 8). The data were best fitted with a mono-exponential function.
×
To obtain the steady-state inactivation of hKv1.5 channels, a protocol with 10-s conditioning pulses from −80 to +50 mV and then a 1-s test pulse to +50 mV was used with 20-s pulse interval. Figure 7E shows the mean variables of I/Imax of hKv1.5 current in the absence and the presence of 50 μM propofol. The variables were fitted with a Boltzmann distribution in individual cells to obtain the V0.5 of hKv1.5 inactivation. The inactivation V0.5 was −9.5 ± 0.7 mV in control and −8.7 ± 0.9 mV in 50 μM propofol (n = 9, P = 0.433, paired Student t test). Fractional block by propofol showed that current blockade did not change significantly even in the voltage range of channel inactivation.
Recovery of hKv1.5 current from inactivation was determined with a paired-pulse protocol (a 5,000-ms step to +50 mV from a holding potential of −80 mV, followed by a 300-ms step to +50 mV with variable P1–P2 interval between 2 and 9,000 ms, pulse interval of 25 s). Both of the recovery curves in the absence and the presence of 50 μM propofol were well fitted with a bi-exponential function (fig. 7F). Propofol slightly slowed the recovery of hKv1.5 current from inactivation, but the difference was not significant (fast time constant: 285 ± 15 ms in the control, 469 ± 86 ms in the presence of propofol, P = 0.125, paired Student t test; slow time constant: 3,956 ± 399 ms in the control, 4,075 ± 437 ms in the presence of propofol, n = 8, P = 0.873, paired Student t test).
Effects of Propofol on Cardiac hERG and hKCNQ1/hKCNE1
It has been demonstrated that both IKr and IKs are present in human cardiac myocytes and play an important role in cardiac repolarization in human heart.24,40  It is interesting to investigate whether the inhibition of human cardiac IKr and/or IKs contributes to the antiatrial tachycardia/fibrillation of propofol. It has been difficult to record these two currents in cardiac myocytes from the chunk dissociation method with a small atrial specimen from patients undergoing coronary bypass surgery. Therefore, HEK 293 cells stably expressing hERG (coding for IKr)30  or hKCNQ1/hKCNE1 (coding for IKs)29  were used here to determine the effects of propofol on these two types of currents.
Figure 8A shows the time course of hERG tail current recorded with a voltage step as shown in the inset in the absence and the presence of 30 and 100 μM propofol. The current was inhibited by 30 and 100 μM propofol, and the inhibition was reversed by washout. Similar results were observed in voltage-dependent hERG current. Both hERG tail current and step current were clearly decreased by propofol (fig. 8B). Figure 8, C and D, illustrates the I–V relations of mean values of hERG tail and step current in the absence and the presence of 30, 100, and 300 μM propofol. Propofol at 30 μM inhibited hERG tail and step current from +20 mV to +60 mV (n = 10, P < 0.05 vs. control, ANOVA), while at 100 and 300 μM, propofol significantly decreased the current from 0 mV from +60 mV (P < 0.05 or P < 0.01 vs. control, ANOVA). The concentration–response curves (fig. 8E) of propofol for inhibiting hERG tail current and step current at +40 mV were fitted with a Hill equation. The IC50 of propofol was 84.4 ± 15.5 μM for inhibiting hERG tail current and 73.2 ± 16.6 μM (n = 7) for inhibiting hERG step current.
Fig. 8.
Effect of propofol on hERG channels. (A) Time course of hERG tail current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hERG traces at corresponding time points shown. (B) Voltage-dependent hERG current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relation of hERG tail current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (D) Current–voltage relation of hERG step current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (E) Concentration–response relation for inhibiting hERG current (at +40 mV) by propofol fitted with a Hill equation (n = 7). (F) Mean values of g/gmax were calculated with hERG tail current and fitted with a Boltzmann function in the absence and the presence of 30 and 100 μM propofol.
Effect of propofol on hERG channels. (A) Time course of hERG tail current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hERG traces at corresponding time points shown. (B) Voltage-dependent hERG current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relation of hERG tail current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (D) Current–voltage relation of hERG step current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (E) Concentration–response relation for inhibiting hERG current (at +40 mV) by propofol fitted with a Hill equation (n = 7). (F) Mean values of g/gmax were calculated with hERG tail current and fitted with a Boltzmann function in the absence and the presence of 30 and 100 μM propofol.
Fig. 8.
Effect of propofol on hERG channels. (A) Time course of hERG tail current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hERG traces at corresponding time points shown. (B) Voltage-dependent hERG current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relation of hERG tail current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (D) Current–voltage relation of hERG step current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (E) Concentration–response relation for inhibiting hERG current (at +40 mV) by propofol fitted with a Hill equation (n = 7). (F) Mean values of g/gmax were calculated with hERG tail current and fitted with a Boltzmann function in the absence and the presence of 30 and 100 μM propofol.
×
The steady-state activation (g/gmax) of hERG channels was determined by normalized tail current in the absence and the presence of 30 and 100 μM propofol (fig. 8F). The activation curves were fitted with a Boltzmann function. The V0.5 of hERG channel activation was 4.1 ± 1.6 mV in control, 0.5 ± 2.1 mV in 30 μM propofol (n = 7, P > 0.05 vs. control), and −10.1 ± 2.2 mV in 100 μM propofol (n = 7, P < 0.05 vs. control, one-way ANOVA followed by the Newman–Keuls test). The activation of hERG channels was significantly shifted to the negative potential by propofol.
