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Cardiovascular Research 2003 58(1):46-54; doi:10.1016/S0008-6363(02)00831-3
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Copyright © 2003, European Society of Cardiology

Drug- and mutagenesis-induced changes in the selectivity filter of a cardiac two-pore background K+ channel

Péter Hajdúa, Chris Ulensb, György Panyic and Jan Tytgatb,*

aCell Biophysics Research Group of the Hungarian Academy of Sciences, Department of Biophysics and Cell Biology, University of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, Hungary
bLaboratory of Toxicology, Faculty of Pharmaceutical Sciences, Catholic University of Leuven, E. Van Evenstraat 4, B-3000 Leuven, Belgium chris.ulens{at}farm.kuleuven.ac.be jan.tytgat{at}farm.kuleuven.ac.be
cDepartment of Biophysics and Cell Biology, University of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, Hungary panyi{at}jaguar.dote.hu

* Corresponding author. Tel.: +32-16-323-403; fax: +32-16-323-405. hajdup{at}jaguar.dote.hu

Received 9 August 2002; accepted 25 November 2002


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: As compared with voltage-gated K+ channels (Kv-type), our knowledge of the structure-function and pharmacology of two-pore background K+ channels is still very limited. Here we have used a drug- and mutagenesis-based approach to study the effect of the antidepressant fluoxetine (FL) and analgesic D-norpropoxyphene (NORP) on the cardiac two-pore background K+ channel. Methods: Whole-cell currents of the cTBAK-1 channel expressed in Xenopus laevis oocytes were investigated using conventional two-microelectrode voltage-clamp recording method combined with functional mutagenesis of the channel protein. Results: Both drugs inhibit cTBAK-1 current: FL proved to be a voltage-dependent pore-blocker, while NORP induced a change in the selectivity of cTBAK-1 giving rise to a shift in the reversal potential (Erev) toward more positive voltages due to an increased Na+ permeability. Mutations were introduced into the selectivity filter of the first (Y105F) and the second (F211Y) pore to mimic the P-region of HERG (GFGN) and Kv1.1 (GYGD) channels. Point mutations in the channel resulted in two distinct phenotypes of cTBAK-1: the mutant Y105F channel lost its selectivity and was unaffected by NORP, in contrast to the F211Y mutant. Conclusion: FL and NORP block the current of cTBAK-1 channels differently, the latter modified the selectivity of the channel pore. Our mutagenesis study revealed that NORP interacts with the selectivity filter of cTBAK-1. The significant role of the GYGD motif in this type of K+ channels is emphasized.

KEYWORDS Ion channels; K-channel; Membrane currents; Membrane permeability/physics; Myocytes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Potassium channels, pore-forming integral proteins, are present in all cells of living organisms [1]. They are involved in numerous cellular processes such as control of resting membrane potential, firing of neurons, contraction of muscles, volume regulation, and immunosuppression. All K+ channels that have been cloned so far contain at least one conservative signature referred to as the pore (P) region which lines the ion conducting pore and critically determines K+ selectivity [2,3]. Point mutations in the putative selectivity filter (GYGD motif) influenced the selectivity of K+ channels and these results also suggested an interaction between the neighboring subunits of the channel to form a functional filter [4–7].

Recently a new, expanding family of K+ channels with two pore (2-P) domains, four transmembrane segments, an extended extracellular loop and intracellular N- and C-termini has been identified in various species (reviewed in Refs. [8,9]). They can be found in excitable or in non-excitable cells such as pancreatic cells and neurons [10]. The cloned members of this family are not voltage-gated, are non-inactivating (except TWIK-2 with slow inactivation kinetics [11]), insensitive to conventional potassium channel inhibitors (4-AP, TEA, peptide blockers) and likely to play a role in setting the membrane potential as a leak K+ conductance. 2-P channels share very low amino-acid sequence homology (25–40%) among each other and show unusual basic electrophysiological properties: there are inward-, Goldman-Hodgkin-Katz- (GHK or open) and outward rectifiers [12–14]. With respect to the pharmacology acid-, stretch- and PUFA-sensitive (polyunsaturated fatty acid) channels can be distinguished. Various anesthetics, neuromodulators and protective agents (bupivacaine, halothane, riluzole, etc.) can inhibit or, on the contrary, activate these channels [12,13,15–25]. Recently, the neuroprotective sipatrigine has been shown to be a very potent and specific blocker of human TREK-1 and TRAAK-1 [15]. It was also demonstrated that a single point mutation in TWIK-1 at residue 69 (C69S) prevented homodimerization of the subunits [26]. Furthermore, the forming of heterodimers of single subunits from different subfamilies was also verified [27].

