Noble gas xenon is a novel adenosine triphosphate-sensitive potassium channel opener

Abstract

Background: Adenosine triphosphate-sensitive potassium (KATP) channels in brain are involved in neuroprotective mechanisms. Pharmacologic activation of these channels is seen as beneficial, but clinical exploitation by using classic K channel openers is hampered by their inability to cross the blood-brain barrier. This is different with the inhalational anesthetic xenon, which recently has been suggested to activate KATP channels; it partitions freely into the brain.

Methods: To evaluate the type and mechanism of interaction of xenon with neuronal-type KATP channels, these channels, consisting of Kir6.2 pore-forming subunits and sulfonylurea receptor-1 regulatory subunits, were expressed in HEK293 cells and whole cell, and excised patch-clamp recordings were performed.

Results: Xenon, in contrast to classic KATP channel openers, acted directly on the Kir6.2 subunit of the channel. It had no effect on the closely related, adenosine triphosphate (ATP)-regulated Kir1.1 channel and failed to activate an ATP-insensitive mutant version of Kir6.2. Furthermore, concentration-inhibition curves for ATP obtained from inside-out patches in the absence or presence of 80% xenon revealed that xenon reduced the sensitivity of the KATP channel to ATP. This was reflected in an approximately fourfold shift of the concentration causing half-maximal inhibition (IC50) from 26 +/- 4 to 96 +/- 6 microm.

Conclusions: Xenon represents a novel KATP channel opener that increases KATP currents independently of the sulfonylurea receptor-1 subunit by reducing ATP inhibition of the channel. Through this action and by its ability to readily partition across the blood-brain barrier, xenon has considerable potential in clinical settings of neuronal injury, including stroke.

Fig. 1

K_ATP channels are activated by xenon. (A) Whole cell recording (holding potential [V_H] −20 mV) from an HEK293 cell-expressing Kir6.2/SUR1 channels demonstrating reversible current activation by 80% xenon and its preclusion by tolbutamide (Tb). Drugs were bath applied as indicated by horizontal bars. Dashed line is zero-current level. (B, C) Current–voltage relationships demonstrating K_ATP current activation by xenon and block by tolbutamide. (D) Mean current amplitude at −20 mV for untransfected and Kir6.2/SUR1-transfected cells in the absence or presence of 80% xenon (Xe). (E) Effects of the SUR1-specific K-channel opener diazoxide (Dz) on Kir6.2/SUR1 whole cell currents. Plotted is the mean holding current at −20 mV every 15 s. (F) Mean effects of the K-channel openers xenon and diazoxide and the blocker tolbutamide on Kir6.2/SUR1 currents. Expressed is the holding current at −20 mV in the presence of the drug as a fraction of the current in the absence of the drug. Xe + Tb: current in the presence of xenon and tolbutamide. Numbers (N) are given above the bars. **P < 0.01 compared with control. ##P < 0.01 compared with lower drug concentration.

Fig. 2

Xenon does not require the SUR1 subunit to activate K_ATP channels. (A) Xenon activation of whole cell Kir6.2ΔC26 currents. Plotted is the mean holding current at −20 mV every 15 s. Note the enhanced rundown compared with channels containing the SUR1 subunit., Dashed line is approximation of slower phase of rundown. (B) Mean data from recordings performed on Kir6.2ΔC26, Kir1.1, and K185Q/SUR1 currents summarizing the effects of activators and inhibitors of these channels. Expressed is the holding current at −20 mV in the presence of the drug as a fraction of the current in the absence of the drug. Note the lack of effect of tolbutamide (Tb) and diazoxide (Dz) on Kir6.2ΔC26 currents. (C) Whole cell Kir6.2-K185Q/SUR1 current is sensitive to tolbutamide but not xenon. Numbers (N) are given above the bars. *P < 0.05 compared with control. **P < 0.01 compared with control.

Fig. 3

Properties of wild-type and mutant K_ATP channels in inside-out patches. (A) Current–voltage relationships obtained from inside-out patches excised from cells expressing Kir6.2/SUR1 revealed macroscopic inwardly rectifying adenosine triphosphate (ATP)-sensitive currents in symmetrical K⁺ concentrations. (B) Mean slope conductance (G) in the presence of ATP or tolbutamide (Tb) expressed as a fraction of the conductance in the absence of the drug (G_c). ATP inhibition was concentration dependent for Kir6.2/SUR1 and absent for Kir6.2-K185Q/SUR1 currents. Numbers (N) are given above the bars. **P < 0.01 compared with control. (C, D) Current–voltage relationships obtained for Kir6.2/SUR1 and Kir6.2-K185Q/SUR1 currents, respectively, in the absence and presence of 0.1 mm tolbutamide.

Fig. 4

Xenon activates wild-type but not mutant K_ATP channels in inside-out patches. (A, B) Xenon enhanced Kir6.2/SUR1 currents, but not Kir6.2-K185Q/SUR1 currents, in the presence of adenosine triphosphate (ATP). Note that 1 mm ATP increased Kir6.2-K185Q/SUR1 currents because of MgATP-dependent refreshment, and because of its interaction with the SUR1 subunit in the presence of MgCl₂. (C) Mean data from recordings as shown in A and B. The slope conductance (G) in the presence of ATP and xenon is expressed as a fraction of the conductance in the absence of the drug (G_c). Xenon activation of Kir6.2/SUR1 increased with increasing ATP concentrations but was absent in the Kir6.2-K185Q mutant both in the absence and presence of ATP. Numbers (N) are given above the bars. ** P < 0.01 compared with control.

Fig. 5

Xenon reduces ATP inhibition of inside-out patches. (A, B) Inside-out patch-clamp recordings of Kir6.2/SUR1 currents in the absence (A) or presence (B) of 80% xenon. Recordings were performed in Mg²⁺-free solution (note the absence of refreshment) and adenosine triphosphate (ATP) was applied as indicated by the bars. (C) ATP concentration–inhibition curves for Kir6.2/SUR1 currents in the absence of MgCl₂ as obtained from experiments as shown in A and B. Experiments were performed either in the absence (open circles) or in the presence of 80% xenon (filled circles). Solutions containing ATP were alternated with ATP-free solutions. Slope conductance (G) in the presence of the drug is expressed as a fraction of the slope conductance in its absence (G_c). The dotted lines and solid lines are the best fit to the data using the modified Hill equation (Eq. 1). Numbers (N) are given above the data points. *P < 0.05 compared with same ATP concentration in the absence of xenon.