Inhibition of cardiac Kv1.5 potassium current by the anesthetic midazolam: mode of action

Abstract

Midazolam is a short-acting benzodiazepine that is widely used in anesthesia. Despite its widespread clinical use, detailed information about cardiac side effects of midazolam is largely lacking. Using the double-electrode voltage clamp technique, we studied pharmacological effects of midazolam on heterologously expressed Kv1.5 channels underlying atrial repolarizing current I(Kur). Midazolam dose-dependently inhibited Kv1.5 current, yielding an IC50 of 17 μM in an HEK cell line and an IC50 of 104 μM in Xenopus oocytes. We further showed that midazolam did not affect the half-maximal activation voltage of Kv1.5 channels. However, a small negative shift of the inactivation curve could be observed. Midazolam acted as a typical open-channel inhibitor with rapid onset of block and without frequency dependence of block. Taken together, midazolam is an open channel inhibitor of cardiac Kv1.5 channels. These data add to the current understanding of the pharmacological profile of midazolam.

Keywords: anesthetics; pharmacology; potassium channels.

Figure 1

Midazolam inhibits cloned cardiac Kv1.5 potassium channels. Notes: (A) Representative current trace of a typical Kv1.5 current elicited by a rectangular voltage protocol in Xenopus oocytes. Midazolam (100 μM) results in a strong inhibition of Kv1.5 current. (B) Dose–response curves for inhibition of Kv1.5 by midazolam established in Xenopus oocytes. (C) Representative current trace of a typical Kv1.5 current elicited by a rectangular voltage protocol in an HEK cell line using the patch-clamp technique. Midazolam (30 μM) results in a pronounced reduction of Kv1.5 current. (D) Dose–response curves established in an HEK cell line stably expressing Kv1.5 channels.

Figure 2

Pharmacological properties of Kv1.5 current inhibition. Notes: Typical families of Kv1.5 current traces elicited by a double-step voltage protocol (inset in [A]) before (A) and after (B) incubation with 100 μM midazolam in Xenopus oocytes. (C) Current–voltage relationship of Kv1.5 current under control conditions (filled boxes) and after incubation with midazolam (open circles) measured at peak current (n=6). (D) Kv1.5 activation curves established by dividing peak current amplitude by the electrochemical driving force. Midazolam did not significantly influence the half-maximal activation voltage (V_1/2) (n=6). (E) Kv1.5 channel inactivation curves established by plotting tail current amplitude versus the potential of the first voltage step. Midazolam resulted in a small but significant shift of the inactivation curve (n=6).

Figure 3

Time constants of channel inhibition. Notes: Exemplary current traces elicited by a rectangular voltage step to +50 mV under control conditions and after application of 30 μM (A) or 1,000 μM (B) midazolam in Xenopus oocytes. Time course of block development was determined by division. In both cases, development of block was fast, yielding time constants (τ) of 9.0 ms for 30 μM (C) and 4.1 ms for 1,000 μM (D) midazolam.

Figure 4

Frequency dependence of Kv1.5 channel block. Notes: Frequency dependence of block was analyzed in Xenopus oocytes using three different pacing rates (1 Hz, 2 Hz, and 4 Hz). Having obtained a control measurement, 300 μM midazolam was washed-in and the measurement was repeated. (A–C) Exemplary experiments showing the first (t=0 seconds) and last (t=16 seconds) current traces after application of 300 μM midazolam. (D) The degree of inhibition was calculated for the last test pulse (at 16 seconds), yielding no significant dependence on the pacing rate (n=6). Abbreviations: t, time; s, seconds.