Figure 9A shows the time course of human cardiac hKCNQ1/hKCNE1 channels expressed in HEK 293 cells in a typical experiment with the voltage step as shown in the inset. Propofol at 30 and 100 μM significantly inhibited hKCNQ1/hKCNE1 step current, and the inhibition was partially reversed by washout. The voltage-dependent hKCNQ1/hKCNE1 current was also suppressed by propofol (fig. 9B). Figure 9C illustrates the I–V relations of mean values of hKCNQ1/hKCNE1 current density in the absence and the presence of 10, 30, and 100 μM propofol. Propofol significantly inhibited hKCNQ1/hKCNE1 current at test potential of 0 to +60 mV (n = 6, P < 0.05 or P < 0.01 vs. control, ANOVA). The concentration–response curves (fig. 9D) of propofol for decreasing hKCNQ1/hKCNE1 current at +40 mV were fitted with a Hill equation. The IC50 of propofol was 32.4 ± 6.0 μM (n = 6) for inhibiting hKCNQ1/hKCNE1 channels.
Fig. 9.
Blockade of hKCNQ1/hKCNE1 channels by propofol. (A) Time course of hKCNQ1/hKCNE1 current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hKCNQ1/hKCNE1 current traces at corresponding time points shown. (B) Voltage-dependent hKCNQ1/hKCNE1 current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relations of hKCNQ1/hKCNE1 current density in the absence and presence of 10, 30, and 100 μM propofol (n = 6 for 10 and 100 μM, and n = 9 for 30 μM, *P < 0.05, 10 μM vs. control at +10 to +60 mV; **P < 0.01, 30 or 100 μM vs. control at +10 to +60 mV, ANOVA). (D) Concentration–response relations for inhibiting hKCNQ1/hKCNE1 current (at +50 mV) by propofol fitted with a Hill equation (n = 6). (E) Mean values of g/gmax were calculated with hKCNQ1/hKCNE1 tail current and fitted with the Boltzmann function in the absence and the presence of 30 μM propofol.
Blockade of hKCNQ1/hKCNE1 channels by propofol. (A) Time course of hKCNQ1/hKCNE1 current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hKCNQ1/hKCNE1 current traces at corresponding time points shown. (B) Voltage-dependent hKCNQ1/hKCNE1 current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relations of hKCNQ1/hKCNE1 current density in the absence and presence of 10, 30, and 100 μM propofol (n = 6 for 10 and 100 μM, and n = 9 for 30 μM, *P < 0.05, 10 μM vs. control at +10 to +60 mV; **P < 0.01, 30 or 100 μM vs. control at +10 to +60 mV, ANOVA). (D) Concentration–response relations for inhibiting hKCNQ1/hKCNE1 current (at +50 mV) by propofol fitted with a Hill equation (n = 6). (E) Mean values of g/gmax were calculated with hKCNQ1/hKCNE1 tail current and fitted with the Boltzmann function in the absence and the presence of 30 μM propofol.
Fig. 9.
Blockade of hKCNQ1/hKCNE1 channels by propofol. (A) Time course of hKCNQ1/hKCNE1 current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hKCNQ1/hKCNE1 current traces at corresponding time points shown. (B) Voltage-dependent hKCNQ1/hKCNE1 current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relations of hKCNQ1/hKCNE1 current density in the absence and presence of 10, 30, and 100 μM propofol (n = 6 for 10 and 100 μM, and n = 9 for 30 μM, *P < 0.05, 10 μM vs. control at +10 to +60 mV; **P < 0.01, 30 or 100 μM vs. control at +10 to +60 mV, ANOVA). (D) Concentration–response relations for inhibiting hKCNQ1/hKCNE1 current (at +50 mV) by propofol fitted with a Hill equation (n = 6). (E) Mean values of g/gmax were calculated with hKCNQ1/hKCNE1 tail current and fitted with the Boltzmann function in the absence and the presence of 30 μM propofol.
×
The steady-state activation (g/gmax) of hKCNQ1/hKCNE1 channels was determined by normalized tail current in the absence and the presence of 30 μM propofol (fig. 9E). The activation curves were fitted with the Boltzmann function. The V0.5 of hKCNQ1/hKCNE1 current activation was 16.3 ± 4.5 mV in control and 20.1 ± 4.1 mV in 30 μM propofol (n = 7, P = 0.088 vs. control, paired Student t test). The activation conductance of hKCNQ1/hKCNE1 channels was not significantly affected by propofol.
Effects of Propofol on Human Atrial Action Potential
The effect of propofol on cardiac action potential was determined in human atrial myocytes at 36°C. Figure 10A displays the action potential traces recorded in a representative cell at 2 Hz. Propofol at 30 μM slightly prolonged action potential duration. Action potential duration at 90% repolarization was significantly increased by the application of propofol (fig. 10B, n = 6, P = 0.024 vs. control, paired Student t test). The measured dV/dT of action potential phase 0 was reduced from 78.2 ± 5.7 V/s to 72.7 ± 5.9 V/s (n = 6, P = 0.034 vs. control, paired Student t test), which may be related to the inhibition of INa as reported previously.13,14  However, resting membrane potential was not affected by propofol (−73.0 ± 1.7 mV vs. −72.4 ± 1.1 mV, n = 6, P = 0.528, paired Student t test).
Fig. 10.
