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Anesth Analg 2003;96:1340-1344
© 2003 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

Modulation of Noninactivating K+ Channels in Rat Cerebellar Granule Neurons by Halothane, Isoflurane, and Sevoflurane

Woo-Jong Shin, MD PhD, and Bruce D. Winegar, PhD

Department of Anesthesia and Perioperative Care, University of California, San Francisco, California

Address correspondence and reprint requests to Bruce Winegar, Pherin Pharmaceuticals, 350 N. Bernardo Ave., Mountain View, CA 94043-5207. Address e-mail to bwinegar{at}pherin.com


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
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 µM) 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 µM), which increased outward currents by 22%, 47%, 52%, and 60%, respectively (n = 5; P < 0.05; ANOVA). Sevoflurane 518 µM 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.

IMPLICATIONS: The volatile anesthetics halothane, isoflurane, and sevoflurane, reversibly enhanced a noninactivating outwardly rectifying K+ current in rat cerebellar granule neurons. These findings support a model of anesthesia that includes a site of action at baseline K+ channels.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The basic mechanism of anesthetic action by volatile anesthetics is not completely understood. The early hypothesis that the anesthetic state was dependent on nonspecific interactions of anesthetics with the membrane bilayer has largely given way to the current idea that membrane-associated proteins, particularly ion channels, are specifically modulated by volatile anesthetics (1). Many ligand- and voltage-gated ion channels are inhibited by volatile anesthetics (2–4). However, these drugs potentiate the inhibitory {gamma}-aminobutyric acid (GABA)A and glycine receptors (5–7). Inhibition of neuronal activity via membrane hyperpolarization can be achieved not only by an influx of chloride anions (Cl-) through GABAA and glycine receptor channels, but also by the opening of K+ channels to allow an efflux of K+. In fact, background K+ channels are recognized to regulate neuronal excitability. In addition to the actions of volatile anesthetics on GABAA and glycine receptors, accumulating evidence indicates that neuronal background K+ channels are positively modulated by volatile anesthetics (8–11). These findings suggest that background K+ channels may be important sites at which inhaled anesthetics act to produce the anesthetic state.

Over the last few years, a new subfamily of K+ channels (KCNK) has been identified. The members of this subfamily have structural features that suggest a dimeric arrangement, with each subunit comprising four transmembrane segments and two pore-forming regions (12). The KCNK subfamily is also distinguished by unique electrophysiological properties and can produce background K+ currents to establish the resting membrane potential, as well as modify neuronal excitability. Volatile anesthetics modulate several members of the KCNK family, including KCNK2, KCNK3, and KCNK5 (11,13).

We used cerebellar granule neurons (CGNs) to test the hypothesis that endogenous neuronal background K+ channels are positively modulated by volatile anesthetics. CGNs are a relevant choice because the KCNK3 subfamily has been identified in the cerebellum (14,15) and because CGNs are abundant in diverse types of K+ channels (16). In addition, the involvement of the cerebellum in sensorimotor coordination and maintenance of muscle tone, posture, and balance makes it a structure that is relevant to investigations of the central effects of volatile anesthetics.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The University of California-San Francisco Committee on Animal Research approved these studies. CGNs were cultured from the cerebella of 7-day-old male Sprague-Dawley rats that were anesthetized with 2% halothane and killed by decapitation. Cerebella were aseptically washed with phosphate-buffered saline (Invitrogen, Carlsbad, CA) at 4°C and minced into 500-µm or smaller sections with a No. 11 scalpel blade. Minced tissue was incubated at room temperature for 10 min in trypsin/EDTA (0.05% trypsin, 0.53 mM EDTA; Invitrogen). The trypsin/EDTA was warmed to 37°C before the incubation was started. After trypsinization, the minced tissue was resuspended in 2 mL of isolation media that contained deoxyribonuclease I (0.05% or 1000 Kunitz/mL; Sigma, St. Louis, MO). Isolation media contained minimal essential media with Earle’s salts, 2 mM glutamine, 10% heat-inactivated horse serum, 6 mg/mL of glucose, 0.5 U/mL of penicillin, 0.5 µg/mL of streptomycin, and 25 mM KCl. The suspension was kept on ice and triturated with fire-polished glass pipettes with the minimal number of strokes to obtain a single-cell suspension, by following the procedure of Slesinger and Lansman (17). CGNs were initially plated in isolation media at a density of ~1 x 105 cells per square centimeter. After a 5-h period for cell attachment, the medium was changed to a serum-free culture medium. The cultures were maintained in serum-free media for up to 2 wk, with media changes every few days. The serum-free medium was composed of neurobasal-SFM (serum-free) media (Invitrogen) supplemented with B-27 (Invitrogen), 0.1 U/mL of penicillin, 0.1 µg/mL of streptomycin, 25 mM KCl, and 2 mM glutamine. Cells were plated on poly L-lysine-coated coverslips and maintained at 37°C in a 95% air/5% CO2 humidified atmosphere.

