17802693
The TASK-1 Two-Pore Domain K+ Channel Is a Molecular Substrate for Neuronal Effects of Inhalation Anesthetics
Despite widespread use of volatile general anesthetics for well over a century, the mechanisms by which they alter specific CNS functions remain unclear. Here, we present evidence implicating the two-pore domain, pH-sensitive TASK-1 channel as a target for specific, clinically important anesthetic effects in mammalian neurons. In rat somatic motoneurons and locus coeruleus cells, two populations of neurons that express TASK-1 mRNA, inhalation anesthetics activated a neuronal K+ conductance, causing membrane hyperpolarization and suppressing action potential discharge. These membrane effects occurred at clinically relevant anesthetic levels, with precisely the steep concentration dependence expected for anesthetic effects of these compounds. The native neuronal K+ current displayed voltage- and time-dependent properties that were identical to those mediated by the open-rectifier TASK-1 channel. Moreover, the neuronal K+ channel and heterologously expressed TASK-1 were similarly modulated by extracellular pH. The decreased cellular excitability associated with TASK-1 activation in these cell groups probably accounts for specific CNS effects of anesthetics: in motoneurons, it likely contributes to anesthetic-induced immobilization, whereas in the locus coeruleus, it may support analgesic and hypnotic actions attributed to inhibition of those neurons.
14988452
Effects of general anaesthetics on ligand-gated ion channels.
The site at which anaesthetics act within the central nervous system (CNS) has been the subject of research for almost 100 years. Most success has been obtained in defining the physical nature of the site [70]. For example, the well established correlation of anaesthetic potency to fat solubility indicates that the site of action is hydrophobic. Research using anaesthetics with unusual solubility properties (sulphur hexafluoride and carbon tetrafluoride) failed to provide support for the alternative hypothesis that interaction within the aqueous phase of the CNS was responsible for anaesthesia [45,46]. It is now generally agreed that interaction at a hydrophobic site is involved, but debate continues as to whether or not this is within the membrane lipids or at a hydrophobic region within specific proteins. Evidence for the latter view has been provided by the fact that bacterial and firefly luciferases are sensitive to anaesthetics with potency ratios comparable to those of mammalian anaesthesia [21, 22, 32, 42, 43]. However, there is no evidence for a substance related to the luciferases within the mammalian CNS and for the protein model of general anaesthesia to be advanced such a target site must be identified. General anaesthetics may affect signal transmission by altering action potential propagation or synaptic transmission or, indeed, both. At clinically relevant concentrations, general anaesthetics appear primarily to affect synaptic processes [5, 53, 58-61], although impulse conduction in small unmyelinated fibres is reduced [4]. There is some evidence that anaesthetics may inhibit neurotransmitter release also [56], which if it were to occur at an excitatory synapse could produce the necessary depression of synaptic transmission. Anaesthetics also have effects on the postsynaptic neurotransmitter receptor/ion channel complex. Much of the early work concentrated on the nicotinic acetylcholine receptor as a model for ligand-gated ion channels in general. However, attention is now being devoted to the receptors activated by excitatory amino acids, which probably form the majority of the fast excitatory synapses in the CNS, and to the principal inhibitory receptors, activated by yaminobutyric acid (GABA) and glycine.
4393717
Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors
Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as γ-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABAA and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA ρ1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABAA and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.
36417260
General anesthetics hyperpolarize neurons in the vertebrate central nervous system.
The effect of general anesthetics on frog motoneurons and rat hippocampus pyramidal cells was examined with sucrose gap and intracellular recording, respectively. A number of volatile and intravenous anesthetics directly hyperpolarized the motoneurons. The potency of these agents in hyperpolarizing motoneurons was strongly correlated with their anesthetic potency. While the responses to barbiturates and alpha-chloralose were blocked by gamma-aminobutyric acid antagonists and were dependent on the chloride gradient, the responses to all the other anesthetics tested were generated by a separate mechanism. Intracellular recording from hippocampal pyramidal cells suggested that an increase in potassium conductance accounts for these responses. Such a nonsynaptic action would contribute to the decreased neuronal responsiveness observed for these compounds and thus to their anesthetic action.
34418327
Mechanisms of halothane action on synaptic transmission in motoneurons of the newborn rat spinal cord in vitro
Action of halothane on synaptic transmission was studied on the isolated newborn rat spinal cord. Clinical doses of halothane (less than or equal to 3%) suppressed mono- and polysynaptic reflexes, dorsal root reflexes, excitatory and inhibitory postsynaptic potentials as well as the spontaneous synaptic potentials caused by impulse bombardment. However, the spontaneous miniature inhibitory postsynaptic potentials observed after blocking impulse activities by tetrodotoxin were not all suppressed by halothane. During halothane administration, the membrane potential of motoneurons was hyperpolarized by several millivolts, associated with an increase in input conductance. However, the threshold potential level for spike generation was virtually unaffected. Depression of synaptic transmission in spinal motoneurons by halothane is suggested to be due to two factors: a reduction in the amount of transmitter release secondary to interference with Ca2+ entry into nerve terminals, either by partial blockade of impulse invasion or voltage-dependent Ca2+ channels; and an increase in the depolarizing current necessary for excitation of motoneurons owing to hyperpolarization and decreased input resistance.
4340661
Volatile general anaesthetics activate a novel neuronal K+ current
Although it is still controversial whether the primary target sites underlying general anaesthesia are proteins1,2 or lipids3–5, it is generally thought that the ultimate targets are ion channels in nerve membranes. One approach to finding these targets is to study the effects of general anaesthetics on identified neurons, where differential effects on neuronal activity can be pursued to the molecular level. Here we report that amongst a group of apparently identical molluscan neurons having endogenous firing activity, a single cell displays an unusual sensitivity to volatile agents (which, at surgical levels, completely inhibit its activity). We further show that this sensitivity is due to a novel anaesthetic-activated K+ current, which is found in the sensitive cell but not in the surrounding insensitive cells. This K+ conductance is not appreciably voltage-gated and persists for as long as the anaesthetic is present. The response to anaesthetics is completely reversible and saturates at low anaesthetic partial pressures: the half-maximal response for halothane occurs at 0.0063 atm, close to its minimum alveolar concentration (0.0075 atm) in man6.