Screening a mouse heart cDNA library a new type of 2-P K+ channels was isolated by Kim et al.: the cTBAK-1 (cardiac Two-pore Background K+ channel) [28]. cTBAK-1 as well as mouse TASK-1 are extremely acid-sensitive in the narrow physiological range and both are GHK-rectifying K+ channels. TASK-1 contains nine additional amino acids in the N-terminus (reviewed in Refs. [8,20,29,30]). This small ‘extra’ chain of amino acid residues at the N-terminus does not result in a significant difference between the two channel types: their intrinsic gating mechanism is very similar [30]. While TASK-1 can be blocked specifically by anadamide in the submicromolar range, no such selective inhibitor exists for cTBAK-1 [31].

In the present study we investigated two inhibitors of cTBAK-1: D-norpropoxyphene (NORP) and fluoxetine (FL) (Fig. 2A and B). It was shown earlier that NORP, a phase I metabolite of propoxyphene, affected the ion-selectivity and gating of HERG channels. This effect was absent when voltage gated Kv1.1 channels were studied [32]. FL is an anti-depressant and blocks the serotonin reuptake in the central nervous system [33]. Moreover, FL inhibits various ion channels in a non-specific manner [34–38]. Xenopus laevis oocytes, as a heterologous expression system, were used to express in vitro synthesized wild-type (WT) and mutant cTBAK-1 encoding cRNA and by means of the conventional two-microelectrode voltage-clamp (TEVC) technique currents through the 2-P channels were measured.


Figure 2
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  		Fig. 2 NORP and FL affect the current of the cTBAK-1. (A and B) Structures of D-norpropoxyphene and fluoxetine, respectively. (C) Representative current traces evoked upon a voltage-ramp in ND-96 (solid line) and in the presence of 250 µM NORP in ND-96 (dashed line). The inset shows the current values at +50 mV during repetitive voltage ramps, arrows indicate the start of application of drug (250 µM) and washout with ND-96, respectively. (D) Extracellular application of FL inhibited the K+ currents. Traces show a current–voltage relationship in ND-96 solution (solid line) and in the presence of 50 µM of FL dissolved in ND-96 (dashed line). The inset shows the current values at +50 mV during repetitive voltage ramps, arrows indicate the start of application of drug (50 µM) and washout, respectively. (E) Extracellular application of NORP proved to be dose-dependent. Normalized shift in the reversal potential ({Delta}Erel, bullet) and the remaining fraction (RF, {blacksquare}) of the current are shown as a function of NORP concentration (RF = INORP/IND-96 where INORP and IND-96 are the currents measured at +50 mV in the presence and absence of NORP, respectively). Superimposed lines show the best fits using the appropriate Hill equations (see text for the equations and the fitted parameters). (F) Normalized shift in the reversal potential ({Delta}Erel, bullet) and the remaining fraction (RF, {blacksquare}) of the current are shown as a function of FL concentration. Superimposed lines show the best fits using the appropriate equations (see text for the equations and the fitted parameters).

 

   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Mutation of the cTBAK-1 clone and expression
First the mouse cTBAK-1 cDNA (acc. # AB008537.1) was subcloned into a pGEMHE vector from its original pBLUEscript (Stratagene, USA) vector [39]. The mutations Y105F and F211Y were introduced into the pGEMHE/cTBAK-1 with the application of Quickchange site directed mutagenesis kit (Stratagene, USA). The following primers for mutating the first (position 105) and the second pore (position 211) were used: 5'-CACCACCATCGGCTTTGGCCATGCGGCGC-3' and 5'-CACCACCATCGGCTACGGAGACTATGTGG-3', respectively. PCR conditions were set according to the protocols issued by the manufacturer. The results were analyzed by enzyme restriction digestion with Alw26 I (Y105F) and Bal I (F211Y) as a consequence of the appearance of the silent mutation sites. For in vitro transcription of pGEMHE/cTBAK-1 (both wild-type and mutants), expression vectors were linearized with Nhe I according to standard protocols, and using the large-scale T7 mMessage mMachine transcription kit (Ambion, USA), capped cRNAs were synthesized from the linearized plasmids. Oocytes were injected with 50 nl of cRNA at a concentration of 0.1 ng/nl.