Effect of propofol on cardiac action potential. (A) Action potentials recorded in a representative human atrial myocyte at 2 Hz in the absence (black line) and the presence of 30 μM propofol (red line). (B) Mean values of human atrial action potential duration at 25, 50, and 90% repolarization during control and after application of 30 μM propofol (n = 6, *P = 0.024 vs. control, paired Student t test). APD = action potential duration.
Effect of propofol on cardiac action potential. (A) Action potentials recorded in a representative human atrial myocyte at 2 Hz in the absence (black line) and the presence of 30 μM propofol (red line). (B) Mean values of human atrial action potential duration at 25, 50, and 90% repolarization during control and after application of 30 μM propofol (n = 6, *P = 0.024 vs. control, paired Student t test). APD = action potential duration.
Fig. 10.
Effect of propofol on cardiac action potential. (A) Action potentials recorded in a representative human atrial myocyte at 2 Hz in the absence (black line) and the presence of 30 μM propofol (red line). (B) Mean values of human atrial action potential duration at 25, 50, and 90% repolarization during control and after application of 30 μM propofol (n = 6, *P = 0.024 vs. control, paired Student t test). APD = action potential duration.
×
Discussion
The current study demonstrates that the intravenous anesthetic propofol blocks human atrial native Ito, IKur, hERG, and hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells with concentrations ranging from 3 to 100 μM and it slightly prolongs human atrial action potential duration.
Previous reports demonstrated that propofol inhibits several cardiac ion channels.1  Propofol at 1 to 100 μM decreases the cardiac depolarization currents including ICa.L in cardiac ventricular myocytes from rat12  and guinea pig,11  rabbit,14  and human atrial myocytes,10 INa in rat and rabbit ventricular myocytes,13,14  and pacemaker current (i.e., hyperpolarization-activated cyclic nucleotide–regulated channels) expressed in HEK 293 cells.16,41  These effects may count for the ionic mechanisms of sinus arrest, bradycardia, and/or atrioventricular blockade observed in patients with propofol as an intravenous anesthetic.4,5,9 
For cardiac repolarization currents, the reports for cardiac inward rectifier potassium current (IK1) are controversial. One study reported that propofol at 3 to 30 μM decreased IK1 in rabbit ventricular myocytes,14  whereas other reports demonstrated that propofol (28 to 60 μM) had no effect on IK1 in ventricular myocytes from guinea pig42  and canine17  hearts. Our results support the notion that propofol has no significant effect on cardiac IK1 because propofol at 30 μM only slightly inhibited human cardiac Kir2.1 channels expressed in HEK 293 cells without statistical significance (Supplemental Digital Content 1, http://links.lww.com/ALN/B107, figure 1, n = 8, inhibited by approximately 3.3% at −120 mV, P = 0.124 vs. control, paired Student t test). This may explain why no change is observed in the resting membrane potential of human atrial myocytes with application of propofol.
The transient outward potassium current Ito plays a role in the early rapid repolarization of cardiac action potential in mammalian heart, including that of humans. Propofol (3 to 60 μM) inhibited Ito in ventricular myocytes from rat,43  rabbit,14  and canine17  hearts. The current study demonstrated the novel information that propofol decreased Ito by binding to the open channels in human atrial myocytes. However, propofol showed no use- or rate-dependent blockade of Ito, which is similar to the effect of propafenone35  and allitridi33  on cardiac Ito.
The ultrarapid delayed rectifier potassium current IKur is present in human atria but not in the ventricles of the human heart.24  It is believed that IKur is a target for developing atrial-selective antiatrial fibrillation drugs.24,44,45  No information is available in the literature regarding the effect of propofol on IKur/Kv1.5. In this study, we provide the novel information that propofol inhibits human atrial IKur with an IC50 of 35.3 μM. We found that the blocking fraction of propofol for IKur was slightly but significantly higher at potentials positive to +20 mV than that at 0 mV, which suggests a stronger inhibition of the current at positive potential of action potential. Because Ito and IKur play a crucial role in the repolarization of human atrial myocytes,25,32,45,46  blockade of Ito and IKur would significantly prolong atrial action potential duration and therefore would exert the antiatrial arrhythmic effect.25,45  Therefore, the antiatrial tachycardia and antiatrial fibrillation observed in patients with propofol21–23  is likely related to the inhibition of Ito and IKur.
Although propofol did not show the development blockade of IKur using short depolarization pulses with a prepulse in human atrial myocytes, the development blockade was observed in HEK 293 cells expressing hKv1.5 channels with a longer duration of depolarization pulses without prepulse. Propofol enhanced the inactivation of hKv1.5 current elicited by 5-s pulses, suggesting that propofol inhibits hKv1.5 by preferentially binding to the open channels. It is interesting that propofol is more sensitive to inhibit native IKur than hKv1.5 expressed in HEK 293 cells.
The delayed rectifier potassium currents IKr and IKs are also important repolarization currents in human heart.24,40  Blockade of IKr may be antiarrhythmic or proarrhythmic.47–49  The proarrhythmic effect of IKr/hERG blockade is mainly related to induction of prolonged QT interval of electrocardiography or Torsades de Pointes.47,48  An earlier report did not find the inhibition of IKr in guinea pig cardiac myocytes,50  while in Xenopus oocytes expressing hERG channels, propofol significantly inhibited hERG current at a high concentration of 100 μM.51  The current study showed that significant inhibition of hERG channels expressed in HEK 293 cells was observed at 30 μM. The IC50 of propofol was 73.2 μM for hERG step current and 84.4 μM for hERG tail current, which is much greater than plasma concentrations in patients.52,53  Experimental study did not find the prolonged QT interval in guinea pigs.51  Clinical reports demonstrated that QTc interval was unaffected54,55  or shortened by propofol.56  Interestingly, propofol reversed the QT prolongation induced by sevoflurane20  and reduced the QT dispersion.57  These clinical reports suggest that the slight hERG/IKr inhibition would not induce a prolonged QT interval.