36837987
Halothane-induced hyperpolarization and depression of postsynaptic potentials of guinea pig thalamic neurons in vitro
Intralaminar thalamic nuclei have been considered to be a component of the non-specific sensory system which is involved in physiological functions related to consciousness and pain sensation. The effect of halothane on membrane potentials and synaptic properties of neurons of the parafascicular (Pf) nucleus in guinea pig brain slices was investigated using intracellular recording methods. Halothane at concentrations of 0.4-1.0 mM, which are in the range of clinical concentrations, produced hyperpolarizations of 2-8 mV in approximately 50% of the cells. The halothane-induced hyperpolarization was nullified at a membrane potential close to the K+ equilibrium potential. The amplitude of the hyperpolarization was dependent on the external K+ concentration, and was decreased by either Ba2+, or 4-aminopyridine, or intracellular injection of Cs+. All these results indicate that the hyperpolarization was due to an increase in K+ conductance. Halothane at clinical concentrations depressed both excitatory and inhibitory postsynaptic potentials in a concentration-dependent manner. On the other hand hyperpolarizing responses to exogenous gamma-aminobutyric acid (GABA) in the presence of bicuculline were suppressed by halothane, but depolarizing responses to L-glutamate were not altered. The results indicate that the depressant action of the anesthetic on the excitatory postsynaptic potential (EPSP) may occur presynaptically, whereas the blocking action on the inhibitory postsynaptic potential (IPSP) may occur postsynaptically.
36297866
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.
24789950
Multiple ionic mechanisms mediate inhibition of rat motoneurones by inhalation anaesthetics
1 We studied the effects of inhalation anaesthetics on the membrane properties of hypoglossal motoneurones in a neonatal rat brainstem slice preparation. 2 In current clamp, halothane caused a membrane hyperpolarization that was invariably associated with decreased input resistance; in voltage clamp, halothane induced an outward current and increased input conductance. Qualitatively similar results were obtained with isoflurane and sevoflurane. 3 The halothane current reversed near the predicted K+ equilibrium potential (EK) and was reduced in elevated extracellular K+ and in the presence of Ba2+ (2 mm). Moreover, the Ba2+‐sensitive component of halothane current was linear and reversed near EK. The halothane current was not sensitive to glibenclamide or thyrotropin‐releasing hormone (TRH). Therefore, the halothane current was mediated, in part, by activation of a Ba2+‐sensitive K+ current distinct from the ATP‐ and neurotransmitter‐sensitive K+ currents in hypoglossal motoneurones. 4 Halothane also inhibited Ih, a hyperpolarization‐activated cationic current; this was primarily due to a decrease in the absolute amount of current, although halothane also caused a small, but statistically significant, shift in the voltage dependence of Ih activation. Extracellular Cs+ (3 mm) blocked Ih and a component of halothane‐sensitive current with properties reminiscent of Ih. 5 A small component of halothane current, resistant to Ba2+ and Cs+, was observed in TTX‐containing solutions at potentials depolarized to ∼−70 mV. Partial Na+ substitution by N‐methyl‐D‐glucamine completely abolished this residual current, indicating that halothane also inhibited a TTX‐resistant Na+ current active near rest potentials. 6 Thus, halothane activates a Ba2+‐sensitive, relatively voltage‐independent K+ current and inhibits both Ih and a TTX‐insensitive persistent Na+ current in hypoglossal motoneurones. These effects of halothane decrease motoneuronal excitability and may contribute to the immobilization that accompanies inhalation anaesthesia.
10104014
Ionic mechanism of isoflurane's actions on thalamocortical neurons.
We studied the actions of isoflurane (IFL) applied in aqueous solutions on ventrobasal neurons from thalamic brain slices of juvenile rats. By using the whole cell, patch-clamp method with current- and voltage-clamp recording techniques, we found that IFL increased a noninactivating membrane conductance in a concentration-dependent reversible manner. In an eightfold concentration range that extended into equivalent in vivo lethal concentrations, IFL did not produce a maximal effect on the conductance; this is consistent with a nonreceptor-mediated mechanism of action. TTX eliminated action potential activity but did not alter IFL effects. The effects on the membrane potential and current induced by IFL were voltage independent but depended on the external [K+], reversing near the equilibrium potential for K+. External Ba2+ or internal Cs+ applications, which block K+ channels, suppressed the conductance increase caused by IFL. External applications of the Ca2+ channel blockers Co2+ or Cd2+ or internal application of the Ca2+ chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N, N',N'-tetraacetic acid did not prevent the effects of IFL, implying little involvement of Ca2+-dependent K+ currents. A contribution of inwardly rectifying K+ channels to the increased steady-state conductance seemed unlikely because IFL decreased inward rectification. An involvement of ATP-mediated K+ channels also was unlikely because application of the ATP-mediated K+ channel blocker glibenclamide (1-80 microM) did not prevent IFL's actions. In contrast to spiking cells, IFL depolarized presumed glial cells, consistent with an efflux of K+ from thalamocortical neurons. The results imply that a leak K+ channel mediated the IFL-induced increase in postsynaptic membrane conductance in thalamic relay neurons. Thus a single nonreceptor-mediated mechanism of IFL action was responsible for the hyperpolarization and conductance shunt of voltage-dependent Na+ and Ca2+ spikes, as reported in the preceding paper. Although anesthetics influence various neurological systems, an enhanced K+ leak generalized in thalamocortical neurons alone could account for anesthesia in vivo.
2337187
Sequence and function of the two P domain potassium channels: implications of an emerging superfamily
Abstract A new superfamily of K+ channels has emerged in the past 2 years. Notable for possessing two pore-forming P domains in each subunit, members of the superfamily have been recognized through phylogeny from micro-organisms to humans. Four subfamilies of two P domain channels have been isolated thus far; among these are the first cloned examples of outward rectifier and open rectifier (or leak) K+ channels. The two P domain K+ channels offer a new perspective from which to glimpse the molecular basis for function and dysfunction of K+-selective ion channels.
14688154
TASK, a human background K+ channel to sense external pH variations near physiological pH
TASK is a new member of the recently recognized TWIK K+ channel family. This 395 amino acid polypeptide has four transmembrane segments and two P domains. In adult human, TASK transcripts are found in pancreas
32769600
TBAK-1 and TASK-1, two-pore K(+) channel subunits: kinetic properties and expression in rat heart.