2.2 Electrophysiology and data analysis
Isolation of X. laevis oocytes was performed as previously described [39]. Procedures for the animal experiments were approved by the Ethical Commission for Animal Experiments of the University of Leuven. Whole-cell recordings in oocytes were carried out 2–4 days post-injection with the TEVC technique (GeneClamp 500, Axon Instruments, USA). The resistances of the electrodes were kept as low as possible (approximately 200 k{Omega}–1 M{Omega}). Until the measurements the oocytes were incubated and held in ND-96 (composition in mM: KCl 2, NaCl 96, MgCl2 1, CaCl2 1.8, Hepes 5, pH 7.4) supplemented with 50 mg/l gentamicin sulfate at room temperature of 18–20°C. For electrophysiological measurements, the following solutions were used: ND-96 lacking gentamicin, high-potassium (HK) solution (composition in mM: KCl 96, NaCl 2, MgCl2 1, CaCl2 1.8, Hepes 5, pH 7.4) and HK solution containing 300 µM BaCl2 (Ba2+ solution). The Na+-free solution was achieved by substitution of Na+ for Tris (Tris solution, composition in mM: KCl 2, Tris–Cl 96, MgCl2 1, CaCl2 1.8, Hepes 5, pH 7.4). The bath solutions were exchanged by a gravity driven perfusion system, and the electrodes were filled with 3 M KCl. The sampling rate was 250 Hz and the currents were filtered at 100 Hz with a four-pole low-pass Bessel-filter in accordance with the Nyquist theorem. The pCLAMP 5.0 program was used for data acquisition and Clampfit 5.0 and 8.0 to analyze the curves obtained off-line. Fits were evaluated visually, as well as by the residuals and the sum of squared differences between the measured and calculated data points.

Statistical comparisons were made using the t-test and where applicable, one-way ANOVA. Dunett's and Student-Newman-Keul's methods were used for multiple comparisons versus control and all pairwise multiple comparisons, respectively. The Pearson product moment correlation was calculated during correlation analysis. For all experiments, the mean and the standard error of the mean (S.E.M.) are reported. Statistical analysis was performed using SPSS software (SPSS, Chicago, IL, USA). The reported errors of the fitted parameters are the standard errors of the parameter estimates in the fitting procedure (Marquard-Levemberg algorithm).

2.3 Compounds
Both D-norpropoxyphene maleate (Sigma, USA) and fluoxetine HCl (ProzacTM, USA) were dissolved in ND-96 or Tris solution. Stock solutions were stored at 4°C until measurements and extracellularly applied at appropriate concentrations.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 cTBAK-1 current in X. laevis oocytes
Fig. 1 shows current traces recorded under different ionic conditions from an oocyte injected with cRNA encoding cTBAK channels. Throughout the electrophysiological experiments in this study oocytes were clamped at 0 mV and 1-s long voltage ramps ranging from –150 to +50 mV were delivered every 10 s. The traces in Fig. 1 demonstrate that in ND-96 solution the current shows outward rectification that diminishes in HK solution. The Ba2+ solution blocks only slightly the current in contrast to Kir channels [40]. These observations confirm the expression of a K+ selective, open rectifying, background channel identical to cTBAK-1 [28].


Figure 1
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  		Fig. 1 Basic biophysical properties of cTBAK-1 channels in Xenopus oocytes studied with TEVC. The cells injected with cTBAK-1 cRNA were clamped at 0 mV and a voltage-ramp pulse ranging from –150 to +50 mV with 1-s duration was delivered every 10 s. The traces were obtained in ND-96 (solid line), in HK solution (dotted line) and in Ba2+ solution (dashed line).

 
3.2 NORP but not FL induces shift in the reversal potential of the current
Fig. 2C illustrates cTBAK-1 current traces obtained in ND-96 in the presence and absence of 250 µM NORP, and current values at +50 mV for the same cell were plotted against time to illustrate the wash-in and wash-out kinetics of NORP (inset in the graph). NORP exerted its effect in a complex manner: it shifted the current–voltage relationship toward depolarizing voltages, inhibited the outward current component and potentiated the inward current. The NORP-induced change in the reversal potential ({Delta}Erev) showed significant correlation with the reversal potential in ND-96 (r = –0.972, P = 0.00572). Thus, to express unambiguously the effect of NORP and eliminate cell-by-cell variances due to different expression levels we normalized ({Delta}Erev) as follows:

