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CELLULAR AND MOLECULAR
C. V. Starr Laboratory for Molecular Neuropharmacology, Department of Anesthesiology (L.P.C., N.T., N.L.H., P.A.G.), Department of Pharmacology (U.R.S., R.L., N.L.H., G.W.A.), and Greenberg Division of Cardiology, Department of Medicine (G.W.A.), Weill Medical College, Cornell University, New York, New York; and Departments of Anesthesiology and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York (G.R.T.)
Received June 29, 2005; accepted July 18, 2005.
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Abstract |
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). An undesirable side effect associated with its use is bradycardia, including asystole, with a reported bradycardia-related death rate of 1.4 per 100,000 patients (Tramèr et al., 1997). Oscillations entrained by the intrinsic electrical activity of neurons in thalamocortical networks are thought to contribute to different levels of consciousness (Steriade and Llinás, 1988; Llinás and Paré, 1991; Bal and McCormick, 1997; Steriade, 2000), and comparable intrinsic activity in cardiac pacemaker cells is thought to establish baseline heart rate (Schram et al., 2002).
One ionic current has been identified in both neurons and cardiac pacemaker cells that appears to regulate this intrinsic electrical activity. Depending on the tissue in which it was characterized, this current has been termed If, Iq, or Ih (Pape, 1996; Ludwig et al., 1999a; Kaupp and Seifert, 2001; Robinson and Siegelbaum, 2003). These currents are conducted by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which are voltage-activated cation channels that belong to the voltage-gated K+ channel superfamily (Kaupp and Seifert, 2001; Robinson and Siegelbaum, 2003). Genes coding for four distinct channel isoforms have been cloned (HCN1–4), and HCN channel transcripts and proteins are widely and variably distributed throughout the mammalian central nervous system (Monteggia et al., 2000; Santoro et al., 2000; Notomi and Shigemoto, 2004) and in cardiac sinoatrial node and Purkinje cells (Ludwig et al., 1999b, 2003; Shi et al., 1999; Moosmang et al., 2001; Marionneau et al., 2005).
In central neurons, Ih contributes to resting membrane potential, action potential firing, dendritic integration, neuronal automaticity, and temporal summation and determines periodicity and synchronization of oscillations in a number of neuronal networks (Robinson and Siegelbaum, 2003). In cardiac pacemaker cells, If contributes to the cardiac pacemaker depolarization (Yanagihara and Irisawa, 1980; DiFrancesco, 1981) that establishes heart rate and rhythm (DiFrancesco, 1993; Irisawa et al., 1993). Both volatile (Tokimasa et al., 1990; Sirois et al., 1998) and intravenous (Wan et al., 2003) anesthetics have been reported to modulate native Ih currents at clinically relevant concentrations.
HCN1 and HCN2 channel conductances have been shown to contribute to the resting membrane potential, action potential firing (excitability), and the stabilization of integrative properties in central neurons (Ludwig et al., 2003; Nolan et al., 2003). In isolated sinoatrial node cells, the HCN1, HCN2, and HCN4 channels generate cardiac pacemaking activity, their relative contributions depending upon species (see Discussion) (Ludwig et al., 2003). Thus, the ability of propofol to modulate Ih in central neurons may contribute to its general anesthetic properties, whereas its effects on Ih in cardiac pacemaker cells may contribute to the bradycardia that has been associated with its use (Tramèr et al., 1997). To clarify the issue of propofol modulation of Ih, we studied the effects of propofol on HCN1, HCN2, and HCN4 channels expressed in Xenopus laevis oocytes and HEK 293 cells. In addition, the effect of propofol on heart rate was examined using the isolated guinea pig heart preparation.