A mammalian K(+) channel subunit (TBAK-1/TASK-1) containing two pore domains and four transmembrane segments and whose mRNA is highly expressed in the heart has been cloned recently. TBAK-1 and TASK-1 are identical except for the additional nine amino acids in the NH(2) terminus of TBAK-1. We examined their kinetic properties, pH sensitivity, and regional cardiac mRNA expression and determined whether a native cardiac K(+) channel with similar kinetic properties was present. When TBAK-1 or TASK-1 was transiently expressed in COS-7 cells, time- and voltage-independent whole cell currents were observed. Single-channel conductances of TBAK-1 and TASK-1 were 14.6 +/- 1.0 and 13.8 +/- 2.8 pS, respectively, at -80 mV in 140 mM extracellular K(+), and the mean open times were 0.8 +/- 0.1 and 0.6 +/- 0.1 ms, respectively. Both TBAK-1 and TASK-1 were highly sensitive to extracellular pH such that a decrease from 7.2 to 6.4 reduced their open probability (P(o)) by 81 +/- 14% and 80 +/- 16%, whereas a decrease in intracellular pH from 7.2 to 6.4 reduced the P(o) by 42 +/- 10% and 47 +/- 12%, respectively. TBAK-1/TASK-1 mRNA was expressed in all regions of the rat heart, with the highest level of expression in the right atrium. A 14-pS K(+) channel with kinetic properties similar to those of TBAK-1/TASK-1 was identified in rat atrial and ventricular cells. These results indicate that TBAK-1/TASK-1 represents a functional native K(+) channel in the rat heart.
17998103
An Open Rectifier Potassium Channel with Two Pore Domains in Tandem Cloned from Rat Cerebellum
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 elegansK+ 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 Zn2+(IC50 = 175 μm), the local anesthetic bupivacaine (IC50 = 68 μm), and the anti-convulsant phenytoin (∼50% inhibition at 200 μm). By demonstrating open rectification and open probability independent of voltage, we have established that rTASK is a baseline potassium channel.
26224816
Proton Block and Voltage Gating Are Potassium-dependent in the Cardiac Leak Channel Kcnk3*
Potassium leak conductances were recently revealed to exist as independent molecular entities. Here, the genomic structure, cardiac localization, and biophysical properties of a murine example are considered. Kcnk3 subunits have two pore-forming P domains and unique functional attributes. At steady state, Kcnk3 channels behave like open, potassium-selective, transmembrane holes that are inhibited by physiological levels of proton. With voltage steps, Kcnk3 channels open and close in two phases, one appears to be immediate and one is time-dependent (τ = ∼5 ms). Both proton block and gating are potassium-sensitive; this produces an anomalous increase in outward flux as external potassium levels rise because of decreased proton block. Single Kcnk3 channels open across the physiological voltage range; hence they are “leak” conductances; however, they open only briefly and rarely even after exposure to agents that activate other potassium channels.
16687514
TASK-1, a Two–Pore Domain K+ Channel, Is Modulated by Multiple Neurotransmitters in Motoneurons
Inhibition of "leak" potassium (K+) channels is a widespread CNS mechanism by which transmitters induce slow excitation. We show that TASK-1, a two pore domain K+ channel, provides a prominent leak K+ current and target for neurotransmitter modulation in hypoglossal motoneurons (HMs). TASK-1 mRNA is present at high levels in motoneurons, including HMs, which express a K+ current with pH- and voltage-dependent properties virtually identical to those of the cloned channel. This pH-sensitive K+ channel was fully inhibited by serotonin, norepinephrine, substance P, thyrotropin-releasing hormone, and 3,5-dihydroxyphenylglycine, a group I metabotropic glutamate receptor agonist. The neurotransmitter effect was entirely reconstituted in HEK 293 cells coexpressing TASK-1 and the TRH-R1 receptor. Given its expression patterns and the widespread prevalence of this neuromodulatory mechanism, TASK-1 also likely supports this action in other CNS neurons.
43402949
The locus coeruleus: neurobiology of a central noradrenergic nucleus
2. Anatomy of the nucleus locus coeruleus 2.
19789144
Double- and triple-labeling of functionally characterized central neurons projecting to peripheral targets studied in vitro
The use of in vitro preparations such as brain slices poses difficulties in determining the correct identity of cells under study. To circumvent this problem, we first used a fluorescence pre-labeling technique (rhodamine-dextran-lysine) to identify cranial motoneurons projecting to the tongue (hypoglossal motoneurons) in the guinea-pig. Following preparation of slices, cells were recorded intracellularly and their electrophysiological properties determined. The cells were then intracellularly stained with both a fluorescence label (Lucifer Yellow) and with the stable, non-fading label biocytin. Under fluorescent illumination, the great majority of recorded cells within the hypoglossal nucleus were double-labeled (rhodamine and Lucifer Yellow) suggesting that most are indeed motoneurons. Biocytin injected into the same motoneurons provided permanent and detailed images of their morphology. Intracellularly stained cells surrounding the hypoglossal nucleus were not labeled with rhodamine and had distinct electro-physiological properties. The use of the retrogradely transported marker rhodamine-dextran-lysine allows the unambiguous identification of motoneurons in a brainstem slice. The combined intracellular injection of Lucifer Yellow and biocytin provides a simple means of melding the advantages of a fluorescent label (compatible with other fluorescence labels and with immunocytochemistry) with the benefits of a stable, non-fading, electron-dense marker. Application of this technique should prove useful in establishing morphological and functional correlates in other areas of the CNS.
20133469
Interstitial PCO2 and pH in rat hippocampal slices measured by means of a novel fast CO2/H+-sensitive microelectrode based on a PVC-gelled membrane
We describe here the construction and properties of a double-barrelled microelectrode (tip diameter 4–10 μm) which permits simultaneous measurements of PCO2 and pH, and which has a 90% response time of only one or a few seconds for a step change in PCO2. The fast response of the CO2-sensitive barrel is due to (i) the use of a PVC-gelled (tridodecylamine-containing) membrane solution which enables the construction of extremely short (≥ 4 μm), yet mechanically stable, membrane columns, and (ii) the presence of carbonic anhydrase in the filling solution. Recordings made in the pyramidal layer of area CA1 in rat hippocampal slices showed that the deviation in the acid direction of the basal interstitial pH (pH0) from that of the perfusion solution was attributable to a higher PCO2 level within the tissue. Most of the late acid shift evoked by stimulation of the Schaffer collaterals (5- to 20-s trains at 10 Hz) could also be explained on the basis of an accumulation of interstitial CO2 at a constant HCO3−concentration. This conclusion was supported by the finding that inhibition of extracellular carbonic anhydrase activity by 10 μM benzolamide completely abolished the activity-induced fall in pH0, but not the increase in PCO2. The initial stimulus-induced alkalosis was accompanied by a slight decrease in PCO2 only, implying a parallel increase in the interstitial HCO3−concentration. Benzolamide produced a dramatic enhancement of the early alkaline shift as well as of the simultaneous fall in PCO2. The latter effect of the drug unmasks a cellular CO2 sink that is induced by neuronal activity.