Formula
where ENORP and END-96 are the Erev in the presence and absence of NORP, respectively. Fig. 2E shows {Delta}Erel as a function of the NORP dose. The Hill equation Formula was fitted to the data points yielding an EC50-value of 122.1±29 µM, a maximum change in {Delta}Erel ({Delta}Erel,ND-96,max) of 0.36±0.03, and a Hill coefficient (n) of –1.45±0.23 [23]. Since the inhibition of cTBAK-1 currents by NORP was partial, the dose–response data for the remaining fraction (RF) of the current at different NORP concentrations could be well fitted using a Hill equation with non-zero offset value: Formula The fitted parameters were 167.8±42 µM for IC50, 0.51±0.09 for the maximum blocked fraction (B), 0.49±0.1 for the unblocked fraction (UB), and 1.1±0.3 for the Hill coefficient (n) (n≥4).

Fig. 2D shows representative cTBAK-1 current traces recorded in ND-96 from a cell in the absence and presence of 50 µM FL. FL inhibited primarily the outward K+ current component: block by FL proved to be voltage-dependent. The blockade of FL could be partially eliminated by washing with drug-free solution for several minutes, like for NORP (inset in Fig. 2D). The dose–response curve obtained for various FL concentrations could be described with a Hill equation and an IC50 value of 226.5±20.5 µM, and a Hill coefficient of 1.2±0.15 (n≥3; Fig. 2F). Interestingly and in contrast to NORP, FL did not alter Erev. A straight line was obtained for the {Delta}Erel–[FL] dose–response curve with a slope of 2x10–5 µM–1 and intercept of 2.9x10–2.

3.3 Effect of NORP is reduced in the absence of Na+
A possible mechanism that might be responsible for the enhanced inward current and the altered Erev in the presence of NORP (Fig. 2C) is an enhanced flux of Na+ ions through cTBAK-1 channels. To clarify the role of Na+ ions in this process we substituted Na+ with Tris which is impermeable to the membrane and the cTBAK-1 channels.

The cTBAK-1 current traces shown in Fig. 3A were recorded in Tris solution in the absence (solid line) and presence (dashed line) of 250 µM NORP. The effect of NORP on the reversal potential did not disappear completely but was diminished significantly compared to the data obtained in ND-96. The fitted parameters of the dose–response curve for NORP induced {Delta}Erel were {Delta}Erel,TRIS,max=0.083±0.001 and EC50,TRIS=61±1.4 µM (Fig. 3B). The block of cTBAK-1 currents by NORP at +50 mV could also be fitted with a Hill equation with a non-zero offset value yielding an IC50-value of 38.4±5.3 µM, n of 1.46±0.2, UB of 0.87±0.1 and B of 0.13±0.05 (n≥4).


Figure 3
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  		Fig. 3 Effect of NORP in sodium-free solution. (A) The cells expressing cTBAK-1 channels were clamped at 0 mV and a voltage-ramp pulse ranging from –150 to +50 mV with 1-s duration was delivered every 10 s. Traces were recorded in Tris solution (solid line) and Tris solution containing 250 µM NORP (dashed line). (B) Normalized shift in the reversal potential ({Delta}Erel, bullet) and the remaining fraction (RF, {blacksquare}) of the current are shown as a function of NORP concentration (RF = INORP,TRIS/ITRIS, where INORP,TRIS and ITRIS are the currents measured at +50 mV in the presence and absence of NORP in Tris solution, respectively). Superimposed lines show the best fits using the appropriate Hill equations (see text for the equations and the fitted parameters).

 
3.4 NORP interacts with the selectivity filter
In the majority of K+ channels, the P-region contains a signature sequence whose essential part is the GYGD motif that is regarded as the selectivity filter [2]. Ulens et al. showed for HERG channels with a pore motif GFGN that NORP was effective in the same manner as demonstrated here for cTBAK-1. Interestingly, Kv1.1, a member of Shaker-type voltage-gated K+ channels having a selectivity region of GYGD, was not affected by NORP [32]. In the cTBAK-1 channel the selectivity motifs of the first and second pore are GYGH and GFGD, respectively [28]. To study the interaction of NORP with cTBAK-1 channels, we introduced mutations into the selectivity filter of the first (position 105, Y105F) and the second P-region (position 211, F211Y) to mimic the pore arrangement of the HERG and Kv1.1 channels, respectively (Fig. 4).