In Xenopus oocytes expressing the cardiac IKs gene IsK, propofol showed less sensitivity for the current inhibition with an estimated IC50 value of 250 μM,58  while in guinea pig cardiac myocytes propofol at 300 μM fully inhibited IKs.50  However, a recent report demonstrated that propofol inhibited IKs in guinea pig ventricular myocytes with an IC50 of 23 μM,11  which is close to that observed in the current study. We found that propofol inhibited human cardiac hKCNQ1/hKCNE1 channels expressed in HEK 293 cells with an IC50 of 32.4 μM. Reduction of human cardiac IKs may also contribute to the inhibition of supraventricular tachycardia/fibrillation in patients with propofol.21–23 
Pharmacokinetic studies have shown that an intravenous dose of propofol for anesthesia may reach a peak level at 44 μM in blood plasma and generally 10 to 20 μM for anesthetic maintenance.52,53  In the current study, we found that significant inhibition of Ito, IKur, and hKCNQ1/hKCNE1 current by propofol was observed at 3 to 10 μM, which is in the range of anesthetic maintenance. Therefore, the conversion of supraventricular tachycardia/fibrillation to sinus rhythm in patients with propofol21–23  may be related to its inhibition of Ito, IKur, and IKs.
It should be noted that the current study demonstrated that significant inhibition of Ito, IKur, and hKCNQ1/hKCNE1 current was observed with 30 μM propofol. The effect was supposed to significantly prolong action potential duration. However, we found that action potential duration was slightly prolonged by propofol in human atrial myocytes. It is well recognized that for the effect of a drug on action potential duration, ICa.L blockade has a counter effect with potassium current inhibition. The slight increase in action potential duration with propofol is most likely due to the ICa.L blockade observed previously in human atrial myocytes10  and other species.12  This phenomenon is similar to that of Ca2+ channel blocker diltiazem on potassium currents and cardiac action potential.36  The conversion of supraventricular tachycardia/fibrillation to sinus rhythm in patients with propofol21–23  may be related to inhibiting more for potassium currents than for ICa.L.
The limitation of the current study was that we were unable to record IKr and IKs in human atrial myocytes isolated from small atrial specimens with the tissue chunk method. It is unclear whether the channels are digested by enzymes. If so, it may lead to an alteration of the action potential waveform and action potential duration prolongation and induce an underestimation of propofol effect on the action potential. The observed slight prolongation of action potential duration by propofol may be related partially to the loss of these two channels.
In summary, the current study reported for the first time that propofol inhibited human cardiac atrial repolarization potassium currents including human atrial Ito and IKur, and slightly prolonged human atrial action potential duration. Propofol also inhibited hERG and hKCNQ1/hKCNE1 channels expressed in HEK 293 cells. Reduction of Ito, IKur, and IKs likely contributes to the suppression of supraventricular tachycardia and atrial fibrillation observed in patients given propofol.
Acknowledgments
The work was supported by a grant from the Important National Science and Technology Specific Projects (grant no. 2009ZX09301-014), Beijing, China.
Competing Interests
The authors declare no competing interests.
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Fig. 1.
Inhibition of Ito by propofol. (A) Membrane currents were recorded with a voltage protocol shown in the inset in a representative human atrial myocyte in the absence (control) and the presence of 50 μM propofol. (B) Membrane currents recorded in another myocyte in the absence and the presence of 2 μM diphenyl phosphine oxide-1 (DPO-1; to inhibit IKur), DPO-1 plus 30 μM propofol, and washout of propofol. (C) Time course of Ito recorded in a representative cell pretreated with 2 μM DPO-1. Propofol at 30 μM gradually inhibited Ito, and the inhibition was reversed by washout.
Inhibition of Ito by propofol. (A) Membrane currents were recorded with a voltage protocol shown in the inset in a representative human atrial myocyte in the absence (control) and the presence of 50 μM propofol. (B) Membrane currents recorded in another myocyte in the absence and the presence of 2 μM diphenyl phosphine oxide-1 (DPO-1; to inhibit IKur), DPO-1 plus 30 μM propofol, and washout of propofol. (C) Time course of Ito recorded in a representative cell pretreated with 2 μM DPO-1. Propofol at 30 μM gradually inhibited Ito, and the inhibition was reversed by washout.
Fig. 1.
Inhibition of Ito by propofol. (A) Membrane currents were recorded with a voltage protocol shown in the inset in a representative human atrial myocyte in the absence (control) and the presence of 50 μM propofol. (B) Membrane currents recorded in another myocyte in the absence and the presence of 2 μM diphenyl phosphine oxide-1 (DPO-1; to inhibit IKur), DPO-1 plus 30 μM propofol, and washout of propofol. (C) Time course of Ito recorded in a representative cell pretreated with 2 μM DPO-1. Propofol at 30 μM gradually inhibited Ito, and the inhibition was reversed by washout.
×
Fig. 2.
Concentration-dependent effects of propofol on Ito. (A) Current–voltage relations of Ito density in cells pretreated with 2 μM diphenyl phosphine oxide-1 (control) and in the co-presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited Ito at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (B) Mean percent inhibition of Ito from 0 to + 60 mV by propofol at 3 to 100 μM. (C) Concentration–response relation for reducing total Ito charge (n = 6) and peak current (n = 6) at + 50 mV. The data were fitted with a Hill equation.
