C. Spencer Yost

Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

Abstract: Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as Δ9‐tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA‐induced calcium flux (ΔCa2+NMDA) in rat brain slices. The presence of anandamide reduced ΔCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting ΔCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences ΔCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated ΔCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA‐stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor‐dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

TWIK-2, a new weak inward rectifying member of the tandem pore domain potassium channel family.

Potassium channels are found in all mammalian cell types, and they perform many distinct functions in both excitable and non-excitable cells. These functions are subserved by several different families of potassium channels distinguishable by primary sequence features as well as by physiological characteristics. Of these families, the tandem pore domain potassium channels are a new and distinct class, primarily distinguished by the presence of two pore-forming domains within a single polypeptide chain. We have cloned a new member of this family, TWIK-2, from a human brain cDNA library. Primary sequence analysis of TWIK-2 shows that it is most closely related to TWIK-1, especially in the pore-forming domains. Northern blot analysis reveals the expression of TWIK-2 in all human tissues assayed except skeletal muscle. Human TWIK-2 expressed heterologously in Xenopus oocytes is a non-inactivating weak inward rectifier with channel properties similar to TWIK-1. Pharmacologically, TWIK-2 channels are distinct from TWIK-1 channels in their response to quinidine, quinine, and barium. TWIK-2 is inhibited by intracellular, but not extracellular, acidification. This new clone reveals the existence of a subfamily in the tandem pore domain potassium channel family with weak inward rectification properties.

A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems

We have combined molecular genetic and cell-free reconstitution approaches to study the mechanism of membrane assembly. The coding region for the carboxy-terminal transmembrane sequence of membrane IgM heavy chain has been inserted between the coding regions for lactamase and globin domains of a fusion protein previously shown to be completely translocated across microsomal membranes in a cell-free transcription-linked translation system. The resulting fusion protein behaves as an integral transmembrane protein of predicted asymmetry: all of the membrane integrated copies display lactamase within the lumen and globin on the cytoplasmic face of the vesicles. In another construction, this transmembrane coding region replaces that of the signal sequence. The resulting fusion protein is not translocated across membranes. These data provide strong evidence that there are stop transfer sequences whose ability to arrest chain translocation and achieve asymmetric transmembrane orientation is independent of the size of the subsequent carboxy-terminal domain to be localized in the cytosol; and that signal and stop transfer sequences are functionally distinct.

Potent Activation of the Human Tandem Pore Domain K Channel TRESK with Clinical Concentrations of Volatile Anesthetics

The tandem pore domain K channel family mediates background K currents present in excitable cells. Currents passed by certain members of the family are enhanced by volatile anesthetics, thus suggesting a novel mechanism of anesthesia. The newest member of the family, termed TRESK (TWIK [tandem pore domain weak inward rectifying channel]-related spinal cord K channel), has not been studied for anesthetic sensitivity. We isolated the coding sequence for TRESK from human spinal cord RNA and functionally expressed it in Xenopus oocytes and transfected COS-7 cells. With both whole-cell voltage-clamp and patch-clamp recording, TRESK currents increased up to three-fold by clinical concentrations of isoflurane, halothane, sevoflurane, and desflurane. Nonanesthetics (nonimmobilizers) had no effect on TRESK. Various IV anesthetics, including etomidate, thiopental, and propofol, have a minimal effect on TRESK currents. Amide and ester local anesthetics inhibit TRESK in a concentration-dependent manner but at concentrations generally larger than those that inhibit other tandem pore domain K channels. We also determined that TRESK is found not only in spinal cord, but also in human brain RNA. These results identify TRESK as a target of volatile anesthetics and suggest a role for this background K channel in mediating the effects of inhaled anesthetics in the central nervous system.

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.

Two-Pore Domain Potassium Channels: New Sites of Local Anesthetic Action and Toxicity

Potassium (K+) channels form the largest family of ion channels with more than 70 such genes identified in the human genome. They are organized in 3 superfamilies according to their predicted membrane topology: (1) subunits with 6 membrane-spanning segments and 1-pore domain, (2) subunits with 2 membrane-spanning segments and 1-pore domain, and (3) subunits with 4 membrane-spanning segments and 2-pore domains arrayed in a tandem position. The last family has most recently been identified and comprises the so-called 2-pore domain potassium (K2P) channels, believed responsible for background or leak K+ currents. Despite their recent discovery, interest in them is growing rapidly with more than 270 references in the literature reported (http://www.ipmc.cnrs.fr/~duprat/2p/ref2p.htm#2P, accessed October 30, 2004). K2P channels are widely expressed in the central nervous system and are involved in the control of the resting membrane potential and the firing pattern of excitable cells. This article will therefore review recent findings on actions of local anesthetics with respect to K2P channels. It begins with an overview of the role of background K+ channels in neuronal excitability and nerve conduction and is followed by a description of the K2P channel family including experimental evidence for the contribution of K2P channels to the mechanism of action and toxicity of local anesthetics.

The ventilatory stimulant doxapram inhibits TASK tandem pore (K2P) potassium channel function but does not affect minimum alveolar anesthetic concentration.