Figure 4
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  		Fig. 4 Amino acid sequence of the pore of wild-type (WT) and mutated channels. The sequences of the two pore regions are shown; GYGH and GFGD selectivity motifs are emphasized with boxes. Note the position of Y105F in the first pore, and F211Y in the second pore (capitals are underlined).

 
In Fig. 5 current traces of wild-type (WT, panel A) and mutant channels (Y105F, panel B and F211Y, panel C) are demonstrated in the presence and absence of 250 µM NORP in ND-96. Panel B of Fig. 5 illustrates that Y105F channels have lost outward rectification in ND-96, and that Erev was moved toward depolarized voltages with respect to the WT channels (even without NORP). NORP did not affect significantly either the reversal potential or the amplitude of the inward and outward components of the current (Table 1A).


Figure 5
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  		Fig. 5 Effect of NORP on the current of mutant c-TBAK-1 channels. Oocytes expressing WT and different mutant cTBAK-1 channels were clamped at 0 mV and a voltage-ramp pulse ranging from –150 to +50 mV with 1-s duration was delivered every 10 s. Solid lines represent recordings in ND-96 whereas dashed lines show traces obtained in the presence of 250 µM NORP in ND-96 from an oocyte expressing (A) WT cTBAK-1, (B) Y105F mutant (mutation in the selectivity filter of the first pore motif) and (C) F211Y mutant current (mutation in the selectivity filter of the second pore motif).

 

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  		Table 1 The effect of 250 µM NORP on WT and two new phenotypes of cTBAK-1 (A) and basic properties of WT and mutant channels (B)

 
Panel C of Fig. 5 shows the effect of NORP on the F211Y mutant channels. Comparison of panels A and C shows that both the change in Erev and reduction of the current (RF) induced by the application of 250 µM NORP are very small compared to the data obtained for WT channels (Table 1A). The statistical comparison of {Delta}Erel and RF at +50 mV showed that the effect of NORP was significantly smaller for the F211Y mutant compared to the effect on WT channels (P<0.001). The drastic reduction in {Delta}Erev means that NORP exerts its selectivity-modifying effect through an interaction with the residue in position Y in the GYGD motif.

3.5 Y105F mutation altered K+ selectivity
To reveal the influence of the mutations on the channel function we determined the basic electrophysiological properties of all channel phenotypes using ion substitution. The cells expressing various channels were perfused with ND-96, HK and Tris solutions, and the difference in Erev or Erev itself was used to compare the modified selectivity of each phenotype, especially the Y105F mutant (see legend of Table 1B).

The Erev-values measured in ND-96 (Erev,ND-96) were analyzed for all channel phenotypes (Table 1B). The rightward shift in Erev,ND-96 for Y105F channels (Fig. 5B) already suggested that the Y105F channel became non-selective for K+ ions. The decreased sensitivity of {Delta}Erev to the application of HK solution ({Delta}Erev,HK; Table 1B) and increased sensitivity of {Delta}Erev to the application of Tris solution ({Delta}Erev,TRIS; Table 1B) for the Y105F channels confirmed that this mutation ruptured the selectivity filter. In clear contrast, the statistical comparison showed no significant difference between the results obtained for {Delta}Erev,HK for WT and F211Y channels (P = 0.248) indicating the existence of an intact selectivity filter in F211Y channels.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In this paper we demonstrate that FL and NORP modified the current of mouse cTBAK-1 channels in X. laevis oocytes. Utilizing mutagenesis experiments we proved that modification of certain amino acid residues in the selectivity filter of cTBAK-1 resulted in phenotypes possessing distinct pharmacological and basic properties. The high concentration of both drugs used by us may raise the question of unspecific modification of cTBAK-1. However, the fact that the NORP- and FL-induced phenotypes are significantly different, points to a drug-specific interaction with cTBAK-1 channels.

Several studies on the effect of FL confirmed that it is a potent inhibitor of various channels present in native cells or expressed in heterologous expression systems [34–38]. We investigated the block of FL in ND-96 on the cTBAK-1 currents and we found that (i) both outward and inward currents were inhibited but not to the same degree, (ii) the washout of the compound was not complete, and most interestingly, (iii) the current–voltage relation curve was not shifted (Fig. 2D and F). These results coincide with previous published data that FL is a conventional, voltage-dependent pore blocker without effect on the selectivity of channels. The irreversibility of block may suggest that FL diffuses into the cells and binds to the intracellular vestibule of the channel pore [34]. This phenomenon may explain the apparent voltage dependence of the block: the inward stream of K+ ions prevents the occlusion of the conducting pore, consequently a higher RF-value can be detected. In our series of experiments, FL was very useful to demonstrate the specific effect of NORP, i.e. modification of the selectivity of cTBAK-1.

