Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The two-pore-domain potassium (2PK⁺) channels give rise to background (leak) K⁺ conductance in resting cells and stabilize the membrane potential at hyperpolarized values, near the potassium equilibrium potential. In addition, they regulate the membrane potential in electrically active cells, in that they remain open during depolarization (in contrast to the inward rectifiers) and conduct robust repolarizing currents. The activity of 2PK⁺ channels is not influenced markedly by the membrane potential, and the majority of 2PK⁺ currents do not exhibit activation and inactivation kinetics. However, the channels can be regulated by various physicochemical alterations (such as membrane tension, extra- or intracellular pH, or temperature changes) and by different signaling pathways [including protein kinases A and C, arachidonic acid, and phospholipase C activation (for review, see Lesage and Lazdunski, 2000)]. These pathways are likely to play a major role in control of 2PK⁺ channel activity and consequently in the excitability of cells.

In concert with the wide spectrum of regulation, 2PK⁺ channels are the most diverse family of potassium channels. Apart from their similar molecular architecture, characterized by intracellular N- and C-terminals, four transmembrane segments (TMS), and two K⁺ channel pore-forming domains (one P domain between the first and second TMS and the other P domain between the third and fourth TMS), some members of the family are remarkably different and show only limited homology, which is unusual in other K⁺ channel families. This is why the 15 mammalian 2PK⁺ channel genes, cloned until now, have been divided into subfamilies, in which the amino acid sequence of the channels and also their regulatory properties are more similar. At present, five subfamilies of 2PK⁺ channels are known: TWIK (tandem pore domain in a weakly inwardly rectifying K⁺ channel), TREK (TWIK related K⁺ channel), THIK (tandem pore domain halothane-inhibited K⁺ channel), TASK (TWIK-related acid-sensitive K⁺ channel), and TALK (TWIK related alkaline pH activated K⁺ channel) (for review, see Patel and Honore, 2001).

The TASK subfamily of 2PK⁺ channels comprises TASK-1 (Duprat et al., 1997), TASK-3 (Kim et al., 2000), and TASK-5 (Kim and Gnatenco, 2001). [TASK-2 (Reyes et al., 1998) and TASK-4, also termed TALK-2 (Decher et al., 2001; Girard et al., 2001) belong to the TALK subfamily despite sharing the name with the other TASK channels]. TASK-1 and TASK-3 have been studied in different expression systems, and TASK-1 subunits have been shown to function as homodimers (Lopes et al., 2001). Recently, formation of functional heterodimeric channels by TASK-1 and TASK-3 subunits in Xenopus laevis oocytes has also been reported (Czirják and Enyedi, 2002a). In contrast with TASK-1 and TASK-3, expression of TASK-5 failed to produce current in heterologous expression systems. In the absence of specific inhibitors and because of the wide and partially overlapping distribution of expression, the relative contribution of the different TASK channels to the background K⁺ conductance of different cell types is difficult to determine.

Inhibition of channel activity by extracellular acidification was often used to differentiate TASK channels from members of the other 2PK⁺ subfamilies, and also between TASK-1 and TASK-3 (Hartness et al., 2001; Czirják and Enyedi, 2002b; Washburn et al., 2002). TASK-1 is extremely sensitive to pH in the 6.5 to 7.5 range (Duprat et al., 1997), whereas the inhibitory curve of TASK-3 is shifted to more acidic values by about 0.8 pH (Rajan et al., 2000). Discrimination between TASK-1 and TASK-3 currents was also attempted by applying Zn²⁺ and local anesthetics (Buckler et al., 2000; Hartness et al., 2001), based on the observation that the heterologously expressed TASK homodimers were inhibited with slightly different half-maximal inhibitory concentrations. However, in native tissues, these interventions may also have affected the possibly present TASK-1/TASK-3 heterodimers having different inhibitory curve from the homodimers [e.g., the pH dependence of the heterodimer is intermediate between the homodimers (Czirják and Enyedi, 2002a)]. Therefore, the proportion of TASK-1 and TASK-3 homodimers cannot be unequivocally deduced from the intermediate inhibition by pH, Zn²⁺, or local anesthetics.

