Blockade of voltage-gated sodium (Na+) channels by local anesthetics represents the main mechanism for inhibition of impulse propagation. Local anesthetic-induced potassium (K+) channel inhibition is also known to influence transmission of sensory impulses and to potentiate inhibition. K+ channels involved in this mechanism may belong to the emerging family of background tandem pore domain K+ channels (2P K+ channels). To determine more precisely the effects of local anesthetics on members of this ion channel family, we heterologously expressed the 2P K+ channels TASK-2 (KCNK5), TASK-1 (KCNK3), and chimeric TASK-1/TASK-2 channels in oocytes of Xenopus laevis. TASK-2 cDNA-transfected HEK 293 cells were used for single-channel recordings. Local anesthetic inhibition of TASK-2 was dose-dependent, agent-specific, and stereoselective. The IC50 values for R-(+)-bupivacaine and S-(-)-bupivacaine were 17 and 43 micro M and for R-(+)-ropivacaine and S-(-)-ropivacaine, 85 and 236 micro M. Lidocaine (1 mM) inhibited TASK-2 currents by 55 +/- 4%, whereas its quaternary positively charged analog N-ethyl lidocaine (QX314) had no effect. Bupivacaine (100 micro M) decreased channel open probability from 20.8 +/- 1.6% to 5.6 +/- 2.2%. Local anesthetics [300 micro M R-(+)-bupivacaine] caused significantly greater depolarization of the resting membrane potential of TASK-2-expressing oocytes compared with water-injected control oocytes (15.8 +/- 2.5 mV versus 0.1 +/- 0.05 mV; p < 0.001). Chimeric TASK-1/TASK-2 2P K+ channel subunits that retained pH sensitivity demonstrated that the carboxy domain of TASK-2 mediates the greater local anesthetic sensitivity of TASK-2. These results show that clinically achievable concentrations of local anesthetics inhibit background K+ channel function and may thereby enhance conduction blockade.

Several reports suggest that clinically used concentrations of inhaled anesthetics can increase conductance through noninactivating potassium channels and that the resulting hyperpolarization might decrease excitability, thereby leading to the anesthetic state. We speculated that animals deficient in such potassium channels might be resistant to the effects of anesthetics. Thus, in the present study, we measured the minimum alveolar anesthetic concentration (MAC) needed to prevent movement in response to a noxious stimulus in 50% of adult mice lacking functional KCNK5 potassium channel subunits and compared these results with those for heterozygous and wild-type mice. We also measured MAC in weaver mice that had a mutation in the potassium channel Kir3.2 and compared the resulting values with those for wild-type mice. MAC values for desflurane, halothane, and isoflurane for KCNK5-deficient mice and isoflurane MAC values for weaver mice did not differ from MAC values found in control mice. Our results do not support the notion that these potassium channels mediate the capacity of inhaled anesthetics to produce immobility. In addition, we found that the weaver mice did not differ from control mice in their susceptibility to convulsions from the nonimmobilizers flurothyl [di-(2,2,2,-trifluoroethyl)ether] or 2N (1,2-dichlorohexafluorocyclobutane).

UNLABELLED: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 approximately 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 micro 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 micro M), which increased outward currents by 22%, 47%, 52%, and 60%, respectively (n = 5; P < 0.05; ANOVA). Sevoflurane 518 micro 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. 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.

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.

