Bruce D. Winegar

Volatile anesthetics activate the human tandem pore domain baseline K+ channel KCNK5.

Background Previous studies have identified a volatile anesthetic–induced increase in baseline potassium permeability and concomitant neuronal inhibition. The emerging family of tandem pore domain potassium channels seems to function as baseline potassium channels in vivo. Therefore, we studied the effects of clinically used volatile anesthetics on a recently described member of this family. Methods A cDNA clone containing the coding sequence of KCNK5 was isolated from a human brain library. Expression of KCNK5 in the central nervous system was determined by Northern blot analysis and reverse-transcription polymerase chain reaction. Functional expression of the channel was achieved by injection of cRNA into Xenopus laevis oocytes. Results Expression of KCNK5 was detected in cerebral cortex, medulla, and spinal cord. When heterologously expressed in Xenopus oocytes, KCNK5 currents exhibited delayed activation, outward rectification, proton sensitivity, and modulation by protein kinase C. Clinical concentrations of volatile general anesthetics potentiated KCNK5 currents by 8–30%. Conclusion Human KCNK5 is a tandem pore domain potassium channel exhibiting delayed activation and sensitivity to volatile anesthetics and may therefore have a role in suppressing cellular excitability during general anesthesia.

Volatile general anesthetics produce hyperpolarization of Aplysia neurons by activation of a discrete population of baseline potassium channels.

Background The mechanism by which volatile anesthetics act on neuronal tissue to produce reversible depression is unknown. Previous studies have identified a potassium current in invertebrate neurons that is activated by volatile anesthetics. The molecular components generating this current are characterized here in greater detail. Methods The cellular and biophysical effects of halothane and isoflurane on neurons of Aplysia californica were studied. Isolated abdominal ganglia were perfused with anesthetic‐containing solutions while membrane voltage changes were recorded. These effects were also studied at the single‐channel level by patch clamping cultured neurons from the abdominal and pleural ganglia. Results Clinically relevant concentrations of halothane and isoflurane produced a slow hyperpolarization in abdominal ganglion neurons that was sufficient to block spontaneous spike firings. Single‐channel studies revealed specific activation by volatile anesthetics of a previously described potassium channel. In pleural sensory neurons, halothane and isoflurane increased the open probability of the outwardly rectifying serotonin‐sensitive channel (S channel). Halothane also inhibited a smaller noninactivating channel with a linear slope conductance of approximately 40 pS. S channels were activated by halothane with a median effective concentration of approximately 500 micro Meter (0.013 atm), which increased channel activity about four times. The mechanism of channel activation involved shortening the closed‐time durations between bursts and apparent recruitment of previously silent channels. Conclusions The results demonstrate a unique ability of halothane and isoflurane to activate a specific class of potassium channels. Because potassium channels are important regulators of neuronal excitability within the mammalian central nervous system, background channels such as the S channel may be responsible in part for mediating the action of volatile anesthetics.

TOK1 Is a Volatile Anesthetic Stimulated K+Channel

Background Volatile anesthetic agents can activate the S channel, a baseline potassium (K sup +) channel, of the marine mollusk Aplysia. To investigate whether cloned ion channels with electrophysiologic properties similar to the S channel (potassium selectivity, outward rectification, and activation independent of voltage) also are modulated by volatile anesthetic agents, the authors expressed the cloned yeast ion channel TOK1 (tandem pore domain, outwardly rectifying K sup + channel) in Xenopus oocytes and studied its sensitivity to volatile agents. Methods Standard two‐electrode voltage and patch clamp recording methods were used to study TOK1 channels expressed in Xenopus oocytes. Results Studies with two‐electrode voltage clamp at room temperature showed that halothane, isoflurane, and desflurane increased TOK1 outward currents by 48–65% in barium Frog Ringers perfusate. The concentrations at which 50% potentiation occurred (EC50 values) were in the range of 768–814 micro meter (0.016–0.044 atm) and had a rank order of potency in atm in which halothane > isoflurane > desflurane. The potentiation of TOK1 by volatile anesthetic agents was rapid and reversible (onset and offset, 1–20 s). In contrast, the non‐anesthetic 1,2‐dichlorohexafluorocyclobutane did not potentiate TOK1 currents in concentrations up to five times the MAC value predicted by the Meyer‐Overton hypothesis based on oil/gas partition coefficients. Single TOK1 channel currents were recorded from excised outside‐out patches. The single channel open probability increased as much as twofold in the presence of isoflurane and rapidly returned to the baseline values on washout. Volatile anesthetic agents did not alter the TOK1 single channel current‐voltage (I‐V) relationship, however, suggesting that the site of action does not affect the permeation pathway of the channel. Conclusion TOK1 is a potassium channel that is stimulated by volatile anesthetic agents. The concentrations over which potentiation occurred (EC50 values) were higher than those commonly used in clinical practice (approximately twice MAC).