Recently, we applied the cationic dye ruthenium red (RR; [(NH₃)₅Ru-O-Ru-(NH₃)₄-O-Ru(NH₃)₅]Cl₆) to discriminate between TASK channels in X. laevis oocytes (Czirják and Enyedi, 2002a) and glomerulosa cells of rat adrenal cortex (Szabadkai et al., 1999; Czirják and Enyedi, 2002b). Although RR is not specific for 2PK⁺ channels, it proved to be remarkably selective within the TASK subfamily. TASK-3 homodimer is inhibited by RR in the range of low micromolar concentrations, whereas the compound does not affect TASK-1 homodimer and the TASK-1/TASK-3 heterodimer (Czirják and Enyedi, 2002a). In the present study, we elucidate the major molecular determinant of RR inhibition of TASK channels and present an explanation for this peculiar asymmetric inhibitory profile.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. Enzymes and kits for molecular biological studies were purchased from Ambion (Austin TX), Amersham Biosciences (Little Chalfont, United Kingdom), Fermentas (Vilnius, Lithuania), New England Biolabs (Beverly, MA), Pharmacia (Uppsala, Sweden), Promega (Madison, WI), and Stratagene (La Jolla, CA). Fine chemicals of analytical grade were obtained from Fluka (Buchs, Switzerland), Promega, and Sigma Chemical Co. (St. Louis, MO).

In Vitro Site-Directed Mutagenesis and Construction of Concatenated Channels. The human pEXO-TASK-1 (Duprat et al., 1997) and the rat pEXO-TASK-3 (Czirják and Enyedi, 2002b) plasmids were used in the molecular biological experiments. In vitro site-directed mutagenesis was performed according to the manufacturers instructions using the QuikChange site-directed mutagenesis kit (Stratagene). Complementary primer pairs were designed coding for the desired mutation together with discriminating silent mutations (introducing or eliminating restriction enzyme sites). The forward primers were: 5'-TCCCAGTGCGAACGTTGGAGTTTCTTCCAC-3' for the TASK-3 D183R mutation; 5'-GTAATCCTCCAGTCTCGGCCCCACCGC-3' for the TASK-3 E70R mutation, and 5'-CTGCGCCTCGAACCGCACAAGGCAGGCGTGC-3' for the TASK-1 K70E mutation. The resulting clones were screened for the newly introduced or eliminated restriction enzyme site and sequenced.

Synthesis of Ion Channel cRNA. The cRNAs coding for TASK-1, TASK-3, and the mutated and/or concatenated 2PK⁺ constructs were synthesized in vitro using the Ambion mMESSAGE mMACHINE T7 in vitro transcription kit. All DNA templates were linearized at the XbaI site of pEXO. For increasing the RNA stability in the oocytes, the linear template contained also the 5' and 3' untranslated regions of the X. laevis globin gene flanking the insert.

Animals and Tissue Preparation and X. laevis Oocyte Injection. Mature female X. laevis frogs were obtained from Amrep Reptielen (Breda, Netherlands). Frogs were anesthetized by immersing them in benzocaine solution (0.03%). Ovarian lobes were removed, the tissue was dissected and treated with collagenase (1.45 mg/ml, 148 U/mg, type I; Worthington Biochemical Corp. (Freehold, NJ) and continuous mechanical agitation in Ca²⁺-free OR2 solution containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 5 mM HEPES, pH 7.5) for 1.5 to 2 h. Stage V and VI oocytes were defolliculated manually, and kept at 18°C in modified Barth's saline containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.82 mM MgSO₄, 0.33 Ca(NO₃)₂, 0.41 mM CaCl₂, 20 mM HEPES buffered to pH 7.5 with NaOH and supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), sodium pyruvate (4.5 mM), and theophyllin (0.5 mM). Oocytes were injected 1 day after defolliculation. Fifty nanoliters of the appropriate cRNA solution was delivered with Nanoliter Injector (World Precision Instruments, Saratosa, Florida). Electrophysiological experiments were performed 3 or 4 days after the injection.

