Limits...
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 - Drug Des Devel Ther (2015)

Bottom Line: However, steady-state inactivation was not significantly affected.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine III, University Hospital Heidelberg, Heidelberg, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4338257&req=5

f7-dddt-9-867: 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.

Mentions: Several inhibitors of hERG channels have been associated with a reduction in the surface expression of hERG channels.20,27 To analyze the effects of midazolam on hERG channel trafficking, we used the Western blot technique. HEK cells stably expressing hERG channels were incubated for 24 hours with either control medium, 100 μM As2O3 as a positive control, or increasing midazolam concentrations (1, 3, 10, 30, 100 μM). Figure 7 (upper panel) shows a representative result of Western blot analysis. Under control conditions, two protein bands could be observed: a first band at 135 kDa representing the core glycosylated hERG protein and a second band at 155 kDa representing complex glycosylated hERG protein. To determine the effect of midazolam on hERG channel surface expression, the amount of complex glycosylated hERG protein was divided by the amount of core glycosylated hERG protein. Compared with control conditions, treatment with As2O3 (100 μM) resulted in a strong reduction of channel surface expression by 77.83%±3.4% (Figure 7, lane 1, n=3 blots). In contrast, incubation with increasing midazolam concentrations (1, 3, 10, 30, 100 μM) did not result in a significant reduction of the 155 kDa hERG (Figure 7, lines 3–8, n=3, P<0.05, analysis of variance).


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 - Drug Des Devel Ther (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4338257&req=5

f7-dddt-9-867: 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.
Mentions: Several inhibitors of hERG channels have been associated with a reduction in the surface expression of hERG channels.20,27 To analyze the effects of midazolam on hERG channel trafficking, we used the Western blot technique. HEK cells stably expressing hERG channels were incubated for 24 hours with either control medium, 100 μM As2O3 as a positive control, or increasing midazolam concentrations (1, 3, 10, 30, 100 μM). Figure 7 (upper panel) shows a representative result of Western blot analysis. Under control conditions, two protein bands could be observed: a first band at 135 kDa representing the core glycosylated hERG protein and a second band at 155 kDa representing complex glycosylated hERG protein. To determine the effect of midazolam on hERG channel surface expression, the amount of complex glycosylated hERG protein was divided by the amount of core glycosylated hERG protein. Compared with control conditions, treatment with As2O3 (100 μM) resulted in a strong reduction of channel surface expression by 77.83%±3.4% (Figure 7, lane 1, n=3 blots). In contrast, incubation with increasing midazolam concentrations (1, 3, 10, 30, 100 μM) did not result in a significant reduction of the 155 kDa hERG (Figure 7, lines 3–8, n=3, P<0.05, analysis of variance).

Bottom Line: However, steady-state inactivation was not significantly affected.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine III, University Hospital Heidelberg, Heidelberg, Germany.

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.

No MeSH data available.


Related in: MedlinePlus