Joseph F. Cotten

CYSTIC FIBROSIS-ASSOCIATED MUTATIONS AT ARGININE 347 ALTER THE PORE ARCHITECTURE OF CFTR : EVIDENCE FOR DISRUPTION OF A SALT BRIDGE

Arginine 347 in the sixth transmembrane domain of cystic fibrosis transmembrane conductance regulator (CFTR) is a site of four cystic fibrosis-associated mutations. To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Every Arg-347 mutation examined, except R347K, had a destabilizing effect on the pore, causing the channel to flutter between two conductance states. Chloride flow through the larger conductance state was similar to that of wild-type CFTR, suggesting that the residue at position 347 does not interact directly with permeating anions. We hypothesized that Arg-347 stabilizes the channel through an electrostatic interaction with an anionic residue in another transmembrane domain. To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. These data suggest that Arg-347 plays an important structural role in CFTR, at least in part by forming a salt bridge with Asp-924; cystic fibrosis-associated mutations disrupt this interaction.

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.

Methoxycarbonyl-etomidate: a novel rapidly metabolized and ultra-short-acting etomidate analogue that does not produce prolonged adrenocortical suppression.

Background:Etomidate is a rapidly acting sedative-hypnotic that provides hemodynamic stability. It causes prolonged suppression of adrenocortical steroid synthesis; therefore, its clinical utility and safety are limited. The authors describe the results of studies to define the pharmacology of (R)-3-methoxy-3-oxopropyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate (MOC-etomidate), the first etomidate analogue designed to be susceptible to ultra-rapid metabolism. Methods:The &ggr;-aminobutyric acid type A receptor activities of MOC-etomidate and etomidate were compared by using electrophysiological techniques in human &agr;1&bgr;2&ggr;2l receptors. MOC-etomidate’s hypnotic concentration was determined in tadpoles by using a loss of righting reflex assay. Its in vitro metabolic half-life was measured in human liver S9 fraction, and the resulting metabolite was provisionally identified by using high-performance liquid chromatography/mass spectrometry techniques. The hypnotic and hemodynamic actions of MOC-etomidate, etomidate, and propofol were defined in rats. The abilities of MOC-etomidate and etomidate to inhibit corticosterone production were assessed in rats. Results:MOC-etomidate potently enhanced &ggr;-aminobutyric acid type A receptor function and produced loss of righting reflex in tadpoles. Metabolism in human liver S9 fraction was first-order, with an in vitro half-life of 4.4 min versus more than 40 min for etomidate. MOC-etomidate’s only detectable metabolite was a carboxylic acid. In rats, MOC-etomidate produced rapid loss of righting reflex that was extremely brief and caused minimal hemodynamic changes. Unlike etomidate, MOC-etomidate produced no adrenocortical suppression 30 min after administration. Conclusions:MOC-etomidate is an etomidate analogue that retains etomidate’s important favorable pharmacological properties. However, it is rapidly metabolized, ultra–short-acting, and does not produce prolonged adrenocortical suppression after bolus administration.

Carboetomidate: a pyrrole analog of etomidate designed not to suppress adrenocortical function.

Background:Etomidate is a sedative hypnotic that is often used in critically ill patients because it provides superior hemodynamic stability. However, it also binds with high affinity to 11&bgr;-hydroxylase, potently suppressing the synthesis of steroids by the adrenal gland that are necessary for survival. The authors report the results of studies to define the pharmacology of (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate), a pyrrole analog of etomidate specifically designed not to bind with high affinity to 11&bgr;-hydroxylase. Methods:The hypnotic potency of carboetomidate was defined in tadpoles and rats using loss of righting reflex assays. Its ability to enhance wild-type &agr;1&bgr;2&ggr;2l and etomidate-insensitive mutant &agr;1&bgr;2M286W&ggr;2l human &ggr;-aminobutyric acid type A receptor activities was assessed using electrophysiologic techniques. Its potency for inhibiting in vitro cortisol synthesis was defined using a human adrenocortical cell assay. Its effects on in vivo hemodynamic and adrenocortical function were defined in rats. Results:Carboetomidate was a potent hypnotic in tadpoles and rats. It increased currents mediated by wild-type but not etomidate-insensitive mutant &ggr;-aminobutyric acid type A receptors. Carboetomidate was a three orders of magnitude less-potent inhibitor of in vitro cortisol synthesis by adrenocortical cells than was etomidate. In rats, carboetomidate caused minimal hemodynamic changes and did not suppress adrenocortical function at hypnotic doses. Conclusions:Carboetomidate is an etomidate analog that retains many beneficial properties of etomidate, but it is dramatically less potent as an inhibitor of adrenocortical steroid synthesis. Carboetomidate is a promising new sedative hypnotic for potential use in critically ill patients in whom adrenocortical suppression is undesirable.