Electrophysiological Recordings. Membrane currents were recorded by two-electrode voltage clamp (OC-725-C; Warner Instrument Corporation, Hamden, CT) using microelectrodes made of borosilicate glass (Clark Electromedical Instruments, Pangbourne, UK) with resistance of 0.3 to 1 M when filled with 3 M KCl. Currents were filtered at 1 kHz, digitally sampled at 1 to 2.5 kHz with a Digidata Interface (Axon Instruments, Union City, CA), and stored on a PC-compatible computer. Recording and data analysis were performed using pClamp software 6.0.4 (Axon Instruments). Experiments were carried out at room temperature, and solutions were applied by a gravity-driven perfusion system. Low [K⁺] solution contained 95.4 mM NaCl, 2 mM KCl, 1.8 CaCl₂, and 5 mM HEPES. High [K⁺] solution contained 80 mM K⁺ (78 mM Na⁺ of the low [K⁺] solution was replaced with K⁺). Unless otherwise stated, the pH of every solution was adjusted to 7.5 with NaOH. Perfusing solutions with pH <6.5 were buffered by including 5 mM MES in addition. RR stock solution (20 mM) was prepared in distilled water, and the inhibitor was diluted further in the perifusing solutions before the measurement. Background K⁺ currents were measured in high-extracellular [K⁺] at the end of 300-ms voltage steps to 100 mV applied in every 3 s. The holding potential was 0 mV. For estimating the amplitude of the background current, the inward current in high [K⁺] was corrected for the small nonspecific leak measured in 2 mM EC [K⁺] at 100 mV.

Statistics and Calculations. Data are expressed as means ± S.E.M. Normalized dose-response curves were fitted by least-squares method (Sigmaplot; SPSS Science, Chicago, IL) to a modified Hill equation of the form: y = /(1 + (c/K_1/2)^n_H) + (1  ), where c is the concentration, K_1/2 is the concentration at which half-maximal inhibition occurs, ^n_H is the Hill coefficient, and  is the fraction inhibited by the treatment. Significance between the pH sensitivity of different TASK-3 constructs was calculated by two-way repeated measures analysis of variance and Scheffé's F test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Mutation of Glutamate 70 to Arginine in TASK-3 Abolishes the Ruthenium Red Sensitivity. TASK-3 K⁺ channel was expressed in X. laevis oocytes, and its currents were measured at 100 mV by two-electrode voltage clamp as the difference of inward currents in high (80 mM) and low (2 mM) [K⁺], as described previously (Czirják et al., 2001). The high level of expression resulted in background potassium currents in the microampere range at 100 mV (9.0 ± 1.8 µA, n = 13), exceeding the endogenous oocyte currents by at least 1 order of magnitude. This allowed reliable estimation of the effects of pharmacological manipulations on TASK-3 currents (Czirják and Enyedi, 2002a,b). Application of RR into the perifusing medium of the oocyte inhibited TASK-3 current in a concentration-dependent manner (Fig. 1.). The half-maximal effect was achieved by 0.7 µM RR. The Hill coefficient of the inhibition was found to be 1.0 (Fig. 2B, ), indicating the binding of one RR molecule to one functional TASK-3 homodimer. The onset of the inhibition was rapid, suggesting an action on the extracellular side, and the inhibition was reversible by extensive washout (not shown). We reported previously that the inhibition of TASK-3 by the positively charged RR was not voltage-dependent (Czirják and Enyedi, 2002a), indicating that the RR binding site on the channel is not in the transmembrane electrical field.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent inhibition of TASK-3 homodimer by ruthenium red. Currents of a representative Xenopus oocyte expressing TASK-3 in extracellular solutions of different K+ and ruthenium red concentrations (as indicated above the curve). The plotted inward currents were measured at the end of 300-ms voltage steps to 100 mV applied every 3 s from a holding potential of 0 mV.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Mutation of glutamate 70 to arginine (E70R) in TASK-3 eliminates the ruthenium red sensitivity. A, position of amino acid residue 70 in the transmembrane topology of TASK-3. B, the effect of ruthenium red on wild-type and E70R mutant TASK-3 homodimeric K+ channels. Inhibition by RR was measured as described in Fig. 1. The currents were corrected for the small nonspecific leak in 2 mM extracellular [K+] at 100 mV and normalized to the value without RR. Each data point represents the average of normalized currents of five to eight oocytes. Error bars are smaller than symbols in most cases. Wild-type TASK-3 points were fitted by a modified Hill equation (see Materials and Methods). E70R points were connected with straight lines.

