Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

Q: What are the key findings of this study?

Midazolam inhibited cardiac hERG channels with an IC50 of 170 μM in Xenopus oocytes and 13.6 μM in HEK 293 cells without affecting channel surface expression.

Q: What is the clinical evidence from this study?

Population: Cardiac repolarization / hERG potassium channel inhibition. Intervention: Midazolam vs. Control external solution / pure Dulbecco's Modified Eagle's Medium (and 100 μM As2O3 positive control for trafficking). Primary outcome: Concentration-dependent inhibition of cardiac hERG channel currents (IC50) (p=<0.05).

Q: What does this research mean for the field?

Midazolam inhibits cardiac hERG channels in a concentration- and state-dependent manner without altering channel surface expression. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

Q: What question did this study set out to answer?

To examine the inhibitory effects, gating kinetics, binding sites, and trafficking impacts of the anesthetic midazolam on cardiac human ether-à-go-go-related gene (hERG) channels.

Eberhard P. Scholz; Nadine Vonderlin; Fathima Fischer; Edgar Zitron; Claudia Seyler; D. Scherer; Dierk Thomas; Hugo A. Katus

doi:10.2147/dddt.s72765

February 1, 2015Drug Design Development and TherapyOpen Access

Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

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Key result

Midazolam inhibits cardiac hERG channels with an IC50 of ~14 μM without altering surface expression.

P<0.05

Why the study?

Does the anesthetic midazolam inhibit cardiac hERG potassium channels or alter hERG channel surface trafficking in vitro?

Key Points

To examine the inhibitory effects, gating kinetics, binding sites, and trafficking impacts of the anesthetic midazolam on cardiac human ether-à-go-go-related gene (hERG) channels.
Assessed hERG channel electrophysiology using two-electrode voltage clamp recordings in Xenopus oocytes and whole-cell patch-clamp techniques in HEK 293 cells stably expressing hERG channels.
Evaluated channel gating, state dependence, and binding site interactions using site-directed pore mutants (F656A and Y652A).
Analyzed subacute channel trafficking and surface expression in HEK 293 cells incubated with midazolam for 24 hours using Western blot assays.
Midazolam blocked hERG currents in a concentration-dependent manner, with an IC50 of 170±22.3 μM in Xenopus oocytes and 13.6±30 μM in HEK 293 cells.
Application of 200 μM midazolam reduced peak tail current amplitude by 52.1%±1.9% (n=6) and shifted the half-maximal activation voltage from -9.2±0.59 mV to -12.6±1.0 mV (P<0.05, n=6).
Channel blockade occurred selectively in the open and inactivated states without frequency dependence, depended on pore residues F656 and Y652, and caused no changes in hERG surface expression.

Structured PICO

Does the anesthetic midazolam inhibit cardiac hERG potassium channels or alter hERG channel surface trafficking in vitro?

P

Population

In vitro electrophysiological and Western blot study using Xenopus oocytes and HEK 293 cells heterologously expressing wild-type and mutant (Y652A, F656A) cardiac hERG channels incubated for 30 minutes (electrophysiology) or 24 hours (trafficking).

I

Intervention

Midazolam incubation at concentrations of 10, 30, 100, 200, 300, and 1,000 μM for 30 minutes in Xenopus oocytes; 1, 10, 30, and 100 μM for 30 minutes in HEK 293 cells (patch-clamp); and 1, 3, 10, 30, and 100 μM for 24 hours in HEK 293 cells (channel trafficking)

C

Comparator

Pre-incubation control measurements in standard external bath solution for electrophysiology; pure Dulbecco's Modified Eagle's Medium (negative control) and 100 μM As2O3 (positive control) for 24-hour trafficking analysis

O

Outcome

Concentration-dependent inhibition (IC50) of hERG potassium channel tail currents measured via double-electrode voltage clamp (Xenopus oocytes) and whole-cell patch-clamp (HEK 293 cells)surrogate

Main Result

p-value: p=<0.05

Limitations

Concentrations required in Xenopus oocytes are up to 30 times higher than in mammalian cells due to diffusion barriers caused by the vitelline membrane and yolk sac
All electrophysiological experiments were performed in vitro at room temperature (22°C) using heterologous expression systems
Exact molecular binding properties within the pore were not evaluated with in silico docking analysis
Diffusion barriers in Xenopus oocytes (vitelline membrane and yolk sac) require drug concentrations up to 30 times higher than in mammalian cells
Electrophysiological recordings were conducted in vitro in heterologous expression systems at room temperature (22°C)
Lack of in silico docking analysis to further characterize exact binding properties

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Implication

Midazolam is a low-affinity open- and inactivated-state pore blocker of cardiac hERG channels (IC50 13.6 μM in HEK 293 cells) that does not impair channel trafficking, making clinically significant QTc prolongation unlikely at standard therapeutic plasma concentrations.

Abstract

Midazolam is a short-acting benzodiazepine that is in wide clinical use as an anxiolytic, sedative, hypnotic, and anticonvulsant. Midazolam has been shown to inhibit ion channels, including calcium and potassium channels. So far, the effects of midazolam on cardiac human ether-à-go-go-related gene (hERG) channels have not been analyzed. The inhibitory effects of midazolam on heterologously expressed hERG channels were analyzed in Xenopus oocytes using the double-electrode voltage clamp technique. We found that midazolam inhibits hERG channels in a concentration-dependent manner, yielding an IC50 of 170 μM in Xenopus oocytes. When analyzed in a HEK 293 cell line using the patch-clamp technique, the IC50 was 13.6 μM. Midazolam resulted in a small negative shift of the activation curve of hERG channels. However, steady-state inactivation was not significantly affected. We further show that inhibition is state-dependent, occurring within the open and inactivated but not in the closed state. There was no frequency dependence of block. Using the hERG pore mutants F656A and Y652A we provide evidence that midazolam uses a classical binding site within the channel pore. Analyzing the subacute effects of midazolam on hERG channel trafficking, we further found that midazolam does not affect channel surface expression. Taken together, we show that the anesthetic midazolam is a low-affinity inhibitor of cardiac hERG channels without additional effects on channel surface expression. These data add to the current understanding of the pharmacological profile of the anesthetic midazolam.

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Cite This Study

Scholz et al. (2015) studied Cardiac repolarization / hERG potassium channel inhibition. Midazolam vs. Control external solution / pure Dulbecco's Modified Eagle's Medium (and 100 μM As2O3 positive control for trafficking) was evaluated on Concentration-dependent inhibition of cardiac hERG channel currents (IC50) (p=<0.05). Midazolam inhibited cardiac hERG channels with an IC50 of 170 μM in Xenopus oocytes and 13.6 μM in HEK 293 cells without affecting channel surface expression.

synapsesocial.com/papers/6a90227781f23260182b1163 https://doi.org/10.2147/dddt.s72765

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Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

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