Volatile anesthetics directly activate baseline S K+ channels in Aplysia neurons

The actions of halothane on serotonin-sensitive potassium channels (S K+ channels) were studied in sensory neurons of Aplysia. The normalized open probability of S K+ channels was increased by clinical concentrations of halothane in cell-attached and excised patches from neurons of the pleural ventrocaudal cluster. No voltage-dependence of channel activation by halothane was observed. Pre-treatment of neurons with 8-bromo-cAMP (8-Br-cAMP) or nordihydroguaiaretic acid (NDGA) had no effect on the relative level of channel activation by halothane. S K+ channels that were activated by arachidonic acid could also be activated by halothane and exhibited closely similar amplitude distributions of open channel current. Results from these experiments showed that S K+ channel activation by halothane did not depend on second messenger modulation of channel activity. We conclude that it is likely that halothane directly activates S K+ channels.

Modulation of noninactivating K+ channels in rat cerebellar granule neurons by halothane, isoflurane, and sevoflurane

Neuronal baseline K+ channels were activated by several volatile anesthetics. Whole-cell recordings from cultured cerebellar granule neurons of 7-day-old male Sprague-Dawley rats showed outward-rectifying K+ currents with a conductance of ∼1.1 ± 0.3 nS (n = 20) at positive potentials. The channel activity was noninactivating, exhibited no voltage gating, and was insensitive to conventional K+ channel blockers. Clinically relevant concentrations of halothane (112, 224, 336, and 448 &mgr;M) dissolved in Ringer’s solution increased outward currents by 29%, 50%, 63%, and 94%, respectively (n = 5;P < 0.05; analysis of variance [ANOVA]). Similar increases in currents were produced by isoflurane (274, 411, 548, and 822 &mgr;M), which increased outward currents by 22%, 47%, 52%, and 60%, respectively (n = 5;P < 0.05; ANOVA). Sevoflurane 518 &mgr;M increased outward currents by 225% (n = 10;P < 0.05; ANOVA). In all experiments, channel activity quickly returned to baseline levels during wash. The outward-rectifying whole-cell current-voltage curves were consistent with the properties of anesthetic-sensitive KCNK channels. These results support the idea that noninactivating baseline K+ channels are important target sites of volatile general anesthetics.

Baseline K+ channels as targets of general anesthetics: studies of the action of volatile anesthetics on TOK1.

A large body of evidence has accumulated in recent years pointing towards the GABA(A) receptor as a primary determinant of volatile anesthetic action (Franks and Lieb, 1994). Nevertheless, our understanding of the function of the central nervous system (CNS) remains sufficiently incomplete that other mechanisms of CNS depression remain to be examined. We have studied a new family of potassium (K+) channels which function as regulators of the baseline excitability of neuronal tissue. As such they must be considered potential targets for volatile anesthetic action and as a possible mechanism by which volatile anesthetics act to allow patients to undergo noxious surgical stimulation.

Activation of single potassium channels in rat cerebellar granule cells by volatile anesthetics.

1. We recently reported that volatile anesthetics activate a potassium channel (S channel) in neurons of the marine mollusk, Aplysia (Winegar et al., 1996. Anesthesiology 85(4) 889-900). 2. These studies were extended to investigate volatile anesthetic actions on potassium channels in rat cerebellar granule cells. 3. Noninactivating potassium channels were observed across a wide range of potentials. 4. Channel activity increased during volatile anesthetic perfusion while the i-V relations were unchanged and remained weakly inward-rectifying with a conductance at negative potentials of approximately 30 pS. 5. Frequent opening of inward rectifiers by volatile anesthetics may stabilize the resting potential near E(K) to resist depolarizing stimuli.