CGNs were identified by their small soma (5–10 µm in diameter) and the presence of bipolar neurites that become apparent by approximately 24–48 h in vitro. Gigaohm seals were formed on CGNs, and they were perfused with aqueous solutions of volatile anesthetics delivered from a gas-tight perfusion system. Saturated stock solutions of halothane, isoflurane, and sevoflurane were stored in gas-tight bottles by dissolving excess amounts in Ringer’s solution. The stock solutions were diluted to the final experimental concentrations with a calibrated gas-tight syringe. The experimental solutions were stored in gas-tight containers and delivered to the cell at 20 µL/min from a Rheodyne (Rohnert Park, CA) high-performance liquid chromatography injector connected to an infusion pump (10). The perfusion rate was fast enough to allow undiluted perfusion of the cell. Perfusion with bathing solution alone had no effect on current amplitudes and other measures of channel activity. The volatile anesthetic concentrations in both the stock solutions and the experimental solutions were calibrated by gas chromatography.

Patch electrodes were pulled from borosilicate pipettes. The shanks were coated with Sylgard®, and the tips were heat-polished. The recording micropipette resistances ranged from 4 to 10 M{Omega}, and seal resistances ranged from 1 to 10 G{Omega}. The electrode filling solution contained (in mM) 150 KOH, 105 aspartic acid, 3 NaCl, 10 HEPES, 85 glucose, 1 EGTA, and 5 MgCl2 (pH 7.4). Ringer’s solution was used as the bath solution, which contained (in mM) 150 NaCl, 3 KCl, 10 HEPES, 14 glucose, 1 EGTA, and 5 MgCl2 (pH 7.4). Whole-cell currents were recorded with an Axopatch 200A amplifier, filtered at 2 kHz, digitized at a sample rate of 100 µs with an Instrutech ITC-16 analog-digital converter, and recorded to disk. The resting membrane potential was first measured in current-clamp mode and then added to the holding potential during whole-cell recordings. The holding potential was initially set to -20 mV, and currents were recorded during voltage pulses from -90 to +90 mV in 10-mV increments of 300-ms duration. Before seal formation, the voltage offset between the patch electrode and the bath solution was adjusted to produce zero current. Whole-cell conductance was determined by linear least-squares fits to the current-voltage plots at positive potentials. All experiments were performed at room temperature (~21°C–23°C).


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Background currents were easily found in whole-cell patches of CGNs. The currents were noninactivating, outward rectifying, and active across a broad range of potentials and exhibited little or no voltage-dependent gating. The basic features of these currents are shown in Fig. 1. The predominantly outward currents reached a steady state without delay after step voltage changes. These currents had a slope conductance at positive potentials of ~1.1 ± 0.3 nS (n = 20) and a reversal potential of approximately -60 mV, which is consistent with the behavior of a K+ selective leak channel. The noninactivating current appeared to be insensitive to tetraethylammonium, Ba2+, glibenclamide, and 4-aminopyridine and did not exhibit rundown over recording periods as long as half an hour (data not shown).



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  		Figure 1. A, Representative K+ currents recorded from a rat cerebellar granule neuron. The whole-cell currents were recorded during perfusion with Ringer’s solution alone (top), 224 µM halothane (middle), and washout (bottom). Voltage pulses were from -90 to 90 mV in 10-mV increments from a holding potential (Vh) of -20 mV. B, Whole-cell current-voltage relations for noninactivating currents. Whole-cell currents at positive potentials were significantly increased by clinically relevant concentrations of halothane (versus control values; n = 5; P < 0.05; ANOVA).

