Enhanced effects of isoflurane on the long QT syndrome 1-associated A341V mutant

Abstract

Background: The impact of volatile anesthetics on patients with inherited long QT syndrome (LQTS) is not well understood. This is further complicated by the different genotypes underlying LQTS. No studies have reported on the direct effects of volatile anesthetics on specific LQTS-associated mutations. We investigated the effects of isoflurane on a common LQTS type 1 mutation, A341V, with an unusually severe phenotype.

Methods: Whole cell potassium currents (IKs) were recorded from HEK293 and HL-1 cells transiently expressing/coexpressing wild-type KCNQ1 (α-subunit), mutant KCNQ1, wild-type KCNE1 (β-subunit), and fusion KCNQ1 + KCNE1. Current was monitored in the absence and presence of clinically relevant concentration of isoflurane (0.54 ± 0.05 mM, 1.14 vol %). Computer simulations determined the resulting impact on the cardiac action potential.

Results: Isoflurane had significantly greater inhibitory effect on A341V + KCNE1 (62.2 ± 3.4%, n = 8) than on wild-type KCNQ1 + KCNE1 (40.7 ± 4.5%; n = 9) in transfected HEK293 cells. Under heterozygous conditions, isoflurane inhibited A341V + KCNQ1 + KCNE1 by 65.2 ± 3.0% (n = 13) and wild-type KCNQ1 + KCNE1 (2:1 ratio) by 32.0 ± 4.5% (n = 11). A341V exerted a dominant negative effect on IKs. Similar differential effects of isoflurane were also observed in experiments using the cardiac HL-1 cells. Mutations of the neighboring F340 residue significantly attenuated the effects of isoflurane, and fusion proteins revealed the modulatory effect of KCNE1. Action potential simulations revealed a stimulation frequency-dependent effect of A341V.

Conclusions: The LQTS-associated A341V mutation rendered the IKs channel more sensitive to the inhibitory effects of isoflurane compared to wild-type IKs in transfected cell lines; F340 is a key residue for anesthetic action.

Figure 1

The impact of isoflurane on wild-type IKs (KCNQ1+KCNE1) and A341V mutant in HEK293 cells. (A) Representative whole-cell current traces of KCNQ1+KCNE1 in the absence and presence of isoflurane (0.54 ± 0.05 mM; equivalent to 1.14 vol % at 22 °C) are shown. The voltage protocol is shown in the inset. (B) The effects of isoflurane on the voltage dependence of activation of KCNQ1+KCNE1. The isochronal activation curves were fitted with the Boltzmann equation as described in Materials and Methods under Data Analysis and Statistical Analysis (n = 9; Table 1). (C) Representative whole cell current traces of A341V+KCNE1 in control and in the presence of isoflurane are shown. The voltage protocol was as shown in Figure 1A. A341V+KCNE1 exhibited markedly slow activation kinetics compared to the wild-type IKs. The inhibitory effects of isoflurane on A341V+KCNE1 were greater than those on KCNQ1+KCNE1. (D) The effects of isoflurane on the voltage dependence of activation of A341V+KCNE1 (n = 8; Table 1). (E) Summary of the inhibitory effects of isoflurane on KCNQ1+KCNE1 and A341V+KCNE1 in HEK293cells. The inhibition of current by isoflurane was quantified by the reduction of the peak current amplitude measured at the end of the depolarizing test pulses to +60 mV. The mutation of alanine to valine at position of 341 of KCNQ1 significantly enhanced the inhibitory effects of isoflurane on IKs (n = 8–9, *P < 0.01 versus KCNQ1+KCNE1).