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Materials and Methods |
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, 2000) in vectors facilitating in vitro transcription were column-purified, linearized with the appropriate restriction enzyme, purified by precipitation, and then transcribed in vitro using the mMessage mMachine in vitro transcription kit (Ambion, Austin, TX). Transcribed HCN cRNA was purified by precipitation and resuspended in RNase-free water for oocyte injection. X. laevis oocytes (Stage V—VI) were obtained from Nasco (Fort Atkinson, WI). Oocytes were defolliculated by agitation in Ca2+-free Ringer solution containing collagenase (concentration titrated for activity of each batch; Invitrogen, Carlsbad, CA) for 1 h, washed, and then incubated at 15°C in full Ringers solution before injection with cRNA sufficient to give 1 to 10 µA of current at hyperpolarized potentials for each channel type (typically 0.1–100 ng of cRNA in a volume of 25–50 nl). Oocytes were maintained at 15 to 17°C before two-electrode voltage clamp (TEVC) recording.
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Heterologous Expression in HEK 293 Cells. Murine HCN (Santoro et al., 1998, 2000) was subcloned into the pcDNA 3.1 expression vector. The vector was coexpressed with an enhanced GFP plasmid (BD Biosciences Clontech, Palo Alto, CA) in HEK 293 cells (American Type Culture Collection, Manassas, VA). Cells were cotransfected with 0.5 to 1 µg of DNA (mHCN2) and 0.1 µg of enhanced GFP using the calcium phosphate precipitation technique. HEK 293 cells were prepared and maintained as described previously (Topf et al., 2003). Transfected cells were cultured in 3% CO2 for 24 h and then placed in 5% CO2 with fresh medium.
Whole-Cell Patch Clamp Recording in HEK 293 Cells. Currents were recorded from GFP-positive cells 48 to 72 h after transfection using the whole-cell patch clamp technique at room temperature (Krasowski et al., 1997). The intracellular solution contained 130 mM potassium gluconate, 10 mM NaCl, 1 mM EGTA, 0.5 mM MgCl2, 2 mM ATP (sodium salt), 0.1 mM GTP (sodium), 5 mM phosphocreatine, and 5 mM HEPES, pH 7.2 (KOH) at an osmolarity of 295 to 305 mOsm. Patch pipettes had a resistance of 4 to 8 M
when filled with intracellular solution. The extracellular solution contained 110 mM NaCl, 0.5 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, and 5 mM KCl, pH 7.4 (NaOH), at an osmolarity of 290 to 310 mOsm. For some recordings, an extracellular solution containing 130 mM NaCl was substituted and had no effect on the data.
Cells were voltage-clamped at -40 mV, and 10-mV voltage steps lasting 10 s were applied from -60 to -130 mV. To evaluate the effects of propofol on the HCN2 channel, propofol in dimethyl sulfoxide (DMSO) was added to extracellular solution to yield a final concentration of 5 µM. After a baseline recording, the bath solution containing DMSO (at the same final concentration as for propofol) was switched to the solution containing propofol for a duration of 2 min. After 2 min, the voltage step protocol was repeated in the presence of propofol. The protocol was repeated again after 5- to 10-min washes. Data were digitized and acquired with an EPC10 amplifier (HEKA) and Pulse software (version 8.65) with no series resistance compensation. Data were filtered at 1.25 kHz with a low-pass Bessel filter and sampled at 2.5 kHz.
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). Hearts were perfused with warmed Krebs-Henseleit solution (37°C) at a constant flow of 10 ml/min. The perfusate contained 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4-7H2O, 24 mM NaHCO3, 1.1 mM KH2PO4, 10 mM glucose, and 2.5 mM CaCl2-2H2O. Propofol (100 mM in DMSO) was diluted with Krebs-Henseleit solution to a stock perfusate solution of 2 mM.