Tandem pore domain K + channels represent a new family of ion channels involved in the control of background membrane conductances. We report the structural and functional properties of a TWIK-related acid-sensitive K + channel (rTASK), a new member of this family cloned from rat cerebellum. The salient features of the primary amino acid sequence include four putative transmembrane domains and, unlike other cloned tandem pore domain channels, a PDZ (postsynaptic density protein, disk-large, zo-1) binding sequence at the C terminal. rTASK has distant overall homology to a putative Caenorhabditis elegans K + channel and to the mammalian clones TREK-1 and TWIK-1. rTASK expression is most abundant in rat heart, lung, and brain. When exogenously expressed in Xenopus oocytes, rTASK currents activate instantaneously, are noninactivating, and are not gated by voltage. Because rTASK currents satisfy the Goldman–Hodgkin–Katz current equation for an open channel, rTASK can be classified an open rectifier. Activation of protein kinase A produces inhibition of rTASK, whereas activation of protein kinase C has no effect. rTASK currents were inhibited by extracellular acidity. rTASK currents also were inhibited by Zn 2+ (IC 50 = 175 μ m ), the local anesthetic bupivacaine (IC 50 = 68 μ m ), and the anti-convulsant phenytoin (∼50% inhibition at 200 μ m ). By demonstrating open rectification and open probability independent of voltage, we have established that rTASK is a baseline potassium channel.

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.

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 ∼30 pS. 5. Frequent opening of inward rectifiers by volatile anesthetics may stabilize the resting potential near EK to resist depolarizing stimuli.

A large body of evidence has accumulated in recent years pointing towards the GABAA 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.

Background Volatile anesthetic agents can activate the S channel, a baseline potassium (K+) 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+ 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 Ringer's perfusate. The concentrations at which 50% potentiation occurred (EC50 values) were in the range of 768-814 microM (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 nonanesthetic 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).

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 microM (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.

The activity of single L-type Ca2+ channels was recorded from cell-attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were concerned with the mechanism by which aminoglycoside antibiotics inhibit ion flow through the channel. Aminoglycosides produced discrete fluctuations in the single-channel current when added to the external solution. The blocking kinetics could be described as a simple bimolecular reaction between an aminoglycoside molecule and the open channel. The blocking rate was found to be increased when either the membrane potential was made more negative or the concentration of external permeant ion was reduced. Both of these effects are consistent with a blocking site that is located within the channel pore. Other features of block, however, were incompatible with a simple pore blocking mechanism. Hyperpolarization enhanced the rate of unblocking, even though an aminoglycoside molecule must dissociate from its binding site in the channel toward the external solution against the membrane field. Raising the external permeant ion concentration also enhanced the rate of unblocking. This latter finding suggests that aminglycoside affinity is modified by repulsive interactions that arise when the pore is simultaneously occupied by a permeant ion and an aminoglycoside molecule.

The activity of single mechanosensitive channels was recorded from cell-attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were designed to investigate the mechanism of channel block produced by externally applied aminoglycoside antibiotics. Neomycin and other aminoglycosides reduced the amplitude of the single-channel current at negative membrane potentials. The block was concentration-dependent, with a half-maximal concentration of approximately 200 microM. At high drug concentrations, however, block was incomplete with roughly one third of the current remaining unblocked. Neomycin also caused the channel to fluctuate between the open state and a subconductance level that was also roughly one third the amplitude of the fully open level. An analysis of the kinetics of the subconductance fluctuations was consistent with a bimolecular reaction between an aminoglycoside molecule and the open channel (kon = approximately 1 x 10(6) M-1s-1 and koff = approximately 400 s-1 at -60 mV). Increasing the external pH reduced both the rapid block of the open channel and the frequency of the subconductance fluctuations, as if both blocking actions were produced by a single active drug species with a pKa = approximately 7.5. The results are interpreted in terms of a mechanism in which an aminoglycoside molecule partially occludes ion flow through the channel pore.

The blocking actions of Fe2+, Co2+, and Ni2+ on unitary currents carried by Ba2+ through single dihydropyridine-sensitive Ca2+ channels were recorded from cell-attached patches on myotubes from the mouse C2 cell line. Adding millimolar concentrations of blocker to patch electrodes containing 110 mM BaCl2 produced discrete excursions to the closed channel level. The kinetics of blocking and unblocking were well described with a simple model of open channel block. Hyperpolarization speeded the exit of all of the blockers from the channel, as expected if the blocking site resides within the pore. The block by Ni2+ differs from that produced by Fe2+ and Co2+ because Ni2+ enters the channel approximately 20 times more slowly and exits approximately 50 times more slowly. Ni2+ also differs from the other transition metals because at millimolar concentrations it reduces the amplitude of the unitary current in a concentration-dependent manner. The results are consistent with the idea that the rate-limiting step for ion entry into the channel is water loss at its inner coordination sphere; unblocking, on the other hand, cannot be explained in terms of simple coulombic interactions arising from differences in ion size.