 
Patch-clamp experiments showed that whole-cell current amplitudes were reversibly increased when cells were perfused with halothane (Fig. 1), isoflurane (Fig. 2), or sevoflurane (Fig. 3). Clinically relevant concentrations of halothane (112, 224, 336, and 448 µM) increased the outward currents from control values by 29%, 50%, 63%, and 94%, respectively (Fig. 1; n = 5; P < 0.05; analysis of variance [ANOVA]). Recordings of isoflurane (274, 411, 548, and 822 µM) increased outward currents by 22%, 47%, 52%, and 60%, respectively (Fig. 2; n = 5; P < 0.05; ANOVA). Sevoflurane 518 µM increased outward currents by 225% (Fig. 3; n = 10; P < 0.05; ANOVA). Unlike halothane and isoflurane, sevoflurane increased inward currents as well. In all cases, channel activity was enhanced over the duration of the exposure period to volatile anesthetics and returned to the baseline level quickly upon wash.



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  		Figure 2. A, Representative K+ currents recorded from a cerebellar granular neuron during perfusion with Ringer’s solution (top), 548 µM of isoflurane (middle), and washout of isoflurane (bottom). Voltage pulses were from -90 to 90 mV in 10-mV increments from a holding potential (Vh) of -20 mV. B, Whole-cell current-voltage relations for noninactivating currents. Whole-cell currents were significantly increased by clinically relevant concentrations of isoflurane (versus control values; n = 5; P < 0.05; ANOVA)

 


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  		Figure 3. A, Representative K+ currents recorded from a cerebellar granular neuron during perfusion with Ringer’s solution (top), 518 µM sevoflurane (middle), and washout (bottom). Voltage pulses were from -90 to 90 mV in 10-mV increments from a holding potential (Vh) of -20 mV. B, Whole-cell current-voltage relations for noninactivating currents. Whole-cell currents were significantly increased by clinically relevant concentrations of sevoflurane (versus control values; n = 10; P < 0.05; ANOVA).

 
To determine whether the volatile anesthetic modulatory actions exhibited any voltage dependence, the current amplitudes measured in the presence of volatile anesthetics were normalized to the control currents and plotted as a function of voltage (Fig. 4). None of the volatile anesthetics exhibited any voltage dependence of current enhancement. The currents were proportionally increased across all measured holding potentials. Figure 4 shows that the modulatory actions of these drugs were evident at negative potentials well within the normal physiological range.



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  		Figure 4. Voltage independence of volatile anesthetic modulatory action on noninactivating K+ currents. Whole-cell current amplitudes in the presence of volatile anesthetics (IVA) were normalized to control currents (Icontrol). The normalized current amplitudes for halothane (224 µM), isoflurane (548 µM), and sevoflurane (518 µM) did not appreciably change as a function of the holding potential. Note that the modulatory action of all three volatile anesthetics persisted into hyperpolarized potentials.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We report that several volatile anesthetics positively modulate a baseline noninactivating K+ current in CGNs. Halothane, isoflurane, and sevoflurane all positively modulate CGN noninactivating K+ currents in a voltage-independent manner. Although enhancement of background currents at depolarized potentials is easily observed in Figures 1–3, the results in Figure 4 show that positive modulation occurs in generally equal proportion at negative potentials in the physiological range. The voltage-independent modulation we observed is consistent with a direct action of volatile anesthetics on the channel, as expected for uncharged small molecules. Although the exact site of action is unclear, the drugs do not appear to interfere with ion permeation through the pore. In fact, the lack of a change in the reversal potential in the presence of anesthetics (Figs. 1–3) argues that at clinical concentrations, these drugs do not affect ion selectivity.