Data Analysis. Analysis of evoked currents was performed as described previously (Santoro et al., 1998, 2000). Analysis was performed with pClamp8.2 software and Origin 6.1 (OriginLab Corp., Northampton, MA), Pulsefit (HEKA, Lambrecht/Pfalz, Germany), Prism 4 (GraphPad Software, Inc., San Diego, CA), and Igor Pro (Wavemetrics, Lake Oswego, OR). The voltage dependence of activation was determined from the amplitude of tail currents observed after each hyperpolarizing voltage step on return to -40 mV. Normalized tail current values were plotted as a function of the test voltage and fit with the Boltzmann function: I(V) = A2 + (A1 - A2)/[1 + exp[(V1/2 - V)/s], where A1 and A2, respectively, are maximum response and offset amplitudes, V1/2 is the midpoint of activation voltage, and s is the slope factor. Traces were fitted to a biexponential function in Pulsefit. An initial "lag" not well described by a biexponential function was excluded from each fit, as described previously (Santoro et al., 1998, 2000).
Concentration-effect curves were fit using a Hill equation in the form, r = RMax x [agonist]nH/([agonist]nH + EC50nH), where r is the peak response, RMax is the maximum response, [agonist] is the agonist concentration, EC50 is the agonist concentration eliciting a half-maximal response, and nH is the Hill coefficient. Data are represented as mean ± S.E.M. Statistical comparisons were determined using one-way analysis of variance (p < 0.05).
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Results |
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Analysis of HCN1-gating kinetics revealed further effects of propofol (Fig. 2). Fitting with a biexponential function of HCN1 current activation in the absence of propofol at -100 mV (Fig. 2A) gave values of 143 ± 8 ms (
fast) and 904 ± 52 ms (slow) (n = 34). Propofol increased fast and slow in a concentration-dependent manner (Fig. 2, A–C), with respective EC50 values of 5.6 ± 1.0 µM, slope = 1.4 ± 0.2 (Fig. 2B), and 31.5 ± 7.5 µM, slope = 1.6 ± 0.2 (Fig. 2C). At clinically relevant concentrations (
10 µM), propofol did not alter the relative current amplitude of the fast component (Fig. 2D). Propofol also affected HCN1 deactivation kinetics, as is evident in Fig. 1B. Fitting of HCN1 deactivation at -40 mV with a single exponential function revealed a dose-dependent acceleration of deactivation in response to propofol, with an EC50 value of 4.5 ± 0.9 µM, slope = 1.1 ± 0.2 µM (Fig. 2E).
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However, application of propofol at concentrations above 5 µM produced slowing of HCN2 channel activation (Fig. 4A). Fitting of HCN2 channel activation kinetics in the absence of propofol at -120 mV with a double exponential function (Fig. 4B) gave values for fast of 327 ± 25 and a slow of 2504 ± 237 ms (n = 23). Application of 10 µM propofol produced a 
2-fold slowing in fast and a 20% increase in slow (Fig. 4, A–D). The EC50 values for the propofol effects on activation time constants were 23.9 ± 30.7 µM (fast) and 30.0 ± 20.2 µM (slow) (Fig. 4, C and D), with no significant change in the relative amplitude of the fast component of activation, which was 0.8 from 0 to 50 µM propofol (Fig. 4E).
Effects of Propofol on HCN2 Activation Kinetics in HEK Cells. In the oocyte expression system, the effects of propofol on HCN1 and HCN2 channels were essentially irreversible; i.e., washout took long enough to preclude accurate assessment of reversibility. This is a relatively common feature of oocyte-based experiments in which small hydrophobic molecules are bath-superfused and may be due to absorption by the yolk and other intracellular bodies. Therefore, we repeated the analysis of propofol effects on HCN2 expressed in HEK cells and found that the effects on kinetics of activation were reversible using this system and also that propofol was more effective in HEK cells than in oocytes (Fig. 5).
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2-fold increase in fast and a 4-fold increase in slow at -130 mV in the HEK cell system; these effects were fully reversible upon washout of propofol (Fig. 5, B–D). Thus, HCN2 channels expressed in HEK cells were sensitive to a clinically relevant concentration of propofol. As with reversibility, the lower sensitivity of HCN2 channels in oocytes probably relates to the tendency for hydrophobic small molecules to be sequestered intracellularly in this system.