Concentration-dependent effects of propofol on Ito. (A) Current–voltage relations of Ito density in cells pretreated with 2 μM diphenyl phosphine oxide-1 (control) and in the co-presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited Ito at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (B) Mean percent inhibition of Ito from 0 to + 60 mV by propofol at 3 to 100 μM. (C) Concentration–response relation for reducing total Ito charge (n = 6) and peak current (n = 6) at + 50 mV. The data were fitted with a Hill equation.
Fig. 2.
Concentration-dependent effects of propofol on Ito. (A) Current–voltage relations of Ito density in cells pretreated with 2 μM diphenyl phosphine oxide-1 (control) and in the co-presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited Ito at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (B) Mean percent inhibition of Ito from 0 to + 60 mV by propofol at 3 to 100 μM. (C) Concentration–response relation for reducing total Ito charge (n = 6) and peak current (n = 6) at + 50 mV. The data were fitted with a Hill equation.
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Fig. 3.
Effects of propofol on time-dependent kinetics of Ito. (A) Expanded current traces of Ito at +40 mV before and after 30 μM propofol in a representative recording, showing the measurement of the time to peak of Ito. (B) Mean values of the time to the peak of Ito activation at 0 mV to +60 mV under control conditions and in the presence of 10 and 30 μM propofol (n = 6, P < 0.01, 30 μM vs. control, ANOVA). (C) Inactivation raw data (points) of Ito at +40 mV before and after 30 μM propofol fitted to a mono-exponential function (solid lines) with time constants shown. (D) Mean values of time constant of Ito inactivation at 0 mV to +60 mV before and after the application of 10 and 30 μM propofol (n = 6, P < 0.05 or P < 0.01 vs. control, ANOVA). (E) Development of Ito inhibition by propofol after channel activation. The solid lines represent that the mono-exponential function is fitted to the onset blocking data (points) by propofol at 10, 30, and 100 μM. (F) Rate constants for the block of Ito by propofol. 1/τblock is plotted against the concentration of propofol. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6).
Effects of propofol on time-dependent kinetics of Ito. (A) Expanded current traces of Ito at +40 mV before and after 30 μM propofol in a representative recording, showing the measurement of the time to peak of Ito. (B) Mean values of the time to the peak of Ito activation at 0 mV to +60 mV under control conditions and in the presence of 10 and 30 μM propofol (n = 6, P < 0.01, 30 μM vs. control, ANOVA). (C) Inactivation raw data (points) of Ito at +40 mV before and after 30 μM propofol fitted to a mono-exponential function (solid lines) with time constants shown. (D) Mean values of time constant of Ito inactivation at 0 mV to +60 mV before and after the application of 10 and 30 μM propofol (n = 6, P < 0.05 or P < 0.01 vs. control, ANOVA). (E) Development of Ito inhibition by propofol after channel activation. The solid lines represent that the mono-exponential function is fitted to the onset blocking data (points) by propofol at 10, 30, and 100 μM. (F) Rate constants for the block of Ito by propofol. 1/τblock is plotted against the concentration of propofol. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6).
Fig. 3.
Effects of propofol on time-dependent kinetics of Ito. (A) Expanded current traces of Ito at +40 mV before and after 30 μM propofol in a representative recording, showing the measurement of the time to peak of Ito. (B) Mean values of the time to the peak of Ito activation at 0 mV to +60 mV under control conditions and in the presence of 10 and 30 μM propofol (n = 6, P < 0.01, 30 μM vs. control, ANOVA). (C) Inactivation raw data (points) of Ito at +40 mV before and after 30 μM propofol fitted to a mono-exponential function (solid lines) with time constants shown. (D) Mean values of time constant of Ito inactivation at 0 mV to +60 mV before and after the application of 10 and 30 μM propofol (n = 6, P < 0.05 or P < 0.01 vs. control, ANOVA). (E) Development of Ito inhibition by propofol after channel activation. The solid lines represent that the mono-exponential function is fitted to the onset blocking data (points) by propofol at 10, 30, and 100 μM. (F) Rate constants for the block of Ito by propofol. 1/τblock is plotted against the concentration of propofol. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6).
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Fig. 4.
Effects of propofol on voltage-dependent kinetics and recovery of Ito from inactivation. (A) Representative current traces recorded with a protocol (1-s conditioning pulses from −100 to +30 mV at a holding potential of –80 mV, followed by a 300-ms test pulse to +50 mV after a 30-ms interval at –50 mV) for determining steady-state inactivation (availability, I/Imax) in a human atrial cell in the presence of 2 μM diphenyl phosphine oxide-1. (B) Tail current of Ito recorded by the protocol (8-ms voltage steps from −50 mV to potentials between −40 to +50 mV, then to −40 mV) for assessing steady-state activation (g/gmax). (C) Mean variables of I/Imax of Ito were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (D) Mean variables of g/gmax of Ito were fitted with a Boltzmann distribution. (E) Mean values of Ito recovery from inactivation determined with a protocol (a 300-ms step to +50 mV from a holding potential of –80 mV with variable P1–P2 interval) in the absence and the presence of 30 μM propofol in six cells. The data were best fitted with a mono-exponential function. (F) Mean percentage values of use-dependent inhibition of Ito by 30 μM propofol at 0.5, 1, 2, and 3 Hz (n = 6).