The electrophysiological properties of the CGN noninactivating current are similar to the properties of several KCNK background channels. Background currents affect excitability in neurons by their actions to set the resting membrane potential and shape action potentials (18,19). Positive modulation of noninactivating channels by inhaled anesthetics is likely to hyperpolarize neurons and decrease neuronal excitability (10,20,21). Volatile anesthetics exert these actions in many regions of the brain, including motoneurons of the spinal cord (9,22) and cortical and hippocampal neurons (8,23,24). Several candidate channels that are likely to contribute to the noninactivating K+ currents in CGNs are KCNK2, KCNK3, and KCNK5. These channels are expressed in the mammalian central nervous system and are positively modulated by volatile anesthetics (11,13,25,26). When expressed in COS7 cells or Xenopus oocytes, volatile anesthetics activate KCNK2 and KCNK3 (11), as well as KCNK5 (13). Of particular interest is KCNK3, which has biophysical properties that parallel anesthetic-sensitive background currents (14,21). Kindler et al. (27) reported that KCNK3 was expressed in the granular layer and Purkinje layer of the rat cerebellum. Millar et al. (14) suggested that KCNK3 channels are responsible for the "standing outward" current IKSO in CGNs, partly on the basis of a shared lack of voltage gating and sensitivity to extracellular pH. However, we cannot exclude that other channels, such as KCNK5, could contribute to the noninactivating current. Without a specific channel antagonist or a knockout animal model, direct identification of the precise channel protein is difficult.

Our results, together with the above-mentioned findings, support a model of general anesthesia in which positive modulation of background channels by inhaled anesthetics contributes to the anesthetic state. The voltage-independent nature of volatile anesthetic modulation of the CGN background current suggests that neurons are affected independently of their functional state. Enhanced background currents can attenuate the action potential frequency and affect the spike wave form (18,20). Background channels are also negatively modulated by neurotransmitters such as 5-hydroxytryptamine (28). It is interesting to speculate that CGN background K+ currents could have a dual effect when modulated by volatile anesthetics to attenuate spike firings and disrupt postsynaptic responses to neurotransmitters.


   Acknowledgments
 
Supported by National Institutes of Health Grant GM57529 (BDW) and by the Korea Science and Engineering Foundation (W-JS).