Effects of Propofol on HCN4 Channels in X. laevis Oocytes. We next asked whether HCN4, a prevalent cardiac HCN isoform, was sensitive to propofol. Representative records from HCN4 recorded in the absence or presence of 20 µM propofol in Ba2+-substituted bath medium to minimize endogenous currents indicate that propofol both slows activation and reduces the fully activated current (Fig. 6A). Fitting of HCN4 channel activation kinetics in the absence of propofol at -140 mV with a double exponential function (Fig. 6A) gave values for fast of 431 ± 6 and a slow of 1827 ± 127 ms (n = 3). Application of 20 µM propofol produced a 15% increase in fast (494 ± 30 ms) and a 2.6-fold slowing of slow (4743 ± 707 ms) (Fig. 6, C and D). Slowing of the current was accompanied by a decrease in the relative amplitude of the slow component of activation (Fig. 6E) and in the maximally activated current (Fig. 6B). To directly compare the effect of propofol on HCN1, HCN2, and HCN4, we also recorded currents from HCN1 and HCN2 in the Ba2+-substituted bath solution. A comparison of data in Fig. 6, B to E, with data presented in Figs. 2, 3, 4, shows that Mg2+ replacement by Ba2+ did not qualitatively or quantitatively alter the propofol-mediated changes in the gating of either HCN1 or HCN2.
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Propofol Increases the RR Interval but Not the QTC. The combined results suggested that propofol might produce bradyarrhythmias via impaired HCN channel function in the sinoatrial node. Therefore, we tested the effect of propofol on heart rate (Fig. 7). Propofol significantly decreased heart rate at 3 µM, but significant effects on QTC were only observed at 100 µM; at this supraclinical concentration, a decrease in QTC (Fig. 7, A–C) and sinus arrest were observed. The lack of an effect on QTC in the clinical concentration range suggests that the bradycardic effect was not due to inhibition of delayed rectifier K+ channels. The abrupt rise in QT interval above 10 µM propofol (Fig. 7D), albeit associated with a decrease in QTC because of dominant effects on RR interval, may reflect inhibition of the cardiac IKs current by propofol at these supraclinical concentrations; previous studies showed an IC50 of 250 µM propofol for IKs in oocytes (Heath and Terrar, 1997).
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Discussion |
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Propofol reportedly inhibits Ih in central neurons (Funahashi et al., 2001, 2004), with an IC50 for suppressing Ih of 235 µM in CA1 pyramidal neurons and 38 µM in area postrema neurons. It is worth noting that the clinically relevant concentration (Eger et al., 2001) of propofol is on the order of 0.4 to 2 µM after accounting for protein binding (Franks and Lieb, 1994; Reves et al., 2005). We found cloned HCN1, HCN2, and HCN4 channels to be more sensitive than was reported for native Ih. A possible explanation for the discrepancy is the use of different voltage protocols in each case. In the studies of Ih in CA1 pyramidal neurons and area postrema neurons, a 1-s voltage step was used to generate the currents; thus, the resultant currents are unlikely to be fully activated. In contrast, we used 5-s (HCN1) and 10- to 30-s (HCN2 and HCN4) voltage steps to activate these slowly gating channels. We demonstrate that propofol at clinically relevant concentrations (<10 µM) suppresses and slows the activation of Ih generated by HCN1 and HCN2 channel isoforms and significantly slows HCN4 channel activation in oocytes with 20 µM propofol.