Covalent Modification of the Regulatory Domain Irreversibly Stimulates Cystic Fibrosis Transmembrane Conductance Regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is regulated by three cytosolic domains, the regulatory domain (R domain) and two nucleotide binding domains. To learn more about how the cytosolic domains regulate channel activity, we used chemical modification to probe their structure. When we applied the sulfhydryl-modifying reagentN-ethylmaleimide (NEM) and other N-substituted maleimides to the cytosolic domains, we found that they rapidly and irreversibly stimulated channel activity. CFTR contains 14 intracellular cysteine residues that might be targets for NEM modification. We identified one, Cys832, that was essential for the response. Cys832 is located in the R domain. Single channel studies showed that NEM stimulated CFTR by increasing the duration of bursts of activity and by shortening the closed interval between bursts. At the single channel level, CFTR in which Cys832 was mutated to alanine behaved identically to wild-type CFTR, except that it failed to respond to NEM. Additional studies showed that NEM modification increased the potency of ATP-mediated stimulation. Previous work has shown that modification of the R domain by phosphorylation, which introduces negative charge, or replacement of multiple serines by negatively charged aspartates stimulates the channel. Our current data show that covalent modification of the R domain with a neutral, hydrophobic adduct at a site that is not phosphorylated can also stimulate CFTR. This finding suggests that an alteration in the conformation of the R domain may be a key feature that regulates channel activity.

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.

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.

Closed-loop Continuous Infusions of Etomidate and Etomidate Analogs in Rats: A Comparative Study of Dosing and the Impact on Adrenocortical Function

Background: Etomidate is a sedative–hypnotic that is often given as a single intravenous bolus but rarely as an infusion because it suppresses adrenocortical function. Methoxycarbonyl etomidate and (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate) are etomidate analogs that do not produce significant adrenocortical suppression when given as a single bolus. However, the effects of continuous infusions on adrenocortical function are unknown. In this study, we compared the effects of continuous infusions of etomidate, methoxycarbonyl etomidate, and carboetomidate on adrenocortical function in a rat model. Methods: A closed-loop system using the electroencephalographic burst suppression ratio as the feedback was used to administer continuous infusions of etomidate, methoxycarbonyl etomidate, or carboetomidate to Sprague–Dawley rats. Adrenocortical function was assessed during and after infusion by repetitively administering adrenocorticotropic hormone 1–24 and measuring serum corticosterone concentrations every 30 min. Results: The sedative–hypnotic doses required to maintain a 40% burst suppression ratio in the presence of isoflurane, 1%, and the rate of burst suppression ratio recovery on infusion termination varied (methoxycarbonyl etomidate > carboetomidate > etomidate). Serum corticosterone concentrations were reduced by 85% and 56% during 30-min infusions of etomidate and methoxycarbonyl etomidate, respectively. On infusion termination, serum corticosterone concentrations recovered within 30 min with methoxycarbonyl etomidate but persisted beyond an hour with etomidate. Carboetomidate had no effect on serum corticosterone concentrations during or after continuous infusion. Conclusions: Our results suggest that methoxycarbonyl etomidate and carboetomidate may have clinical utility as sedative–hypnotic maintenance agents when hemodynamic stability is desirable.

In vivo and in vitro pharmacological studies of methoxycarbonyl-carboetomidate.