Currents flowing through single stretch-inactivated ion channels were recorded from cell-attached patches on myotubes from mdx mice, Adding micromolar concentrations of gadolinium to patch electrodes containing normal saline produced rapid transitions in the single-channel current between the fully open and closed states. The kinetics of the current fluctuations followed the predictions of a simple model of open channel block in which the transitions in the current arise from the entry and exit of Gd from the channel pore: histograms of the open and closed times were well fit with single exponentials, the blocking rate depended linearly on the concentration of gadolinium in the patch electrode, and the unblocking rate was independent of the concentration of gadolinium. Hyperpolarizing the patch increased the rate of unblocking (approximately e-fold per 85 mV), suggesting the charged blocking particle can exit the channel into the cell under the influence of the applied membrane field. The rate of blocking was rapid and was independent of the patch potential, consistent with the rate of ion entry into the pore being determined by its rate of diffusion in solution. When channel open probability was reduced by applying suction to the electrode, the blocking kinetics were independent of the extent of inactivation, suggesting that mechanosensitive gating does not modify the structure of the channel pore.

1. The blocking actions of Zn2+ on currents carried by Ba2+ through single dihydropyridine‐sensitive Ca2+ channels were recorded from cell‐attached patches on myotubes from the mouse C2 cell line. 2. Adding 100 microM‐Zn2+ to the patch electrode containing 110 mM‐BaCl2 produced an increase in the open channel noise, presumably arising from unresolved blocking and unblocking of the open channel by Zn2+. Adding between 200 and 1000 microM‐Zn2+ to the electrode reduced the amplitude of the unitary current in a concentration‐dependent manner. 3. The single‐channel current‐voltage (i‐V) relations showed that Zn2+ reduced the amplitude of the unitary Ba2+ currents at all potentials more negative than 0 mV. A plot of the amplitude of the unitary current in the presence of Zn2+, normalized to the amplitude in its absence, showed that block of the current depended on voltage, decreasing as the patch potential was made more negative. 4. The normalized amplitudes of the unitary currents were plotted as a function of the logarithm of [Zn2+] in the electrode. The relation for currents recorded at different potentials were fitted to an expression for binding to a single site with a KD at 0 mV of approximately 500 microM. The KD changed approximately e‐fold per 83 mV with hyperpolarization. The results suggest Zn2+ binds to a site located at approximately 15% of the potential drop from the surface membrane. 5. Reducing the concentration of Ba2+ in the patch electrode enhanced the steady‐state block of unitary currents by Zn2+. The inverse of the unitary current was plotted as a function of [Ba2+]o in the presence and absence of Zn2+; both were linear and intersected at the ordinate, indicating Ba2+ and Zn2+ compete for a channel site. 6. The kinetics of Zn2+ block of unitary Ba2+ currents were studied by amplitude distribution analysis. As expected for a simple reaction between blocking ion and open channel, the blocking rate depended linearly on the concentration of Zn2+, while the exit rate was independent of concentration. The second‐order rate coefficient for Zn2+ entry in the presence of 110 mM‐BaCl2 at 0 mV was approximately 2.0 X 10(7) M‐1S‐1, while the exit rate was approximately 16000 s‐1. 7. Both entry and exit rates increased as the membrane potential was made more negative. The entry rate increased approximately e‐fold per 66 mV, while the exit rate increased approximately e‐fold per 41 mV.(ABSTRACT TRUNCATED AT 400 WORDS)