3989602
Determination of extracellular bicarbonate and carbon dioxide concentrations in brain slices using carbonate and pH-selective microelectrodes
The extracellular pH of the brain is subject to shifts during neural activity. To understand these pH changes, it is necessary to measure [H+], [HCO3-], [CO3(2-)] and [CO2]. In principle, this can be accomplished using CO3(2-) and pH-sensitive microelectrodes; however, interference from HCO3- and Cl-, and physiological changes in [HCO3-], complicate measurements with CO3(2-) electrodes. Calibration requires knowledge of slope response, interference constants and corrections for [HCO3-] shifts. We show that when [HCO3-] is altered at constant [CO2] in the absence of Cl-, the HCO3- interference cancels and the Nikolsky equation reduces to the Nernst equation for CO3(2-). Measurement of CO3(2-) slope response by this method yielded a value of 28.5 +/- 0.72 mV per decade change in [CO3(2-)]. In Cl(-)-containing solutions, interference coefficient for HCO3- and Cl- were determined by altering [HCO3-] at constant [CO2], changing [CO2] at constant [HCO3-], then solving the simultaneous Nikolsky equations for each transition. The mean interference constants corresponded to selectivity ratios of 245:1 and 1150:1 for CO3(2-) over HCO3- and Cl- respectively. To correct for possible changes in [HCO3-], the equilibrium relation between CO3(2-) and HCO3- was substituted into the Nikolsky equation to yield an equation in [CO3(2-)] and [H+]. By simultaneously measuring shifts in [H+] with a pH microelectrode, this equation is readily solved for [CO3(2-)]. These methods were tested by measuring [HCO3-] and [CO2] in experimental solutions, and in the extracellular fluid of rat hippocampal slices.
25547214
Modulation of the hyperpolarization‐activated cation current of rat thalamic relay neurones by intracellular pH
1 Properties of the hyperpolarization‐activated cation current (Ih) were investigated in thalamocortical neurones of an in vitro slice preparation of the rat ventrobasal thalamic complex (VB) before and during changes of pipette pH (pHp), intracellular pH (pHi) and bath pH (pHb) using the whole‐cell patch‐clamp technique and fluorescence ratio imaging of the pH indicator 2′,7′‐bis(carboxyethyl)‐5(and ‐6)‐carboxyfluorescein (BCECF). 2 Recording of Ih with predefined pHp revealed significant shifts in the voltage dependence of Ih activation (V½) of 4‐5 mV to more positive values for a pHp of 7.5 and 2‐3 mV to more negative values for a pHp of 6.7 as compared to control values (pHp= 7.1). 3 Application of the weak acid lactate (20 mM), which produced a slow monophasic intracellular acidification, induced a reversible negative shift of V½ of up to 3 mV. Application of 20 mM TMA, which caused a distinct intracellular alkalinization, shifted V½ to 4‐5 mV more positive values. 4 In slices bathed in Hepes‐buffered saline, no significant pHo dependence of Ih was observed. Changing pHo by altering the extracellular [HCO3−] in the presence of constant pCO2 also revealed no significant pHo dependence of Ih. 5 Rhythmic stimulation of thalamocortical neurones with repetitive depolarizing pulse trains caused an intracellular acidification, which reversibly decreased the amplitude and time course of activation of Ih. 6 The results of the present study indicate that shifts in pHi result in a significant modulation of the gating properties of Ih channels in TC neurones. Through this mechanism activity‐dependent shifts in pHi may contribute to the up‐ and downregulation of Ih.
24566453
Myocardial Depressant Effects of Sevoflurane: Mechanical and Electrophysiologic Actions In Vitro
Background The effects of anesthetic concentrations of sevoflurane were studied in isolated myocardial tissue to delineate the mechanisms by which cardiac function is altered. Methods Isometric force of isolated guinea pig ventricular papillary muscle was studied at 37 degrees C in normal and 26 mM Potassium sup + Tyrode's solution at various stimulation rates. Normal and slow action potentials were evaluated using conventional microelectrodes. Effects of sevoflurane on sarcoplasmic reticulum function in situ were also evaluated by its effect on rapid cooling contractures, which are known to activate Calcium2+ release from the sarcoplasmic reticulum, and on contractions of rat papillary muscle. Finally, Calcium2+ and Potassium sup + currents of isolated guinea pig ventricular myocytes were examined using the whole-cell patch clamp technique. Results Sevoflurane equivalent to 1.4% and 2.8% depressed guinea pig myocardial contractions to approximately 85 and approximately 65% of control, respectively, although the maximum rate of force development at 2 or 3 Hz and force in rat myocardium after rest showed less depression. In the partially depolarized, beta-adrenergically stimulated myocardium, sevoflurane selectively depressed late peak force without changing early peak force, whereas it virtually abolished rapid cooling contractures. Sevoflurane did not alter the peak amplitude or maximum depolarization rate of normal and slow action potentials, but action potential duration was significantly prolonged. In isolated guinea pig myocytes at room temperature, 0.7 mM sevoflurane (equivalent to 3.4%) depressed peak Calcium2+ current by approximately 25% and increased the apparent rate of inactivation. The delayed outward Potassium sup + current was markedly depressed, but the inwardly rectifying Potassium sup + current was only slightly affected by 0.35 mM sevoflurane. Conclusions These results suggest that the direct myocardial depressant effects of sevoflurane are similar to those previously described for isoflurane. The rapid initial release of Calcium2+ from the sarcoplasmic reticulum is not markedly decreased, although certain release pathways, specifically those induced by rapid cooling, appear to be depressed. Contractile depression may be partly related to the depression of Calcium2+ influx through the cardiac membrane. The major electrophysiologic effect of sevoflurane seems to be a depression of the delayed outward Potassium sup + current, which appears to underlie the increased action potential duration.
