Background Although accumulating evidence suggests that arousal pathways in the brain play important roles in emergence from general anesthesia, the roles of monoaminergic arousal circuits are unclear. In this study we tested the hypothesis that methylphenidate (an inhibitor of dopamine and norepinephrine transporters) induces emergence from isoflurane anesthesia. Methods Using adult rats we tested the effect of methylphenidate IV on time to emergence from isoflurane anesthesia. We then performed experiments to test separately for methylphenidate-induced changes in arousal and changes in minute ventilation. A dose-response study was performed to test for methylphenidate–induced restoration of righting during continuous isoflurane anesthesia. Surface electroencephalogram recordings were performed to observe neurophysiological changes. Plethysmography recordings and arterial blood gas analysis were performed to assess methylphenidate-induced changes in respiratory function. Droperidol IV was administered to test for inhibition of methylphenidate's actions. Results Methylphenidate decreased median time to emergence from 280 to 91 s. The median difference in time to emergence without compared to with methylphenidate was 200 [155, 331] s (median, [95% confidence interval]). During continuous inhalation of isoflurane, methylphenidate induced return of righting in a dose-dependent manner, induced a shift in electroencephalogram power from delta to theta, and induced an increase in minute ventilation. Administration of droperidol (0.5 mg/kg IV) prior to methylphenidate (5 mg/kg IV) largely inhibited methylphenidate-induced emergence behavior, electroencephalogram changes, and changes in minute ventilation. Conclusions Methylphenidate actively induces emergence from isoflurane anesthesia by increasing arousal and respiratory drive, possibly through activation of dopaminergic and adrenergic arousal circuits. Our findings suggest that methylphenidate may be clinically useful as an agent to reverse general anesthetic-induced unconsciousness and respiratory depression at the end of surgery.

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 wildtype 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. The cystic fibrosis transmembrane conductance regulator (CFTR)1 contains an anion-selective pore (1-6). Earlier work has shown that amino acid residues in the two membranespanning domains, MSD1 and MSD2, determine the properties of this pore and harbor a number of disease-associated mutations (7-12). Despite the importance of this region to CFTR function, an understanding of the pore and MSD structure is limited.Studies of the effect of cystic fibrosis (CF)-associated mutations have been of value in identifying structurally and functionally important regions of CFTR. At least four CF-associated mutations have been identified at position 347 in M6: R347C, R347H, R347L, and R347P, suggesting that Arg-347 is important for CFTR structure and function (13-15).2 Early studies by Sheppard et al. (7) showed that mutation of Arg-347 to proline significantly decreased single-channel conductance with little effect on CFTR trafficking to the plasma membrane. Other work by Tabcharani et al. (8,16) and Linsdell and Hanrahan (17) emphasized the importance of Arg-347 for anomalous mole-fraction behavior, iodide permeability, and voltagedependent block by DIDS, in addition to single-channel conductance. Interestingly, mutation of Arg-347 to a histidine (R347H) produced a channel that displayed pH-dependent conductance and anomalous mole-fraction behavior (8). These studies suggested that Arg-347 may line the pore and that a positive charge at position 347 is sufficient for wild-type conductance. Because mutation of Arg-347 eliminated anomalous mole-fraction behavior, Arg-347 itself was proposed to be an anion binding site in the CFTR pore (8), and the presumed positive charge introduced upon protonation of His-347 was thought to facilitate interactions with permeating a...

TWIK-related acid-sensitive K(+)-1 (TASK-1 [KCNK3]) and TASK-3 (KCNK9) are tandem pore (K(2P)) 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 microM), and TASK-1/TASK-3 heterodimeric channel function (EC50, 9 microM). 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.