Binding of RR to Glu 70 in Both Subunits of Homodimeric TASK-3 Is Required for Inhibition. Given the fact that 2PK⁺ channels work as dimers, next we addressed the question of whether Glu 70 at both subunits was necessary for the high-affinity RR binding. If RR required both glutamates at position 70 for interacting with the channel, converting only one of them to a positively charged amino acid should be sufficient to prevent the binding of the inhibitor and consequently render the mutant channel RR insensitive.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Mutation of glutamate 70 to arginine either in the first or in the second linked subunit renders the TASK-3/TASK-3 tandem construct ruthenium red resistant. A, transmembrane topology of the TASK-3/TASK-3 tandem construct. Amino acid residues 70 and 465 (the latter analogous to location 70 in the second TASK-3 repeat) are indicated with arrows. B, concentration-dependent inhibition of the "wild-type" (), E70R (), and E465R mutant (×) TASK-3/TASK-3 constructs by RR. The method of measurement and representation was the same as in Fig. 2B. "Wild-type" data points were fitted by a modified Hill equation; data points of the mutants were connected with straight lines.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   pH-sensitivity of the TASK-3 constructs. Currents of oocytes expressing TASK-3 (), E70R mutant TASK-3 (), the TASK-3/TASK-3 tandem construct (), the E70R mutant TASK-3/TASK-3 tandem (+), and the E465R mutant TASK-3/TASK-3 tandem (×) were measured at different pH values in high (80 mM) extracellular [K+] at 100 mV. The currents were corrected for the small nonspecific leak in 2 mM extracellular [K+] at 100 mV and normalized to the maximum at pH 7.5. Each point represents the average of normalized currents of five to six oocytes. In most cases, error bars are smaller than symbols. Data points are connected with straight lines.

The E70C Mutant of TASK-3 Is Also RR-Insensitive. To test whether the absence of the negatively charged residue in position 70 or the introduction of the positively charged arginine is responsible for the loss of RR-sensitivity, Glu 70 was mutated to cysteine. The E70C mutant also became RR-insensitive (similarly to the E70R mutant), indicating that the presence of negative charges is required for the inhibition (Fig. 5.). To cross-link the two E70C TASK-3 subunits we perfused the oocytes with the sulfhydryl reagent phenylarsine oxide (100 µM for 3 min); however, this treatment failed to affect the channel activity.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Mutation of glutamate 70 to cysteine renders TASK-3 RR-resistant. Effect of ruthenium red on wild-type and E70C mutant TASK-3 homodimeric K+ channels is shown. The protocol and calculations were identical to those of Fig. 2B. However, higher concentrations of ruthenium red (3, 20, and 80 µM) were used for the E70C mutant. For comparison, the data points of wild-type TASK-3 are repeated in this figure.

K70E Is a Gain-of-RR-Sensitivity Mutation in TASK-1. As discussed above, TASK-1, the close relative of TASK-3, is resistant to RR inhibition (Fig. 6). Because TASK-1 has positively charged Lys 70, we replaced this basic residue with the acidic glutamate. The K70E mutant was functional and the amplitude of its expressed current (3.7 ± 0.5 µA, n = 7) was similar to that of wild-type TASK-1 (3.7 ± 0.3 µA, n = 4). However, as opposed to the wild-type channel, RR inhibited the K70E mutant of TASK-1 (Fig. 6.), indicating that Glu 70 is sufficient to create a RR binding site even in the molecular context of TASK-1. Presumably, the amino acid environment surrounding position 70 (being different in TASK-1 and TASK-3) contributes to the formation of the RR binding site, or the distance of amino acids in position 70 are different in TASK-1 and TASK-3, because higher concentrations of RR (half-maximal effect at 10.6 µM, Hill coefficient = 0.6) were required to reduce the currents of the K70E mutant of TASK-1 compared with TASK-3. For excluding the possibility that RR inhibits TASK channels by two distinct inhibitory mechanisms in different concentration ranges, a high concentration of RR (required for TASK-1 K70E inhibition) was also tested on TASK-3 E70R. A high concentration of RR (80 µM) failed to inhibit the E70R mutant of TASK-3 (0.9 ± 1.4% inhibition, n = 4). Thus, RR sensitivity of the K70E TASK-1 mutant together with the abolished RR sensitivity of TASK-3 mutants lacking glutamates in position 70 in one or both subunits indicate the crucial role of this amino acid residue in RR binding and inhibition.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Mutation of lysine 70 to glutamate renders TASK-1 RR-sensitive. Concentration-dependent inhibition of wild-type and K70E mutant TASK-1 homodimeric K+ channels by RR. The method of measurement and representation was the same as in Fig. 2B; however, higher concentrations of RR were applied (0.1, 0.3, 1, 3, 5, 20, and 80 µM). Wild-type TASK-1 points were connected with straight lines; data points of the K70E mutant were fitted by a modified Hill equation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Ruthenium red, the polycationic trinuclear ruthenium ammine, was originally used as a dye for histological staining. It also became well known to interfere with transmembrane Ca²⁺ or Ca²⁺-related ion transports at intracellular organelles and the plasma membrane. It has been shown that application of RR to subcellular fractions or in the cytosol (the large, positively charged molecule cannot penetrate the membrane easily) inhibits the mitochondrial Ca²⁺-uniporter (Moore, 1971), the Ca²⁺-release channel (ryanodine receptor) (Volpe et al., 1986), and the Ca²⁺-ATPase of the sarcoplasmic reticulum (Vale and Carvalho, 1973). RR also inhibits the plasma membrane Ca²⁺-pump (Missiaen et al., 1990), voltage-dependent Ca²⁺ channels (Cibulsky and Sather, 1999), the highly Ca²⁺-permeable nonspecific cation channel vanilloid (capsaicin) receptors (Garcia-Martinez et al., 2000), and members of the recently described epithelial Ca²⁺-selective channel family, ECaCh (Hoenderop et al., 2001). RR binds to and may also affect the function of several Ca²⁺-binding proteins, including calsequestrin, calreticulin, troponin C, and calmodulin (Charuk et al., 1990). With respect to potassium channels, RR was found to inhibit the high conductance Ca²⁺-activated K⁺ channel (maxi, BK) from the intracellular side, and it was assumed that it interacts with the Ca²⁺-binding region of the channel (Wann and Richards, 1994; Wu et al., 1999).