96149557
Ionic channels of excitable membranes
PART I Classical biophysics of the squid giant axon The superfamily of voltage-gated channels Voltage-gated calcium channels Potassium channels and chloride channels Ligand-gated channels of fast chemical synapses Modulation, slow synaptic action, and second messengers Sensory transduction and excitable cells Calcium dynamics, epithelial transport, and intercellular coupling PART II Elementary properties of ions in solution Elementary properties of pores Counting channels Structure of channel proteins Selective permeability: Independence Selective permeability: Saturation and binding Classical mechanisms of block Structure-function studies of permeation and block Gating mechanisms: Kinetic thinking Gating: Voltage sensing and inactivation Modification of gating in voltage-sensitive channels Cell biology and channels Evolution and origins
45487917
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.
39462331
TASK-3, a New Member of the Tandem Pore K+ Channel Family*
We have isolated from the rat cerebellum cDNA library a complementary DNA encoding a new member of the tandem pore K+ channel family. Its amino acid sequence shares 54% identity with that of TASK-1, but less than 30% with those of TASK-2 and other tandem pore K+ channels (TWIK, TREK, TRAAK). Therefore, the new clone was named TASK-3. Reverse transcriptase-polymerase chain reaction analysis showed that TASK-3 mRNA is expressed in many rat tissues including brain, kidney, liver, lung, colon, stomach, spleen, testis, and skeletal muscle, and at very low levels in the heart and small intestine. When expressed in COS-7 cells, TASK-3 exhibited a time-independent, noninactivating K+-selective current. Single-channel conductance was 27 pS at −60 mV and 17 pS at 60 mV in symmetrical 140 mm KCl. TASK-3 current was highly sensitive to changes in extracellular pH (pH o ), a hallmark of the TASK family of K+channels. Thus, a change in pH o from 7.2 to 6.4 and 6.0 decreased TASK-3 current by 74 and 96%, respectively. Mutation of histidine at position 98 to aspartate abolished pH o sensitivity. TASK-3 was blocked by barium (57%, 3 mm), quinidine (37%, 100 μm), and lidocaine (62%, 1 mm). Thus, TASK-3 is a new member of the acid-sensing K+ channel subfamily (TASK).
25972671
Cloning, functional expression and brain localization of a novel unconventional outward rectifier K+ channel.
Human TWIK‐1, which has been cloned recently, is a new structural type of weak inward rectifier K+ channel. Here we report the structural and functional properties of TREK‐1, a mammalian TWIK‐1‐related K+ channel. Despite a low amino acid identity between TWIK‐1 and TREK‐1 (approximately 28%), both channel proteins share the same overall structural arrangement consisting of two pore‐forming domains and four transmembrane segments (TMS). This structural similarity does not give rise to a functional analogy. K+ currents generated by TWIK‐1 are inwardly rectifying while K+ currents generated by TREK‐1 are outwardly rectifying. These channels have a conductance of 14 pS. TREK‐1 currents are insensitive to pharmacological agents that block TWIK‐1 activity such as quinine and quinidine. Extensive inhibitions of TREK‐1 activity are observed after activation of protein kinases A and C. TREK‐1 currents are sensitive to extracellular K+ and Na+. TREK‐1 mRNA is expressed in most tissues and is particularly abundant in the lung and in the brain. Its localization in this latter tissue has been studied by in situ hybridization. TREK‐1 expression is high in the olfactory bulb, hippocampus and cerebellum. These results provide the first evidence for the existence of a K+ channel family with four TMS and two pore domains in the nervous system of mammals. They also show that different members in this structural family can have totally different functional properties.
11605399
Mechano- or Acid Stimulation, Two Interactive Modes of Activation of the TREK-1 Potassium Channel*
TREK-1 is a member of the novel structural class of K+ channels with four transmembrane segments and two pore domains in tandem (1, 2). TREK-1 is opened by membrane stretch and arachidonic acid. It is also an important target for volatile anesthetics (2, 3). Here we show that internal acidification opens TREK-1. Indeed, lowering pH i shifts the pressure-activation relationship toward positive values and leads to channel opening at atmospheric pressure. The pH i -sensitive region in the carboxyl terminus of TREK-1 is the same that is critically involved in mechano-gating as well as arachidonic acid activation. A convergence, which is dependent on the carboxyl terminus, occurs between mechanical, fatty acids and acidic stimuli. Intracellular acidosis, which occurs during brain and heart ischemia, will induce TREK-1 opening with subsequent K+ efflux and hyperpolarization.
20414039
Cloning and Expression of a Novel pH-sensitive Two Pore Domain K+ Channel from Human Kidney*
A complementary DNA encoding a novel K+ channel, called TASK-2, was isolated from human kidney and its gene was mapped to chromosome 6p21. TASK-2 has a low sequence similarity to other two pore domain K+ channels, such as TWIK-1, TREK-1, TASK-1, and TRAAK (18–22% of amino acid identity), but a similar topology consisting of four potential membrane-spanning domains. In transfected cells, TASK-2 produces noninactivating, outwardly rectifying K+ currents with activation potential thresholds that closely follow the K+equilibrium potential. As for the related TASK-1 and TRAAK channels, the outward rectification is lost at high external K+concentration. The conductance of TASK-2 was estimated to be 14.5 picosiemens in physiological conditions and 59.9 picosiemens in symmetrical conditions with 155 mm K+. TASK-2 currents are blocked by quinine (IC50 = 22 μm) and quinidine (65% of inhibition at 100 μm) but not by the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine, and Cs+. They are only slightly sensitive to Ba2+, with less than 17% of inhibition at 1 mm. As TASK-1, TASK-2 is highly sensitive to external pH in the physiological range. 10% of the maximum current was recorded at pH 6.5 and 90% at pH 8.8. Unlike all other cloned channels with two pore-forming domains, TASK-2 is essentially absent in the brain. In human and mouse, TASK-2 is mainly expressed in the kidney, where in situ hybridization shows that it is localized in cortical distal tubules and collecting ducts. This localization, as well as its functional properties, suggest that TASK-2 could play an important role in renal K+ transport.