TWIK-related acid-sensitive K+-1 (TASK-1 [KCNK3]) and TASK-3 (KCNK9) are tandem pore (K2P) potassium (K) channel subunits expressed in carotid bodies and the brainstem. Acidic pH values and hypoxia inhibit TASK-1 and TASK-3 channel function, and halothane enhances this function. These channels have putative roles in ventilatory regulation and volatile anesthetic mechanisms. Doxapram stimulates ventilation through an effect on carotid bodies, and we hypothesized that stimulation might result from inhibition of TASK-1 or TASK-3 K channel function. To address this, we expressed TASK-1, TASK-3, TASK-1/TASK-3 heterodimeric, and TASK-1/TASK-3 chimeric K channels in Xenopus oocytes and studied the effects of doxapram on their function. Doxapram inhibited TASK-1 (half-maximal effective concentration [EC50], 410 nM), TASK-3 (EC50, 37 &mgr;M), and TASK-1/TASK-3 heterodimeric channel function (EC50, 9 &mgr;M). Chimera studies suggested that the carboxy terminus of TASK-1 is important for doxapram inhibition. Other K2P channels required significantly larger concentrations for inhibition. To test the role of TASK-1 and TASK-3 in halothane-induced immobility, the minimum alveolar anesthetic concentration for halothane was determined and found unchanged in rats receiving doxapram by IV infusion. Our data indicate that TASK-1 and TASK-3 do not play a role in mediating the immobility produced by halothane, although they are plausible molecular targets for the ventilatory effects of doxapram.

Additive Inhibition of Nicotinic Acetylcholine Receptors by Corticosteroids and the Neuromuscular Blocking Drug Vecuronium

Background: Neuromuscular disorders associated with muscular weakness and prolonged paralysis are common in critically ill patients. Acute myopathy has been described in patients receiving a combination therapy of corticosteroids and nondepolarizing neuromuscular blocking drugs for treatment of acute bronchospasm. The cause of this myopathy is not fully established and may involve drug interactions that perturb neuromuscular transmission. To investigate the interaction of corticosteroids with neuromuscular blocking drugs, the authors determined the effects of methylprednisolone and hydrocortisone alone and in combination with vecuronium on fetal (&ggr;-subunit containing) and adult (&egr;-subunit containing) subtypes of the muscle-type nicotinic acetylcholine receptor. Methods: Functional channels were expressed in Xenopus laevis oocytes and activated with 1 &mgr;M acetylcholine. The resulting currents were recorded using a whole cell two-electrode voltage clamp technique. Results: Both forms of the muscle-type acetylcholine receptor were potently inhibited by methylprednisolone and hydrocortisone, with concentrations producing 50% inhibition in the range of 400–600 &mgr;M and 1–2 mM, respectively. The corticosteroids produced noncompetitive antagonism of the muscle-type nicotinic acetylcholine receptor at clinical concentrations. Both receptor forms were also inhibited, even more potently, by vecuronium, with a concentration producing 50% inhibition in the range of 1–2 nM. Combined application of vecuronium and methylprednisolone showed additive effects on both receptor forms, which were best described by a two-site model, with each site independent. Conclusions: The enhanced neuromuscular blockade produced when corticosteroids are combined with vecuronium may augment pharmacologic denervation and contribute to the pathophysiology of prolonged weakness observed in some critically ill patients.

Species-specific differences in response to anesthetics and other modulators by the K2P channel TRESK.

TRESK (TWIK-related spinal cord K+ channel) is the most recently characterized member of the tandem-pore domain potassium channel (K2P) family. Human TRESK is potently activated by halothane, isoflurane, sevoflurane, and desflurane, making it the most sensitive volatile anesthetic-activated K2P channel yet described. Herein, we compare the anesthetic sensitivity and pharmacologic modulation of rodent versions of TRESK to their human orthologue. Currents passed by mouse and rat TRESK were enhanced by isoflurane at clinical concentrations but with significantly lower efficacy than human TRESK. Unlike human TRESK, the rodent TRESKs are strongly inhibited by acidic extracellular pH in the physiologic range. Zinc inhibited currents passed by both rodent TRESK in the low micromolar range but was without effect on human TRESK. Enantiomers of isoflurane that have stereoselective anesthetic potency in vivo produced stereospecific enhancement of the rodent TRESKs in vitro. Amide local anesthetics inhibited the rodent TRESKs at almost 10-fold smaller concentrations than that which inhibit human TRESK. These results identified interspecies differences and similarities in the pharmacology of TRESK. Further characterization of TRESK expression patterns is needed to understand their role in anesthetic mechanisms.

Characterization of the interactions between volatile anesthetics and neuromuscular blockers at the muscle nicotinic acetylcholine receptor.

Volatile anesthetics enhance the neuromuscular blockade produced by nondepolarizing muscle relaxants (NDMRs). The neuromuscular junction is a postulated site of this interaction. We tested the hypothesis that volatile anesthetic enhancement of muscle relaxation is the result of combined drug effects on the nicotinic acetylcholine receptor. The adult mouse muscle nicotinic acetylcholine receptor (&agr;2, &bgr;, &dgr;, &egr;) was heterologously expressed in Xenopus laevis oocytes. Concentration-effect curves for the inhibition of acetylcholine-induced currents were established for vecuronium, d-tubocurarine, isoflurane, and sevoflurane. Subsequently, inhibitory effects of NDMRs were studied in the presence of the volatile anesthetics at a concentration equivalent to half the concentration producing a 50% inhibition alone. All individually tested compounds produced rapid and readily reversible concentration-dependent inhibition. The calculated 50% inhibitory concentration values were 9.9 nM (95% confidence interval [CI], 8.4–11.4 nM), 43.4 nM (95% CI, 33.6–53.3 nM), 897 &mgr;M (95% CI, 699–1150 &mgr;M), and 818 &mgr;M (95% CI, 685–1001 &mgr;M) for vecuronium, d-tubocurarine, isoflurane, and sevoflurane, respectively. Coapplication of either isoflurane or sevoflurane significantly enhanced the inhibitory effects of vecuronium and d-tubocurarine, especially so at small concentrations of NDMRs. Volatile anesthetics increase the potency of NDMRs, possibly by enhancing antagonist affinity at the receptor site. This effect may contribute to the clinically observable enhancement of neuromuscular blockade by volatile anesthetics.