We have reported previously a calcium-independent effect of RR. Extracellular application of the dye inhibited the background K⁺ conductance of rat adrenal glomerulosa cells, in addition to the reduction of L-type voltage-dependent Ca²⁺ current (Szabadkai et al., 1999). Because the glomerulosa background K⁺ conductance has been assigned mainly to the TASK subfamily of the two-pore-domain K⁺ channels (Czirják et al., 2000), it was feasible to examine the effect of RR on different 2P channels. TASK-3 and TRAAK [TWIK-related arachidonic acid-stimulated K⁺ channel (Fink et al., 1998), TREK subfamily] were found to be sensitive to RR, whereas their closely related subfamily members, TASK-1 and TREK-1, respectively, were resistant (Czirják and Enyedi, 2002a). By taking advantage of the RR sensitivity of TASK-3 versus TASK-1, and using also competitive PCR, TASK-3 was found to dominate the background K⁺ conductance of rat adrenal glomerulosa cells (Czirják and Enyedi, 2002b). RR was also used to demonstrate TASK-1/TASK-3 heterodimer formation in Xenopus laevis oocytes (Czirják and Enyedi, 2002a).

In addition to adrenal glomerulosa cells, TASK-1 and TASK-3 are expressed at many other locations and show partially overlapping distribution. TASK-1 and TASK-3 expression (and often also the corresponding pH-sensitive background K⁺ currents) were detected in the central nervous system in cerebellar granule neurons (Millar et al., 2000; Medhurst et al., 2001), somatic motoneurons of the brainstem and spinal cord (Talley et al., 2000), neurons of the locus ceruleus (Bayliss et al., 2001), serotoninergic raphe neurons (Washburn et al., 2002), formatio reticularis, and nuclei of the hypothalamus (Talley et al., 2001). In peripheral tissues, TASK expression and TASK-like currents were demonstrated in type I chemosensitive cells of the rat carotid body (Buckler et al., 2000) and in H-146 cells, an established model of the lung oxygen sensor neuroepithelial bodies (O'Kelly et al., 1999).