Figure 2

The impact of isoflurane on wild-type IKs and A341V mutant under heterozygous conditions. Whole-cell currents were monitored in HEK293 cells expressing A341V+KCNQ1+KCNE1 (2.4 μg of complementary DNA [cDNA] for each subunit) and compared to those with 2KCNQ1+KCNE1 (4.8 μg of KCNQ1 and 2.4 μg of KCNE1 cDNA). (A) Representative current traces of 2KCNQ1+KCNE1 in control and in the presence of isoflurane are shown. (B) The effects of isoflurane on the voltage dependence of activation of 2KCNQ1+KCNE1. The isochronal activation curves were fitted with the Boltzmann equation as described in Materials and Methods under Data Analysis and Statistical Analysis (n = 11; Table 1). (C) Representative current traces of A341V+KCNQ1+KCNE1 in control and in the presence of isoflurane are shown. The inhibitory effects of isoflurane on A341V+KCNQ1+KCNE1 were greater than those on 2KCNQ1+KCNE1, similar to the comparisons between KCNQ1+KCNE1 and A341V+KCNE1. (D) The effects of isoflurane on the voltage-dependence of activation of A341V+KCNQ1+KCNE1 is depicted (n = 13; Table 1).

Figure 3

Summary of the inhibitory effects of isoflurane under heterozygous conditions and the impact of A341V on current density. (A) Summary of the inhibitory effects of isoflurane on 2KCNQ1+KCNE1 and A341V+KCNQ1+KCNE1 at +60 mV. Similar to the homozygous conditions (Figure 2), the A341V mutant was significantly more susceptible to the inhibitory effects of isoflurane (n = 11–13, *P < 0.01 versus 2KCNQ1+KCNE1). (B) The current-voltage relationships under control (isoflurane-free) conditions. Current amplitudes were normalized to cell capacitance to yield current density. A341V+KCNQ1+KCNE1 exhibited significantly smaller current density than the corresponding wild-type IKs, 2KCNQ1+KCNE1, and KCNQ1+KCNE1 (n = 11–13, *P < 0.01 vs. 2KCNQ1+KCNE1, P < 0.05 vs. KCNQ1+KCNE1; §P < 0.05 vs. 2KCNQ1+KCNE1 and KCNQ1+KCNE1). This is suggestive of a dominant negative effect by A341V.

Figure 4

The impact of isoflurane on KCNQ1+KCNE1 and A341V+KCNE1 in a cardiac cell line, HL-1 cells. (A) Representative whole-cell current traces of KCNQ1+KCNE1 in control and in the presence of isoflurane is shown. The voltage protocol is as depicted in Figure 1A. (B) The effects of isoflurane on the corresponding voltage-dependence of activation of KCNQ1+KCNE1 are depicted. The isochronal activation curves were fitted with the Boltzmann equation as described in Materials and Methods under Data Analysis and Statistical Analysis (n = 8; Table 2). In contrast to the results in HEK293 cells, isoflurane did not shift the activation curve of KCNQ1+KCNE1 in HL-1 cells. (C) Representative whole-cell current traces of A341V+KCNE1 in control and in the presence of isoflurane is shown. (D) The effects of isoflurane on the voltage-dependence of activation of A341V+KCNE1 are presented (n = 8; Table 2). (E) Summary of the inhibitory effects of isoflurane on KCNQ1+KCNE1 and A341V+KCNE1 in HL-1 cells. Similar to the results in HEK 293 cells, A341V+KCNE1 was significantly more sensitive to the inhibitory effects of isoflurane compared to those on KCNQ1+KCNE1 (n = 8/group, *P < 0.01 versus KCNQ1+KCNE1).