31190948
Genomic Organization of Nematode 4TM K+ Channelsa
ABSTRACT: As many as 50 genes in the C. elegans genome may encode K+ channels belonging to the novel structural class of two‐pore (4TM) channels. Many 4TM channels can be grouped into channel subfamilies. We analyzed 4TM channels in C. elegans using methods made possible by having complete genomic sequence. Two genes were chosen for comprehensive analysis, n2P16 and n2P17. By comparing the pattern of conservation in genomic DNA sequences between C. elegans and a closely related species, C. briggsae, we were able to identify all coding regions and predict the gene structure for these two genes. Given the extent of the 4TM channel family, we were surprised to discover that n2P17 produced at least six alternative transcripts encoding a constant central region and variable amino‐ and carboxyl‐termini. Blocks of highly conserved DNA sequences in noncoding regions were also apparent and most likely confer important regulatory functions. The interspecies comparison of the deduced channel proteins revealed that the extracellular loop between M1 and P1 is an apparent hot spot for evolutionary change in both channels. This contrasts with the membrane‐spanning domains that are highly conserved. Analysis of intron positions for 36 channels revealed that introns are frequently present at an identical position within the pore region, but very few are located in membrane‐spanning domains.
5694563
Ion Channels and Synaptic Organization Analysis of the Drosophila Genome
The most obvious conclusion from this survey is that neuronal signaling proteins, for the most part, have been highly conserved throughout evolution. Despite the dramatic differences in complexity and organization of nervous systems and behavioral outputs in worms, flies, and mammals, the key proteins, and presumably the functional mechanisms they govern, are remarkably similar. Apparently, the basic framework for neuronal signaling and synaptic organization was completed early in metazoan evolution, and evolution of much more complicated nervous systems did not entail extensive alteration of this framework. Thus, genomic analysis fully confirms the belief that one can obtain fundamental neurobiological insights from studies of simple model organisms.Still, at this point, it is easier to catalog the information obtained from sequence analysis than to decipher all the functional implications. It is not in any way apparent, for example, why C. elegans, with only 302 neurons, encodes more than 90 K+ channels. Drosophila with a nominally more complex nervous system and behavioral repertoire has a set of only about 30 K+ channel genes. Indeed, most ion channel gene families contain more members in worms than flies. Apparently, increased behavioral complexity does not require an increase in molecular complexity but may depend instead on increases in the number of neurons and the complexity of the wiring diagram. Although somewhat counterintuitive, a greater number of ion channels may be required in worms than flies to provide its simple nervous system with the potential for more extensive signaling capabilities than would otherwise be possible.With the identification in this analysis of new channel subfamilies, it is likely that all the major classes of channel pore-forming subunits have now been described and the complete set of evolutionarily conserved ion channels has been defined. Although this would not have been obvious a priori, there is a striking difference between ion channel pore-forming subunits and auxiliary subunits with respect to evolutionary conservation. With the exception of Ca2+ channels, very few of the accessory subunits identified in mammals have homologous counterparts in worms or flies. The acquisition of novel ion channel subunits that modify the biophysical properties of pore-forming subunits may represent one way of adding additional functional complexity to the basic set of channels.It remains an open question whether the more complex nervous systems of vertebrates, particularly mammals, employ any signaling mechanisms that are fundamentally different from those in invertebrates. If so, the strong conservation of the basic set of signaling proteins argues that these mechanisms either depend on an unknown set of proteins or involve the same components that can be incorporated into novel molecular and cellular networks that generate unique functions and outputs.From the perspective of the genome, it seems safe to predict that elucidation of the fundamental molecular mechanisms of neuronal signaling applicable to all organisms will continue to derive enormous benefit from invertebrate model systems. With future emphasis on understanding the distinctive in vivo functions of the array of proteins now defined only by sequence, the availability of forward and reverse genetic techniques in worms and flies will continue to be of critical importance. The identification of the entire set of ion channels and neuronal signaling proteins opens up a new era in which neurobiologists can work back from the complete gene set toward an understanding of how they function to produce behavior.‡To whom correspondence should be addressed (e-mail: troy@mit.edu).
23440364
Spinal Cord Motoneuron Excitability during Isoflurane and Nitrous Oxide Anesthesia
Background Recent evidence suggests that the spinal cord is an important site of anesthesia that is necessary for surgical immobility, but the specific effect of anesthetics within the spinal cord is unclear. This study assessed the effect of isoflurane and nitrous oxide on spinal motoneuron excitability by monitoring the H‐reflex and the F wave. Methods Eight adult patients, categorized as American Society of Anesthesiologists physical status 1 or 2, who were undergoing elective orthopedic surgery were anesthetized with 0.6, 0.8, 1.0, and 1.2 times the estimated minimum alveolar concentration (MAC) of isoflurane. Nitrous oxide was added in graded concentrations of 30%, 50%, and 70%, whereas the isoflurane concentration was decreased to maintain a total MAC of 1. The H‐reflex of the soleus muscle and the F wave of the abductor hallucis muscle were measured before anesthesia and 15 min after each change of anesthetic concentration. Four or more trials of the H‐reflex and 18 trials of the F wave were recorded at each concentration of anesthesia. The effect of the anesthetics on the H‐reflex and F wave was analyzed using Dunnett's test. Results H‐reflex amplitude was decreased to 48.4 +/‐ 18.6% of preanesthesia level at 0.6 MAC isoflurane and to 33.8 +/‐ 19.1% when isoflurane concentration increased from 0.6 MAC to 1.2 MAC. F wave amplitude and persistence decreased to 52.2 +/‐ 33.6% and 44.4 +/‐ 26% of baseline at 0.6 MAC isoflurane, and to 33.8 +/‐ 26% and 21.7 +/‐ 22.8% at 1.2 MAC isoflurane. Isoflurane plus nitrous oxide (total 1 MAC) decreased H‐reflex amplitude to 30.4–33.3% and decreased F wave persistence to 42.8–56.3% of baseline. Conclusions Both isoflurane alone and isoflurane plus nitrous oxide decrease H‐reflex and F‐wave amplitude and F‐wave persistence. These effects suggest that isoflurane and nitrous oxide decrease motoneuronal excitability in the human spinal cord. This may play an important role in producing surgical immobility.