Effects of propofol on voltage-dependent kinetics and recovery of Ito from inactivation. (A) Representative current traces recorded with a protocol (1-s conditioning pulses from −100 to +30 mV at a holding potential of –80 mV, followed by a 300-ms test pulse to +50 mV after a 30-ms interval at –50 mV) for determining steady-state inactivation (availability, I/Imax) in a human atrial cell in the presence of 2 μM diphenyl phosphine oxide-1. (B) Tail current of Ito recorded by the protocol (8-ms voltage steps from −50 mV to potentials between −40 to +50 mV, then to −40 mV) for assessing steady-state activation (g/gmax). (C) Mean variables of I/Imax of Ito were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (D) Mean variables of g/gmax of Ito were fitted with a Boltzmann distribution. (E) Mean values of Ito recovery from inactivation determined with a protocol (a 300-ms step to +50 mV from a holding potential of –80 mV with variable P1–P2 interval) in the absence and the presence of 30 μM propofol in six cells. The data were best fitted with a mono-exponential function. (F) Mean percentage values of use-dependent inhibition of Ito by 30 μM propofol at 0.5, 1, 2, and 3 Hz (n = 6).
Fig. 4.
Effects of propofol on voltage-dependent kinetics and recovery of Ito from inactivation. (A) Representative current traces recorded with a protocol (1-s conditioning pulses from −100 to +30 mV at a holding potential of –80 mV, followed by a 300-ms test pulse to +50 mV after a 30-ms interval at –50 mV) for determining steady-state inactivation (availability, I/Imax) in a human atrial cell in the presence of 2 μM diphenyl phosphine oxide-1. (B) Tail current of Ito recorded by the protocol (8-ms voltage steps from −50 mV to potentials between −40 to +50 mV, then to −40 mV) for assessing steady-state activation (g/gmax). (C) Mean variables of I/Imax of Ito were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (D) Mean variables of g/gmax of Ito were fitted with a Boltzmann distribution. (E) Mean values of Ito recovery from inactivation determined with a protocol (a 300-ms step to +50 mV from a holding potential of –80 mV with variable P1–P2 interval) in the absence and the presence of 30 μM propofol in six cells. The data were best fitted with a mono-exponential function. (F) Mean percentage values of use-dependent inhibition of Ito by 30 μM propofol at 0.5, 1, 2, and 3 Hz (n = 6).
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Fig. 5.
Inhibition of IKur by propofol. (A) Time course of IKur recorded in a typical experiment in the absence and the presence of 30 μM propofol with original IKur traces at corresponding time points shown. (B) Voltage-dependent IKur traces recorded in a representative cell with the voltage protocol shown in the inset. IKur was reversibly inhibited by 30 μM propofol. (C) Current–voltage relations of IKur in the absence and the presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited IKur at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (D) Mean percentage value of IKur at 0 to +60 mV in the presence of 3 to 100 μM propofol. Significant voltage dependence was observed for the drug effect at 10 to 100 μM, and a stronger effect was observed at potentials positive to between +20 and +60 mV (P < 0.05 or P < 0.01 vs. 0 mV, ANOVA). (E) Concentration–response relation for inhibiting IKur (at +50 mV) by propofol fitted with a Hill equation (n = 6).
Inhibition of IKur by propofol. (A) Time course of IKur recorded in a typical experiment in the absence and the presence of 30 μM propofol with original IKur traces at corresponding time points shown. (B) Voltage-dependent IKur traces recorded in a representative cell with the voltage protocol shown in the inset. IKur was reversibly inhibited by 30 μM propofol. (C) Current–voltage relations of IKur in the absence and the presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited IKur at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (D) Mean percentage value of IKur at 0 to +60 mV in the presence of 3 to 100 μM propofol. Significant voltage dependence was observed for the drug effect at 10 to 100 μM, and a stronger effect was observed at potentials positive to between +20 and +60 mV (P < 0.05 or P < 0.01 vs. 0 mV, ANOVA). (E) Concentration–response relation for inhibiting IKur (at +50 mV) by propofol fitted with a Hill equation (n = 6).
Fig. 5.
Inhibition of IKur by propofol. (A) Time course of IKur recorded in a typical experiment in the absence and the presence of 30 μM propofol with original IKur traces at corresponding time points shown. (B) Voltage-dependent IKur traces recorded in a representative cell with the voltage protocol shown in the inset. IKur was reversibly inhibited by 30 μM propofol. (C) Current–voltage relations of IKur in the absence and the presence of 3, 10, 30, and 100 μM propofol. Propofol significantly inhibited IKur at concentrations from 10 to 30 and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (D) Mean percentage value of IKur at 0 to +60 mV in the presence of 3 to 100 μM propofol. Significant voltage dependence was observed for the drug effect at 10 to 100 μM, and a stronger effect was observed at potentials positive to between +20 and +60 mV (P < 0.05 or P < 0.01 vs. 0 mV, ANOVA). (E) Concentration–response relation for inhibiting IKur (at +50 mV) by propofol fitted with a Hill equation (n = 6).
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Fig. 6.
Inhibition of hKv1.5 current by propofol. (A) Voltage-dependent hKv1.5 currents were recorded with a voltage protocol shown in the inset in a representative HEK 293 cell stably expressing KCNA5 gene before (control) and after the application of 50 μM propofol, and washout of propofol. (B) Current–voltage relations of hKv1.5 current density in the absence and the presence of 10, 30, 50, and 100 μM propofol. Propofol significantly inhibited hKv1.5 current at concentrations from 10 to 30, 50, and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (C) Concentration–response relation for inhibiting total hKv1.5 current charge and “quasi”-steady-state current measured at the end of the voltage pulse and current peak (n = 7) at + 50 mV. The data were fitted with a Hill equation.
