Vonderlin N et al. (2015), Anesthetic drug midazolam inhibits cardiac huma...

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Drug Des Devel Ther 2015 Feb 16;9:867-77. doi: 10.2147/DDDT.S72765.

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

Vonderlin N , Fischer F , Zitron E , Seyler C , Scherer D , Thomas D , Katus HA , Scholz EP .

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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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Species referenced: Xenopus
Genes referenced: gnao1 kcnh2

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	Figure 1. Midazolam inhibits heterologously expressed hERG channels.Notes: (A) A typical family of hERG outward currents elicited by a double-stage voltage protocol. (B) Incubation with midazolam resulted in a reduction of hERG currents. (C) The corresponding activating current amplitude measured at the end of the first test pulse (first dashed lines in A and B) as a function of the test pulse potential. (D) hERG activation curve: tail current amplitudes (at second dashed lines in A and B) are shown as a function of the preceding test pulse potential. Midazolam significantly shifted the half-maximal activation voltage of the hERG channels (control −9.17±0.59 mV; midazolam −12.58±0.99 mV). Protocol: holding potential −80 mV, test pulse −70 to +70 mV (2,000 msec) in 10 mV increments, return pulse to −50 mV (2,000 msec). Of note, only every second voltage step is displayed in (A) and (B) in order to achieve a clearer presentation.Abbreviation: hERG, human ether-à-go-go-related gene.
	Figure 2. Concentration dependence of midazolam-induced hERG block.Notes: (A) To determine the concentration dependence of midazolam-induced block, a standard voltage protocol was used, eliciting large inward tail currents (see inset). Exemplary current traces elicited by a voltage step to 70 mV prior and after midazolam incubation (100 μM) are displayed. (B) Midazolam inhibited hERG channels in a concentration-dependent manner, yielding an IC50 of 170 μM and a Hill slope of 1.0. Protocol: repetitive pulsing at a frequency of 0.1 Hz, holding potential −80 mV, variable test pulses between −70 mV and +70 mV (400 msec, 10 mV increment), and return pulses to −120 mV (400 msec). (C) When analyzed in a mammalian cell line (HEK), the inhibitory effects of midazolam were more pronounced, resulting in a current reduction by 49% and 63% (10 and 30 μM, respectively). A representative current trace under control conditions is shown as an inset.Abbreviation: hERG, human ether-à-go-go-related gene.
	Figure 3. Effects of midazolam on channel inactivation.Notes: (A) In order to analyze the effects of midazolam on steady-state inactivation, a double-step voltage protocol was used (see inset). A typical family of current traces is displayed for control conditions (A) and after application of 200 μM midazolam (B). Current traces were corrected for channel deactivation by extrapolation to the beginning of the second voltage step (see dashed lines and arrows in A). (C) Current-voltage relationship of tail current amplitude. Steady-state inactivation was obtained by dividing the current amplitude by the electrochemical driving force (D). Midazolam did not significantly affect the half-maximal inactivation voltage of hERG channels. Protocol: holding potential −80 mV, first test pulse to +40 mV (one second), second test pulse between −140 to +40 mV (20 mV increment, 500 msec).Abbreviation: hERG, human ether-à-go-go-related gene.
	Figure 4. State dependence of midazolam-induced hERG block.Notes: To investigate the state dependence of hERG channel inhibition, two different voltage protocols were chosen. First, oocytes were depolarized from a holding potential of −80 mV using a long pulse to 0 mV (6 seconds). (A) Exemplary activating current traces are displayed for control conditions and after incubation with 200 μM midazolam. Fractional block was then calculated by division and plotted versus time (B). Within the first seconds, fractional block proceeded in a time-dependent manner, pointing to an open channel block by midazolam. (C) Next, a double-step voltage protocol was applied. From a holding potential, channels were activated and inactivated by a long voltage step to +80 mV (3,500 msec). This step was followed by a second step to 0 mV (3,500 msec). Normalized relative current during the second voltage step is displayed in (D). In contrast with (B), no further development of block could be observed.Abbreviation: hERG, human ether-à-go-go-related gene.
	Figure 5. Onset and frequency dependence of block.Notes: (A) Onset of block was recorded over a period of 30 minutes, using a double-step voltage protocol. Protocol: holding potential −80 mV, first step to +30 mV (400 msec), return pulse −60 mV (400 msec), frequency 0.033 Hz. Peak tail current amplitude was followed to determine the degree of inhibition. Block development was fast, reaching half maximal inhibition at approximately 4 minutes after initiation of midazolam application (n=6). (B) To analyze the frequency dependence of block, pulses were applied at frequencies of 1, 0.5, and 0.33 Hz for 60 seconds. Exemplary current traces for the first and last repetitions at a pacing rate of 1 Hz after midazolam incubation as well as the potential protocol are displayed as an inset. Relative current after midazolam incubation is displayed as a function of time. No frequency dependence of block could be observed. Protocol: holding potential −80 mV, first step to +20 mV (300 msec), return pulse −40 mV (300 msec).
	Figure 6. Mutations of the aromatic pore residues F656A and Y652A affect the inhibitory effects of midazolam.Notes: (A–C) Exemplary current traces before and after incubation of midazolam in hERG WT as well as in Y652A hERG and F656A hERG. (D) Gives an overview of the observed effects. Compared with hERG WT, the inhibitory effects of midazolam were significantly attenuated in mutant hERG channels. Protocol: holding potential −80 mV, first pulse to +70 mV (400 msec), second pulse to −120 mV (400 msec).Abbreviations: hERG, human ether-à-go-go-related gene; WT, wild-type.
	Figure 7. Midazolam does not attenuate hERG channel surface expression. Effects of midazolam on channel surface expression analyzed by the Western blot technique (upper panel). Image density of the 155 kDa hERG form divided by the 135 kDa hERG form was determined to quantify channel surface expression (lower panel). Incubation with 100 μM As2O3 served as a positive control. Compared with control conditions (lane 2), increasing midazolam concentrations (lanes 3–7) did not result in a significant change of channel surface expression.Abbreviation: hERG, human ether-à-go-go-related gene.

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