26489372
Suppression of Spinal Cord Motoneuron Excitability Correlates with Surgical Immobility during Isoflurane Anesthesia
Background Recent evidence suggests that the spinal cord is an important site of anesthetic action that produces surgical immobility. Inhalation anesthetics depress the Hoffmann's reflex (H reflex) and F wave, indicating spinal motoneuron suppression. The aim of this study was to assess the correlation between isoflurane‐induced immobility and H‐ and F‐wave suppression. Methods The baseline H reflex and F wave were measured before anesthesia in 15 adult patients. After induction, 1% end‐tidal isoflurane was maintained for 20 min before the H and F waves were reelicited. Using an electric stimulus applied to the forearm and grading the response as movement or no movement, the authors increased or decreased the isoflurane concentration in 0.1% steps, depending on the movement responses. The H and F waves were recorded 20 min after each change of isoflurane concentration. The correlation between H‐ and F‐wave suppression and surgical immobility was analyzed using a paired t test with Bonferroni correction. Results H‐reflex amplitude (2.74 +/‐ 1.63 mV) and F‐wave persistence (70.69 +/‐ 26.19%) at the highest isoflurane concentration that allowed movement response to a stimulus are different (P < 0.01) from these (1.97 +/‐ 1.46 mV; 43.16 +/‐ 22.91%) at the lowest isoflurane concentration that suppressed response. At 0.8% isoflurane, the H‐reflex amplitude was 3.69 +/‐ 1.83 mV with movement and 1.01 +/‐ 1.14 mV without movement (P < 0.01); F‐wave amplitude was 0.29 +/‐ 0.15 mV with movement and 0.11 +/‐ 0.06 mV without movement (P < 0.01); F‐wave persistence was 80 +/‐ 22.36% with movement and 34.9 +/‐ 25.75% without movement (P < 0.01). Conclusions The degree of H‐ and F‐wave amplitude and F‐wave persistence suppression correlates with movement response, suggesting that isoflurane‐suppressive action in the spinal cord plays a significant role in producing surgical immobility.
15062314
Effects of locus coeruleus activation on electroencephalographic activity in neocortex and hippocampus
Experiments were conducted to examine the hypothesis that increased neuronal discharge activity of noradrenergic neurons of the locus coeruleus (LC) above resting discharge rates can alter forebrain electroencephalographic (EEG) activity. Small infusions (70-135 nl) of the cholinergic agonist bethanechol within 500 microns of the LC were used to activate this nucleus reversibly in halothane-anesthetized rats. A combined recording-infusion probe allowed verification of this electrophysiological activation. Simultaneously, EEG activity was recorded from sites in the frontal cortex and hippocampus and subjected to power-spectrum analyses. The findings were (1) LC activation was consistently followed, within 5 to 30 sec, by a shift from low- frequency, high-amplitude to high-frequency, low-amplitude EEG activity in frontal neocortex and by the appearance of intense theta-rhythm in the hippocampus; (2) forebrain EEG changes followed LC activation with similar latencies whether infusions were made lateral or medial to the LC; (3) infusions placed outside the immediate vicinity of the LC were not followed by these forebrain EEG effects; (4) following infusion- induced activation, forebrain EEG returned to preinfusion patterns with about the same time course as the recovery of LC activity (10-20 min for complete recovery). These infusion-induced effects on EEG activity were blocked or severely attenuated by pretreatment with the alpha 2- agonist clonidine, which inhibits LC discharge and norepinephrine release, or the beta-antagonist propranolol. These observations indicate that enhanced LC discharge activity is the crucial mediating event for the infusion-induced changes in forebrain EEG activity observed under these conditions and suggest that LC activation may be sufficient to induce EEG signs of cortical and hippocampal activation.
58449536
A Hypnotic Response to Dexmedetomidine, an α2 Agonist, Is Mediated in the Locus Coerüleus in Rats
Dexmedetomidine, the highly selective alpha 2-adrenergic agonist, produces a dose-dependent hypnotic response in rats through a central mechanism. Because the locus coeruleus (LC) contains pathways involved in the maintenance of vigilance and a high prevalence of alpha 2 adrenoceptors, we investigated the role of this brainstem nucleus in the hypnotic response to dexmedetomidine. The experimental model consisted of chronic, stereotactically cannulated rats (n = 157) in which the hypnotic response to dexmedetomidine was assessed by the duration of the loss of their righting reflex. Correct placement of the cannula was confirmed histologically at necropsy. The hypnotic response to dexmedetomidine 0.3-333.3 micrograms administered into the LC increased in a dose-dependent fashion. Dexmedetomidine 6.6 micrograms injected 2 mm lateral to the LC did not cause the animals to lose their righting response. Atipamezole 0.07 micrograms-12 micrograms, a selective alpha 2-adrenergic antagonist, blocked the hypnotic response to dexmedetomidine 6.6 micrograms when both were administered into the LC. Also, atipamezole 0.7-30 micrograms, administered into the LC, blocked in a dose-dependent manner the hypnotic response to intraperitoneal (ip) dexmedetomidine 50 micrograms.kg-1. Atipamezole injected into the LC did not block the hypnotic response to pentobarbital 40 mg.kg-1 ip. Prazosin, an alpha 1-adrenergic antagonist, 4.2 micrograms into the LC or 1.0 mg.kg-1 ip, did not alter the hypnotic response to dexmedetomidine 6.6 micrograms into the LC. The present data suggest that alpha 2-adrenergic receptors in the LC appear to be a major site for the hypnotic action of dexmedetomidine.(ABSTRACT TRUNCATED AT 250 WORDS)
12936304
Dexmedetomidine Injection into the Locus Ceruleus Produces Antinociception
Background alpha2 -Adrenergic agonists such as clonidine and dexmedetomidine are known to produce sedation and analgesia in humans. The sedative effect of these agents is thought to occur through supraspinal pathways, involving the locus ceruleus (LC) and its projections in rats. While the antinociceptive response to alpha2 agonists, given intrathecally, is mediated predominantly in the spinal cord, other sites of action have not been systematically studied. The authors examined whether alpha2 -adrenergic receptors in the LC mediate an antinociceptive effect. Methods For administration of different drugs into the LC, guide cannulas were placed with their tips in the LC in male Sprague-Dawley rats. Dexmedetomidine (3.5 micro gram/0.2 micro liter) was microinjected into the LC through the cannula, or given systemically by intraperitoneal injection (50 micro gram/kg). The antinociceptive effect of dexmedetomidine was measured using the tail-flick latency response. To determine the sites through which dexmedetomidine injection into the LC produces antinociception, the authors examined whether this response could be perturbed by the specific alpha2 -adrenergic antagonists atipamezole and L659,066 and pertussis toxin administered either into the LC or intrathecally before injection of dexmedetomidine systemically or directly into the LC. To eliminate the possibility that drug administered in one site (LC or intrathecal) could reach the other site, the dispositional characteristics of radiolabeled dexmedetomidine (LC) or atipamezole (intrathecal) were studied. Results Dexmedetomidine placed into the LC produces a dose-dependent increase in the tail-flick latency. This antinociceptive effect was blocked by pertussis toxin and by the alpha2 antagonists atipamezole and L659,066 placed in the LC. Intrathecal administration of atipamezole and pertussis toxin also blocked the antinociceptive effect of dexmedetomidine placed in the LC.3 Hydrogen-dexmedetomidine introduced into the LC did not reach the spinal cord in pharmacologically active concentrations; also, intrathecally administered3 Hydrogen-atipamezole did not reach the LC in appreciable amounts. The systemic administration of dexmedetomidine produced an increase in tail-flick latency, and this effect was attenuated by the injection of atipamezole and L695,066 into the LC. Conclusions Part of the mechanism by which dexmedetomidine produces an antinociceptive effect is by an action directly on the LC, demonstrated by these studies in which antinociception produced by injection of this drug into the LC can be blocked by specific alpha2 antagonists injected into the LC. Furthermore, the action of dexmedetomidine in the LC in turn may result in an increase in activation of alpha2 adrenoceptors in the spinal cord, because the antinociceptive effect of LC dexmedetomidine injection also can be blocked by intrathecal injection of atipamezole and pertussis toxin.