TASK currents of native cells were discriminated by their moderately different pH, Zn²⁺, and local anesthetic sensitivity (Bayliss et al., 2001; Hartness et al., 2001; Czirják and Enyedi, 2002b; Washburn et al., 2002). RR seems to be a more powerful tool to facilitate the separation of TASK currents in native cells. In position 70, TASK-1 and TASK-3 subunits possess a positive lysine and a negative glutamate, respectively. Binding of and inhibition by RR requires glutamates to be present in both subunits at this location. Replacing glutamate 70 by a positive amino acid in even one of the subunits extinguishes RR inhibition. This, together with a Hill coefficient of 1.0 for the inhibition, suggest that RR interconnects two glutamates 70 (for schematic model, see Fig. 7.) All of our present and previous experimental data are consistent with this model. As we have reported recently (Czirják and Enyedi, 2002a), TASK-3 homodimeric K⁺ channel (having glutamates 70 in both subunits) is inhibited by RR. On the contrary, the TASK-1 homodimer (lysines 70) and the TASK-1/TASK-3 heterodimer [lysine 70 (K70) and glutamate 70 (E70), respectively] are not affected. In the present study, we demonstrate that the TASK-3/TASK-3 tandem construct (E70, E465) is inhibited by RR, whereas the homodimer of E70R or E70C mutant TASK-3 subunits [having positive arginines 70 (R70) or neutral cysteines (C70), respectively] and the E70R and E465R mutants of the TASK-3/TASK-3 tandem channel (both of them containing one glutamate and one arginine in the key positions) are RR resistant. Furthermore, even the TASK-1 homodimer became RR sensitive when its lysines 70 were replaced by glutamates. The half-maximal inhibitory concentration of RR on the mutated TASK-1 is higher if compared with the wild-type TASK-3, indicating that the environment around the introduced glutamate may also influence the binding. Still, the most plausible interpretation of these observations is that glutamate 70 residues are cornerstones of the RR binding site formed by the channel subunits, and RR binds to both of these glutamates simultaneously. Binding of RR to the outer surface of the channel is consistent with the voltage-independence of the inhibition (Czirják and Enyedi, 2002a). RR does not enter the transmembrane electrical field, because in this case, the positively charged drug would be concentrated around its binding site and induce stronger inhibition at negative membrane potentials. Instead, RR may bind to both glutamates 70 and may repel K⁺ from the outer aperture of the channel by its positive charge, cause steric hindrance or may fix glutamates 70 at a given distance and stabilize the closed conformation.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Model of TASK inhibition by ruthenium red. RR interconnects glutamates 70 (E) located in the first extracellular loop of the channel subunits. The two subunits are drawn in different shades of gray.

RR is far too nonspecific to be used as a TASK-3 inhibitor in vivo. Intraperitoneal administration of RR to mice, rat, or cat caused flaccid paralysis, an effect ascribed to the inhibition of the Ca²⁺-dependent release of acetylcholine in neuromuscular junctions (Tapia and Velasco, 1997). On the other hand, injection of the dye into the cerebrospinal fluid (mice, rat, and cat) induced intense generalized convulsions accompanied by continuous electroencephalographic discharges characteristic of status epilepticus. Stereotaxic microinjection of RR into the substantia nigra pars reticulata produced long-lasting intense circling movements and head orientation, whereas intrahippocampal microinjection resulted in a well-defined motor abnormality known as "wet-dog shakes". Initially, these abnormal motor activities were explained by inhibition of the release of inhibitory neurotransmitters and by the neurotoxic effect of RR (Tapia and Velasco, 1997). Later, susceptibility of different neuron populations to RR was related to the inhibition of K⁺ currents. Supporting this assumption, the membrane potential of X. laevis oocytes injected with mRNA extracted from cultured cortical and cerebellar granule neurons was depolarized by RR (Velasco et al., 1999), but the molecular target of the drug remained unknown. We propose that the inhibitory effect of RR on the neuronal background K⁺ channels, TASK-3 and TRAAK, contributes to the neuronal excitation and the consequent motor activity induced by intracerebral or intracerebroventricular injection of RR.

Development of specific two-pore-domain K⁺ channel inhibitors would highly facilitate the evaluation of the functional significance of these background K⁺ channels in native tissues and in vivo. In turn, specific two-pore-domain channel agonists might also be clinically useful. Accumulating evidence indicates that activation of 2PK⁺ channels (mainly members of the TASK and TREK subfamilies) by volatile anesthetics contributes to the general central depressive effect of these compounds (Patel et al., 1999; Sirois et al., 2000; Talley and Bayliss, 2002). Regarding the distinct expression pattern of different 2PK⁺ channels in the central nervous system, selective 2PK⁺ channel agonists might have more targeted clinical effects (e.g., hypnotic, analgesic, immobilizing, or neuroprotective) than those of volatile anesthetics. Within the closely related members of the TASK 2PK⁺ channel family, a single amino acid in position 70 profoundly modified the susceptibility of the channel to RR, suggesting that this site may be a possible target for designing selective TASK antagonists of higher overall specificity.