The authors thank Jae H. Shim, MD, PhD; Woo J. Jeon, MD; Sang Y. Cho, MD, PhD; Jong H. Yeom, MD, PhD; Kyo S. Kim, MD, PhD; and Kyung H. Kim, MD, PhD, for their help and encouragement.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Franks NP, Lieb WR. Molecular and cellular mechanisms of general anaesthesia. Nature 1994; 367: 607–14.[Medline]
  2. Yang J, Zorumski CF. Effects of isoflurane on N-methyl-D-aspartate gated ion channels in cultured rat hippocampal neurons. Ann N Y Acad Sci 1991; 625: 287–9.[Web of Science][Medline]
  3. Zorn L, Kulkarni R, Anantharam V, et al. Halothane acts on many potassium channels, including a minimal potassium channel. Neurosci Lett 1993; 161: 81–4.[Web of Science][Medline]
  4. Study RE. Isoflurane inhibits multiple voltage-gated calcium currents in hippocampal pyramidal neurons. Anesthesiology 1994; 81: 104–16.[Web of Science][Medline]
  5. Jones MV, Brooks PA, Harrison NL. Enhancement of gamma-aminobutyric acid-activated Cl- currents in cultured rat hippocampal neurones by three volatile anaesthetics. J Physiol 1992; 449: 279–93.[Abstract/Free Full Text]
  6. Harrison NL, Kugler JL, Jones MV, et al. Positive modulation of human gamma-aminobutyric acid type A and glycine receptors by the inhalation anesthetic isoflurane. Mol Pharmacol I993; 44: 628–32.
  7. Mihic SJ, McQuilkin SJ, Eger EI II, et al. Potentiation of gamma-aminobutyric acid type A receptor-mediated chloride currents by novel halogenated compounds correlates with their abilities to induce general anesthesia. Mol Pharmacol 1994; 46: 851–7.[Abstract]
  8. Nicoll RA, Madison DV. General anesthetics hyperpolarize neurons in the vertebrate central nervous system. Science 1982; 217: 1055–7.[Abstract/Free Full Text]
  9. Takenoshita M, Takahashi T. Mechanisms of halothane action on synaptic transmission in motoneurons of the newborn rat spinal cord in vitro. Brain Res 1987; 402: 303–10.[Web of Science][Medline]
  10. Winegar BD, Owen DF, Yost CS, et al. Volatile general anesthetics produce hyperpolarization of Aplysia neurons by activation of a discrete population of baseline potassium channels. Anesthesiology 1996; 85: 889–900.[Web of Science][Medline]
  11. Patel AJ, Honore E, Lesage F, et al. Inhalational anesthetics activate two-pore-domain background K+ channels. Nat Neurosci 1999; 2: 422–6.[Web of Science][Medline]
  12. Goldstein SAN, Price LA, Losenthal DN, et al. ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 1996; 93: 13256–61.[Abstract/Free Full Text]
  13. Gray AT, Zhao BB, Kindler CH, et al. Volatile anesthetics activate the human tandem pore domain baseline K+ channel KCNK5. Anesthesiology 2000; 92: 1722–30.[Web of Science][Medline]
  14. Millar JA, Barratt L, Southan AP, et al. A functional role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons. Proc Natl Acad Sci U S A 2000; 97: 3614–8.[Abstract/Free Full Text]
  15. Leonoudakis D, Gray AT, Winegar BD, et al. An open rectifier potassium channel with two pore domains in tandem cloned from rat cerebellum. J Neurosci 1998; 18: 868–77.[Abstract/Free Full Text]
  16. Ross CA, Bredt D, Snyder SH. Messenger molecules in the cerebellum. Trends Neurosci 1990; 13: 216–22.[Web of Science][Medline]
  17. Slesinger PA, Lansman JB. Inactivation of calcium currents in granule cells cultured from mouse cerebellum. J Physiol 1991; 435: 101–21.[Abstract/Free Full Text]
  18. Siegelbaum SA, Camardo JS, Kandel ER. Serotonin and cyclic AMP close single K+ channels in Aplysia sensory neurons. Nature 1982; 299: 413–7.[Medline]
  19. Ilan N, Goldstein SAN. KCNK0: single, cloned potassium leak channels are multi-ion pores. Biophys J 2001; 80: 241–53.[Web of Science][Medline]
  20. Sirois JE, Pancrazio JJ, Lynch C III, et al. Multiple ionic mechanisms mediate inhibition of rat motoneurones by inhalation anaesthetics. J Physiol 1998; 512(Pt 3): 851–62.[Abstract/Free Full Text]
  21. Sirois JE, Qiubo L, Talley EM, et al. The TASK-1 two-pore domain K+ channel is a molecular substrate for neuronal effects of inhalation anesthetics. J Neurosci 2000; 20: 6347–54.[Abstract/Free Full Text]
  22. Collins JG, Kendig JJ, Mason P. Anesthetic actions within the spinal cord: contributions to the state of general anesthesia. Trends Neurosci 1995; 18: 549–53.[Web of Science][Medline]
  23. Southan AP, Wann KT. Inhalation anaesthetics block accommodation of pyramidal cell discharge in the rat hippocampus. Br J Anaesth 1989; 63: 581–6.[Free Full Text]
  24. Berg-Johnson J, Langmoen IA. Isoflurane hyperpolarizes neurones in rat and human cerebral cortex. Acta Physiol Scand 1987; 130: 679–85.[Web of Science][Medline]
  25. Talley EM, Lei QB, Sirois JE, et al. TASK-1, a two-pore domain K+ channel, is modulated by multiple neurotransmitters in motoneurons. Neuron 2000; 25: 399–410.[Web of Science][Medline]
  26. Talley EM, Solorzano G, Lei Q, et al. CNS distribution of members of the two-pore-domain (KCNK) potassium channel family. J Neurosci 2001; 21: 7491–505.[Abstract/Free Full Text]
  27. Kindler CH, Pietruck C, Yost CS, et al. Localization of the tandem pore domain K+ channel TASK-1 in the rat central nervous system. Brain Res Mol Brain Res 2000; 80: 99–108.[Medline]
  28. Sirois JE, Lynch C III, Bayliss DA. Convergent and reciprocal modulation of a leak K+ current and Ih by an inhalational anaesthetic and neurotransmitters in rat brainstem motoneurones. J Physiol 2002; 541: 717–29.[Abstract/Free Full Text]
Accepted for publication December 17, 2002.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2003 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press