Propofol has negative chronotropic effects in the isolated guinea pig heart (Stowe et al., 1992; Alphin et al., 1995). In the first detailed study on the effects of propofol on cardiac function (Stowe et al., 1992), propofol produced a biphasic response on heart rate. Over a low micromolar concentration range (0.5–50 µM), propofol decreased the heart rate, with an EC50 of 10.7 ± 0.2 µM (determined post hoc), whereas it produced asystole at exceedingly high concentrations (
500 µM). In a subsequent study by Alphin et al. (1995), propofol slowed the atrial rate, with a reported EC50 of 9.1 ± 0.5 µM. Our data demonstrate that propofol produces slowing in the clinically relevant concentration range and produced ventricular fibrillation progressing to asystole with prolonged application of a supraclinical concentration (100 µM; data not shown). Our data both confirm the original observations and, more importantly, expand on them with respect to the addition of the QT interval analysis. The fact that propofol at concentrations less than 10 µM had no significant effect on the QTC indicates that the observed slowing was not due to propofol-mediated inhibition of the cardiac IKs current, which would prolong QTC because of delayed repolarization (Daleau and Turgeon, 1994), consistent with previous reports of a much lower sensitivity of IKs channels to propofol (IC50 of 250 µM) (Heath and Terrar, 1997). Thus, the slowing of sinus rate observed both by us and others with low micromolar concentrations of propofol is probably due to propofol impairment of HCN channel function. This interpretation is supported by our observations that, over the same concentration range, propofol modulates HCN channel gating in both oocyte and HEK 293 cells.
HCN channel distribution is variable across species. In rabbits, for example, HCN1 and HCN4 proteins and transcripts are abundant in the sinoatrial node, whereas HCN2 protein expression is weak and HCN3 transcript is absent (Ishii et al., 1999; Shi et al., 1999). In contrast, HCN4 transcripts predominate in the mouse sinoatrial node, whereas HCN2 levels are moderate and HCN1 is the least detected (Moosmang et al., 2001). In the human heart, HCN2 may be the predominant isoform (Ludwig et al., 1999b) but detailed information on its precise distribution is lacking. Thus, the effects of propofol on heart rate in different species are probably a product of the relative sensitivities of the different HCN subunits to propofol and the relative contributions of each HCN subunit type to cardiac pacemaker currents. Furthermore, heteromeric HCN channels may generate cardiac Ih in some instances; future studies are necessary to understand the effect of heteromerization on HCN channel sensitivity to propofol.
In conclusion, the present study provides evidence that the intravenous general anesthetic propofol inhibits and slows the activation of HCN1, HCN2, and HCN4 channels. Because of the importance of the Ih current in contributing to neuronal and cardiac pacemaker cell excitability and autorhythmicity, these effects could provide novel insights into the hypnotic, amnestic, and bradycardic properties of propofol. Of equal importance, these results provide a framework for the development of propofol derivatives that retain desirable anesthetic properties but lack adverse cardiac side effects.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: HCN, hyperpolarization-activated, cyclic nucleotide-gated; mHCN, murine HCN; HEK, human embryonic kidney; QTC, corrected QT interval; IKs, slow delayed rectifier potassium current; TEVC, two-electrode voltage clamp; GFP, green fluorescent protein; DMSO, dimethyl sulfoxide.
Address correspondence to: Dr. Geoffrey W. Abbott, Greenberg Division of Cardiology, Department of Medicine, Weill Medical College of Cornell University, Starr 463, 520 East 70th Street, New York, NY 10021. E-mail: gwa2001{at}med.cornell.edu
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, 0.1 µM; 
, 0.5 µM; 
, 1 µM; 
, 2 µM; 
, 5 µM; 
, 20 µM; and 
, 50 µM. D, normalized peak tail conductance versus prepulse voltage at selected propofol concentrations (0.1, 0.5, 5, and 20 µM removed for clarity). [hexagon] and ----, control data. Boltzmann fits were normalized to the fit-determined value of the amplitude in each case to account for the lack of G/V relationship saturation at the higher propofol concentrations. E, shift in V1/2 of activation for HCN1 from control versus all propofol concentrations, calculated from data in D.
RR (s).