Inhibition of hKv1.5 current by propofol. (A) Voltage-dependent hKv1.5 currents were recorded with a voltage protocol shown in the inset in a representative HEK 293 cell stably expressing KCNA5 gene before (control) and after the application of 50 μM propofol, and washout of propofol. (B) Current–voltage relations of hKv1.5 current density in the absence and the presence of 10, 30, 50, and 100 μM propofol. Propofol significantly inhibited hKv1.5 current at concentrations from 10 to 30, 50, and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (C) Concentration–response relation for inhibiting total hKv1.5 current charge and “quasi”-steady-state current measured at the end of the voltage pulse and current peak (n = 7) at + 50 mV. The data were fitted with a Hill equation.
Fig. 6.
Inhibition of hKv1.5 current by propofol. (A) Voltage-dependent hKv1.5 currents were recorded with a voltage protocol shown in the inset in a representative HEK 293 cell stably expressing KCNA5 gene before (control) and after the application of 50 μM propofol, and washout of propofol. (B) Current–voltage relations of hKv1.5 current density in the absence and the presence of 10, 30, 50, and 100 μM propofol. Propofol significantly inhibited hKv1.5 current at concentrations from 10 to 30, 50, and 100 μM (n = 6, *P < 0.05 or **P < 0.01 vs. control, ANOVA). (C) Concentration–response relation for inhibiting total hKv1.5 current charge and “quasi”-steady-state current measured at the end of the voltage pulse and current peak (n = 7) at + 50 mV. The data were fitted with a Hill equation.
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Fig. 7.
Effects of propofol on kinetics and recovery of hKv1.5 current from inactivation. (A) Inactivation data of hKv1.5 current at +50 mV before and after 50 μM propofol fitted to a mono-exponential function with time constants shown. (B) Mean values of time constant of hKv1.5 current inactivation at +10 mV to +60 mV before and after the application of 50 μM propofol (n = 11, *P < 0.05 or **P < 0.01 vs. control, paired Student t test). (C) Currents elicited by 5-s pulses to +50 mV in the absence and the presence of increasing concentrations of propofol. (D) 1/τblock as a function of the propofol concentration for data obtained at concentrations of 30, 50, and 100 μM. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6). (E) Mean variables of I/Imax of hKv1.5 current in the absence and the presence of 50 μM propofol were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (F) Mean values of hKv1.5 current recovery from inactivation determined with a protocol shown in the inset in the absence and the presence of 50 μM propofol (n = 8). The data were best fitted with a mono-exponential function.
Effects of propofol on kinetics and recovery of hKv1.5 current from inactivation. (A) Inactivation data of hKv1.5 current at +50 mV before and after 50 μM propofol fitted to a mono-exponential function with time constants shown. (B) Mean values of time constant of hKv1.5 current inactivation at +10 mV to +60 mV before and after the application of 50 μM propofol (n = 11, *P < 0.05 or **P < 0.01 vs. control, paired Student t test). (C) Currents elicited by 5-s pulses to +50 mV in the absence and the presence of increasing concentrations of propofol. (D) 1/τblock as a function of the propofol concentration for data obtained at concentrations of 30, 50, and 100 μM. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6). (E) Mean variables of I/Imax of hKv1.5 current in the absence and the presence of 50 μM propofol were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (F) Mean values of hKv1.5 current recovery from inactivation determined with a protocol shown in the inset in the absence and the presence of 50 μM propofol (n = 8). The data were best fitted with a mono-exponential function.
Fig. 7.
Effects of propofol on kinetics and recovery of hKv1.5 current from inactivation. (A) Inactivation data of hKv1.5 current at +50 mV before and after 50 μM propofol fitted to a mono-exponential function with time constants shown. (B) Mean values of time constant of hKv1.5 current inactivation at +10 mV to +60 mV before and after the application of 50 μM propofol (n = 11, *P < 0.05 or **P < 0.01 vs. control, paired Student t test). (C) Currents elicited by 5-s pulses to +50 mV in the absence and the presence of increasing concentrations of propofol. (D) 1/τblock as a function of the propofol concentration for data obtained at concentrations of 30, 50, and 100 μM. The line is a regression fit of the equation, 1/τblock = k[D] + l (n = 6). (E) Mean variables of I/Imax of hKv1.5 current in the absence and the presence of 50 μM propofol were fitted with a Boltzmann distribution. Squares represent the fractional block (Ipropofol/Icontrol) at each potential. (F) Mean values of hKv1.5 current recovery from inactivation determined with a protocol shown in the inset in the absence and the presence of 50 μM propofol (n = 8). The data were best fitted with a mono-exponential function.
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Fig. 8.
Effect of propofol on hERG channels. (A) Time course of hERG tail current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hERG traces at corresponding time points shown. (B) Voltage-dependent hERG current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relation of hERG tail current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (D) Current–voltage relation of hERG step current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (E) Concentration–response relation for inhibiting hERG current (at +40 mV) by propofol fitted with a Hill equation (n = 7). (F) Mean values of g/gmax were calculated with hERG tail current and fitted with a Boltzmann function in the absence and the presence of 30 and 100 μM propofol.