Figure 5

The effects of isoflurane on wild-type IKs and A341V mutant under heterozygous conditions in HL-1 cells. The cDNA ratios for each subunit used for transfection were the same for those used in Figure 2. (A) Representative current traces of 2KCNQ1+KCNE1 recorded in control and in the presence of isoflurane are shown. (B) The corresponding effects on the voltage-dependence of activation of 2KCNQ1+KCNE1. The isochronal activation curves were fitted with the Boltzmann equation as described in Materials and Methods under Data Analysis and Statistical Analysis (n = 10; Table 2). Isoflurane shifted the activation curve in the depolarizing direction. (C) Representative current traces of A341V+KCNQ1+KCNE1 in control and in the presence of isoflurane are shown. (D) The corresponding effects on the voltage dependence of activation of A341V+KCNQ1+KCNE1 (n = 8). The isochronal activation curves were not significantly shifted by the application of isoflurane, which was in contrast to the results in HEK293 cells (see Figure 3). (E) Summary of the inhibitory effects of isoflurane on 2KCNQ1+KCNE1 and A341V+KCNQ1+KCNE1 in HL-1 cells. Similar to the results in HEK293 cells, the A341V mutant was significantly more sensitive to the inhibitory effects of isoflurane compared to corresponding wild-type IKs (n = 8–10, *P < 0.01 versus 2KCNQ1+KCNE1).

Figure 6

The effect of isoflurane on KCNQ1 expressed alone in HL-1 cells. (A) Representative current traces of KCNQ1 are shown in the absence and presence of 0.5 mM isoflurane. (B) The effects of isoflurane on the corresponding voltage-dependence of activation of KCNQ1 are depicted. Isoflurane had no significant effect on the KCNQ1 isochronal activation curves that were fitted with the Boltzmann equation as described in Materials and Methods under Data Analysis and Statistical Analysis. (C) Summary of the inhibitory effects of isoflurane on KCNQ1 at various test potentials is shown.

Figure 7

Role of F340 in determining anesthetic sensitivity. The inhibitory effects of isoflurane (0.5 mM) were determined in two KCNQ1 mutants, F340A and F340C, as shown in panels (A) and (B), respectively. The results are summarized in (C). Current inhibition was determined at a test-pulse potential of +60 mV from a −50 mV holding potential. Compared to the wild type KCNQ1, the inhibition by isoflurane on the two mutants were significantly attenuated. However, between the two mutants, a significant difference was observed in the inhibition by isoflurane. *denotes significantly different from F340A, n=4–5/group.

Figure 8

Effects on isoflurane on whole-cell potassium currents (IKs) fusion proteins. A. Schematic of the fusion proteins. In the MK24 protein, the C-terminus of KCNE1 was linked to the N-terminus of KCNQ1, and in the MKK44, the C-terminus of KCNE1 was linked to the N-terminus of a tandem homodimer of KCNQ1. B. Isoflurane effects on MK24 and MKK44. In control, both MK24 and MKK44 exhibited IKs-like activation kinetics. C. Summary of the effects of isoflurane (0.5 mM) on the fusion proteins. Inhibition of current was determined at a test-pulse potential of +60 mV from a −50 mV holding potential. *denotes significantly different from MK24, n=9–12/group.

Figure 9

Simulation of the cardiac action potential using the ten Tusscher model of human ventricular myocyte. (A) Simulated action potential under control condition (wild-type IKs) in the absence and presence (dashed lines) of isoflurane. (B) Simulated action potential under LQT1 (Long QT Syndrome Type 1) conditions with heterozygous expression of A341V in the absence and presence (dashed lines) of isoflurane. In both cases, isoflurane triggered prolongation of the action potential duration.

Figure 10

Simulation of the cardiac action potential at various stimulation frequencies under control and LQT1 conditions with heterozygous expression of A341V. Using the ten Tusscher model, ventricular action potentials under control (wild-type IKs; Panel A) and LQT1 (A341V; Panel B) conditions were stimulated at 1, 2, and 3 Hz in the absence and presence of isoflurane. For the wild-type conditions, the integrity of the action potential profile was maintained at the various stimulation frequencies; no evidence of arrhythmogenicity was evident in the absence or presence of isoflurane. For the LQT1 conditions, no evidence of arrhythmogenicity was evident at stimulation frequencies of 1 and 2 Hz. However, at 3 Hz, early afterdepolarizations were apparent in the absence and presence of isoflurane.