20785244
Substitution of a mutant alpha2a-adrenergic receptor via "hit and run" gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo.
Norepinephrine contributes to antinociceptive, sedative, and sympatholytic responses in vivo, and alpha2 adrenergic receptor (alpha2AR) agonists are used clinically to mimic these effects. Lack of subtype-specific agonists has prevented elucidation of the role that each alpha2AR subtype (alpha2A, alpha2B, and alpha2C) plays in these central effects. Here we demonstrate that alpha2AR agonist-elicited sedative, anesthetic-sparing, and analgesic responses are lost in a mouse line expressing a subtly mutated alpha2AAR, D79N alpha2AAR, created by two-step homologous recombination. These functional changes are accompanied by failure of the D79N alpha2AAR to inhibit voltage-gated Ca2+ currents and spontaneous neuronal firing, a measure of K+ current activation. These results provide definitive evidence that the alpha2AAR subtype is the primary mediator of clinically important central actions of alpha2AR agonists and suggest that the D79N alpha2AAR mouse may serve as a model for exploring other possible alpha2AAR functions in vivo.
37851731
Drug receptors and the inhibition of nerve cells
It would be trite indeed to say much to this audience of the contributions to pharmacology made by Sir John Gaddum. His quantitative approaches to the study of drug antagonism have found their way into most textbooks of pharmacology. I dare say that, if asked to define that aspect of the subject which is uniquely 'pharmacology'-a task of increasing difficulty in these interdisciplinary days-many Society members would think first of those quantitative methods for studying drug-receptor interactions spawned by A.J. Clark, tested with antagonists by John Gaddum, and much popularized by Heinz Schild (Clark, 1933; Gaddum, 1937; 1957; Schild, 1949). I thank the Trustees for providing me with the opportunity to add my own small tribute to the memory of John Gaddum's work; this is particularly so because-as you will see-my own research has been much influenced by his contributions. John Gaddum worked mostly with peripheral tissues. This was convenient because the tissues were readily accessible, easy to maintain in vitro, and in those days devites could be made to measure the appropriate response, such as contraction, relaxation or secretion; furthermore, it was generally not important to distinguish the drug effects on the individual cells within the syncytium. The first efforts to classify receptors on nerves were also made at their terminations in the periphery, following on from the well-known observations of Lindor Brown and John Gillespie (1957). That field, the study of autonomic presynaptic receptors, has matured and has led to novel therapies. But in the ganglia of the autonomic nervous system and in the central nervous system, the individual nerve cell is the functional unit, and information about drug receptors on nerve cells can best be obtained by recording from single cells. Conversely, the demonstration and characterization of drug receptors on nerve cells can itself be used as a way of classifying the cells, particularly when taken in concert with other information regarding the ion channels expressed, the transmitters synthesized and the targets to which the cells project.
11447903
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.
25590755
A functional role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons.
Cerebellar granule neurons (CGNs) are one of the most populous cells in the mammalian brain. They express an outwardly rectifying potassium current, termed a "standing-outward" K(+) current, or IK(SO), which does not inactivate. It is active at the resting potential of CGNs, and blocking IK(SO) leads to cell depolarization. IK(SO) is blocked by Ba(2+) ions and is regulated by activation of muscarinic M(3) receptors, but it is insensitive to the classical broad-spectrum potassium channel blocking drugs 4-aminopyridine and tetraethylammonium ions. The molecular nature of this important current has yet to be established, but in this study, we provide strong evidence to suggest that IK(SO) is the functional correlate of the recently identified two-pore domain potassium channel TASK-1. We show that IK(SO) has no threshold for activation by voltage and that it is blocked by small extracellular acidifications. Both of these are properties that are diagnostic of TASK-1 channels. In addition, we show that TASK-1 currents expressed in Xenopus oocytes are inhibited after activation of endogenous M(3) muscarinic receptors. Finally, we demonstrate that mRNA for TASK-1 is found in CGNs and that TASK-1 protein is expressed in CGN membranes. This description of a functional two-pore domain potassium channel in the mammalian central nervous system indicates its physiological importance in controlling cell excitability and how agents that modify its activity, such as agonists at G protein-coupled receptors and hydrogen ions, can profoundly alter both the neuron's resting potential and its excitability.
44428239
Narcotic Properties of Carbon Dioxide in the Dog
PaCO2 ranging from 15 mm. to 95 mm. of mercury with arterial pH values from 7.64 to 7.10 bad no effect on the minimum anesthetic concentration (MAC) for halothane in dogs. PaCO2 levels above 95 mm. of mercury with arterial pH below 7.10 were progressively narcotic, and replaced the halothane required to maintain a constant depth of anesthesia. Anesthesia was achieved with CO2 alone at PaCO2 of 245 mm. of mercury. The mechanism of CO2 narcosis correlated well (P>0.05) with the pH changes in the brain as measured in the cisternal CSF, and appeared to be independent of arterial pH, PaCO2 and cerebrospinal fluid PCO2