Effect of propofol on hERG channels. (A) Time course of hERG tail current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hERG traces at corresponding time points shown. (B) Voltage-dependent hERG current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relation of hERG tail current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (D) Current–voltage relation of hERG step current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (E) Concentration–response relation for inhibiting hERG current (at +40 mV) by propofol fitted with a Hill equation (n = 7). (F) Mean values of g/gmax were calculated with hERG tail current and fitted with a Boltzmann function in the absence and the presence of 30 and 100 μM propofol.
Fig. 8.
Effect of propofol on hERG channels. (A) Time course of hERG tail current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hERG traces at corresponding time points shown. (B) Voltage-dependent hERG current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relation of hERG tail current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (D) Current–voltage relation of hERG step current in the absence and presence of 30, 100, and 300 μM propofol (n = 10 for 30 and 100 μM, and n = 7 for 300 μM, P < 0.05, 30 μM vs. control at +20 to +60 mV; *P < 0.05 or **P < 0.01, 100 or 300 μM vs. control at 0 to +60 mV, ANOVA). (E) Concentration–response relation for inhibiting hERG current (at +40 mV) by propofol fitted with a Hill equation (n = 7). (F) Mean values of g/gmax were calculated with hERG tail current and fitted with a Boltzmann function in the absence and the presence of 30 and 100 μM propofol.
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Fig. 9.
Blockade of hKCNQ1/hKCNE1 channels by propofol. (A) Time course of hKCNQ1/hKCNE1 current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hKCNQ1/hKCNE1 current traces at corresponding time points shown. (B) Voltage-dependent hKCNQ1/hKCNE1 current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relations of hKCNQ1/hKCNE1 current density in the absence and presence of 10, 30, and 100 μM propofol (n = 6 for 10 and 100 μM, and n = 9 for 30 μM, *P < 0.05, 10 μM vs. control at +10 to +60 mV; **P < 0.01, 30 or 100 μM vs. control at +10 to +60 mV, ANOVA). (D) Concentration–response relations for inhibiting hKCNQ1/hKCNE1 current (at +50 mV) by propofol fitted with a Hill equation (n = 6). (E) Mean values of g/gmax were calculated with hKCNQ1/hKCNE1 tail current and fitted with the Boltzmann function in the absence and the presence of 30 μM propofol.
Blockade of hKCNQ1/hKCNE1 channels by propofol. (A) Time course of hKCNQ1/hKCNE1 current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hKCNQ1/hKCNE1 current traces at corresponding time points shown. (B) Voltage-dependent hKCNQ1/hKCNE1 current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relations of hKCNQ1/hKCNE1 current density in the absence and presence of 10, 30, and 100 μM propofol (n = 6 for 10 and 100 μM, and n = 9 for 30 μM, *P < 0.05, 10 μM vs. control at +10 to +60 mV; **P < 0.01, 30 or 100 μM vs. control at +10 to +60 mV, ANOVA). (D) Concentration–response relations for inhibiting hKCNQ1/hKCNE1 current (at +50 mV) by propofol fitted with a Hill equation (n = 6). (E) Mean values of g/gmax were calculated with hKCNQ1/hKCNE1 tail current and fitted with the Boltzmann function in the absence and the presence of 30 μM propofol.
Fig. 9.
Blockade of hKCNQ1/hKCNE1 channels by propofol. (A) Time course of hKCNQ1/hKCNE1 current recorded in a typical experiment in the absence and the presence of 30 and 100 μM propofol with the original hKCNQ1/hKCNE1 current traces at corresponding time points shown. (B) Voltage-dependent hKCNQ1/hKCNE1 current was recorded in a representative cell with the voltage protocol shown in the inset and was reversibly inhibited by 30 and 100 μM propofol. (C) Current–voltage relations of hKCNQ1/hKCNE1 current density in the absence and presence of 10, 30, and 100 μM propofol (n = 6 for 10 and 100 μM, and n = 9 for 30 μM, *P < 0.05, 10 μM vs. control at +10 to +60 mV; **P < 0.01, 30 or 100 μM vs. control at +10 to +60 mV, ANOVA). (D) Concentration–response relations for inhibiting hKCNQ1/hKCNE1 current (at +50 mV) by propofol fitted with a Hill equation (n = 6). (E) Mean values of g/gmax were calculated with hKCNQ1/hKCNE1 tail current and fitted with the Boltzmann function in the absence and the presence of 30 μM propofol.
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Fig. 10.
Effect of propofol on cardiac action potential. (A) Action potentials recorded in a representative human atrial myocyte at 2 Hz in the absence (black line) and the presence of 30 μM propofol (red line). (B) Mean values of human atrial action potential duration at 25, 50, and 90% repolarization during control and after application of 30 μM propofol (n = 6, *P = 0.024 vs. control, paired Student t test). APD = action potential duration.
Effect of propofol on cardiac action potential. (A) Action potentials recorded in a representative human atrial myocyte at 2 Hz in the absence (black line) and the presence of 30 μM propofol (red line). (B) Mean values of human atrial action potential duration at 25, 50, and 90% repolarization during control and after application of 30 μM propofol (n = 6, *P = 0.024 vs. control, paired Student t test). APD = action potential duration.
Fig. 10.
Effect of propofol on cardiac action potential. (A) Action potentials recorded in a representative human atrial myocyte at 2 Hz in the absence (black line) and the presence of 30 μM propofol (red line). (B) Mean values of human atrial action potential duration at 25, 50, and 90% repolarization during control and after application of 30 μM propofol (n = 6, *P = 0.024 vs. control, paired Student t test). APD = action potential duration.
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