BACKGROUND:We previously developed 2 etomidate analogs that retain etomidates favorable hemodynamic properties but whose adrenocortical effects are reduced in duration or magnitude. Methoxycarbonyl (MOC)-etomidate is rapidly metabolized and ultrashort acting whereas (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate) does not potently inhibit 11&bgr;-hydroxylase. We hypothesized that MOC-etomidates labile ester could be incorporated into carboetomidate to produce a new agent that possesses favorable properties individually found in each agent. We describe the synthesis and pharmacology of MOC-(R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (MOC-carboetomidate), a “soft” analog of carboetomidate. METHODS:MOC-carboetomidates octanol:water partition coefficient was determined chromatographically and compared with those of etomidate, carboetomidate, and MOC-etomidate. MOC-carboetomidates 50% effective concentration (EC50) and 50% effective dose for loss of righting reflexes (LORR) were measured in tadpoles and rats, respectively. Its effect on &ggr;-aminobutyric acid A (GABAA) receptor function was assessed using 2-microelectrode voltage clamp electrophysiological techniques and its metabolic stability was determined in pooled rat blood using high performance liquid chromatography. Its duration of action and effects on arterial blood pressure and adrenocortical function were assessed in rats. RESULTS:MOC-carboetomidates octanol:water partition coefficient was 3300 ± 280, whereas those for etomidate, carboetomidate, and MOC-etomidate were 800 ± 180, 15,000 ± 3700, and 190 ± 25, respectively. MOC-carboetomidates EC50 for LORR in tadpoles was 9 ± 1 &mgr;M and its EC50 for LORR in rats was 13 ± 5 mg/kg. At 13 &mgr;M, MOC-carboetomidate enhanced GABAA receptor currents by 400% ± 100%. Its metabolic half-life in pooled rat blood was 1.3 min. The slope of a plot of the duration of LORR in rats versus the logarithm of the hypnotic dose was significantly shallower for MOC-carboetomidate than for carboetomidate (4 ± 1 vs 15 ± 3, respectively; P = 0.0004123). At hypnotic doses, the effects of MOC-carboetomidate on arterial blood pressure and adrenocortical function were not significantly different from those of vehicle alone. CONCLUSIONS:MOC-carboetomidate is a GABAA receptor modulator with potent hypnotic activity that is more rapidly metabolized and cleared from the brain than carboetomidate, maintains hemodynamic stability similar to carboetomidate, and does not suppress adrenocortical function.

Task-1 (kcnk3) and Task-3 (kcnk9) Tandem Pore Potassium Channel Antagonists Stimulate Breathing in Isoflurane-anesthetized Rats

BACKGROUND:TASK-1 and TASK-3 tandem pore potassium channel subunits provide a constitutive acidic pH- and hypoxia-inhibited potassium conductance. TASK channels are expressed in a number of tissues involved in regulation of breathing, and the TASK-1/TASK-3 heterodimer provides the predominant hypoxia-sensitive potassium conductance in carotid body type 1 glomus chemosensing cells. The carotid bodies have an important role in regulation of breathing. Doxapram is a potent TASK-1 and TASK-3 potassium channel antagonist and a carotid body and breathing stimulant. PK-THPP and A1899 are potent and selective TASK-1 and TASK-3 antagonists. I hypothesized PK-THPP and A1899 are, like doxapram, breathing stimulants. METHODS:I studied rat TASK-3 potassium channel function by Ussing chamber using Fischer rat thyroid monolayers. To quantify breathing effects, I studied male Sprague–Dawley rats spontaneously breathing 1.5% isoflurane in room air by noninvasive plethysmography and by arterial blood gas analysis. RESULTS:PK-THPP, A1899, and doxapram inhibit rat TASK-3 potassium channel function with IC50s of 42 nM (33–52), 1.6 &mgr;M (0.8–3.3), and 22 &mgr;M (18–28) (n = 4–6; 95% confidence limits). IV PK-THPP, A1899, and doxapram stimulated breathing by plethysmography with a peak change in minute ventilation relative to baseline of 84% ± 19% and 226% ± 56% (for PK-THPP at 0.5 and 5 mg/kg; mean ± SEM; n = 3–4; P < 0.05 and P < 0.001, respectively, relative to vehicle); 46% ± 2% and 236% ± 48% (for A1899 at 5 and 25 mg/kg; n = 3–4; P > 0.05 and P < 0.001, respectively); 103% ± 20% (for doxapram at 25 mg/kg; n = 4), and 33% ± 9% (for dimethylsulfoxide vehicle at 1 mL/kg; n = 4). PK-THPP and A1899, unlike doxapram, induced a profound and lasting respiratory alkalosis by arterial blood gas analysis. Thirty minutes after IV drug administration, I observed an arterial pH and carbon dioxide partial pressure of 7.62 ± 0.02 and 23 ± 0.8 mm Hg (for PK-THPP after 5 mg/kg; n = 4; P < 0.001 for both relative to vehicle), 7.49 ± 0.02 and 31 ± 2 mm Hg (for A1899 at 25 mg/kg; n = 6; P < 0.05 and 0.001, respectively), 7.43 ± 0.03 and 39 ± 4 mm Hg (for doxapram after 25 mg/kg; n = 4; P > 0.05 for both), and 7.38 ± 0.03 and 48 ± 4 mm Hg (for dimethylsulfoxide vehicle after 1 mL/kg; n = 3). CONCLUSIONS:PK-THPP and A1899 are potent rat TASK-3 antagonists and effective breathing stimulants. PK-THPP and A1899 effects on breathing were of greater magnitude and/or duration relative to that of doxapram. PK-THPP and A1899 or related compounds may have therapeutic potential for treating breathing disorders.