The effect of NORP on the action potential and ionic currents was demonstrated earlier in cardiac and neuronal cells in mammals (canine Purkinje fibers, GH3 cells) and confirmed that besides the Na+ current, the K+ current is also blocked upon exposure to NORP [41,42]. Recently Ulens et al. have reported that NORP modified selectivity and gating of HERG, but not of Kv1.1 channels in Xenopus oocytes [32]. Since the selectivity filter of a cTBAK-1 channel contains two GYGH and two GFGD sequence motifs, we anticipated observing a qualitatively similar effect as for HERG. We characterized the effect of NORP on cTBAK-1 currents with the relative shift in the Erev and the RF: both parameters were concentration-dependent and could be characterized with EC50- and IC50-values of 122.1 and of 167.8 µM, respectively. The incomplete inhibition of the outward current component could foretell an altered channel ‘gating’. Accordingly, we characterized a NORP-induced change in selectivity of cTBAK-1 besides a possible inhibitory effect. The shift in Erev in the presence of 250 µM NORP was approximately half that for HERG channels, mimicking the 50:50 situation of the GYGH and GFGD motifs in the pore. The ion-substitution experiments revealed that NORP made the channel more permeable to Na+; the presence of a small {Delta}Erev suggests that the selectivity of cTBAK-1 channel for K+ ions may reduce. The dose-dependency of NORP was also determined in Tris solution for both the change in Erev and reduction in current, yielding EC50 values of 61 µM and IC50 of 38.4 µM, respectively. The apparent higher affinity might be explained by the fact that the inward flow of Na+ ions in ND-96 destabilizes the channel–drug complex. Taken together, we believe that NORP acts as selectivity modifier on cTBAK-1, whereby the channel becomes fairly unselective given the rise in the permeability of Na+ over K+.

To reveal the molecular determinants of the altered permeability upon NORP application, we introduced two point mutations into the selectivity filter of cTBAK-1: Y105F mutant mimics the pore motif of the HERG channel; the F211Y follows the Kv1.1 selectivity sequence. The reason for choosing these mutants originates from the functional interaction between selectivity filter sequences of neighboring K+ channel subunits as was shown for some K+ channels: the Y–D interaction in the GYGD motifs of the pore region is very likely to be responsible for the high selectivity of the channel [4–7]. The presence of other residues in the position of Y in one of the domains is likely to reduce the permeability of K+ over Na+, while the appropriate amino acids in the position of D residue can restore the modification or loss of K+ selectivity, maintain rectification properties of current and single channel conductance, and exclude the permeation of larger monovalent cations.

Hence, the absence of outward rectification of our Y105F channels, the more positive Erev and the lower shift in HK solution may all indicate that the stringent K+ selectivity of the pore vanished. Furthermore, experiments in Na+-free solution provide evidence that Na+ can permeate through the channel (Fig. 4B and Table 1B). Based on the results summarized, we are convinced that the mutation Y105F resulted in a tetrameric construct that was unable to maintain a K+-selective channel. NORP is incapable of changing Erev since the mutation disrupted the ion-selectivity filter, thereby increasing Na+ permeability. Hence, the binding of the drug to the channel could not cause a further increase in Erev.

We have to address the question whether the Y to F mutation in the first pore resulted in a non-selective cTBAK-1 channel, despite the fact that the same change of the corresponding residue in KAT1 did not do so [6]. Based on previous experiments with mutant channels, and by comparing the selectivity filters of the channels cloned so far, we hypothesize that position D in the sequence GYGD plays a crucial role in the formation of a K+ selective pore: the presence of a neutral or negatively charged residue (e.g. D,N,T), provided also that there is an F instead of Y two residues upstream, seems to be a prerequisite for K+ selectivity [4–7]. In our mutant Y105F, a histidine resides in the place of D, that is likely to rearrange the amino acid side chains of the selectivity filter and the outer vestibule, hence modifying the high affinity K+ binding sites.

In contrast, the mutation F211Y resulted in a channel that showed similar electrophysiological properties like the WT (Fig. 4 and Table 1B). This Y–F change, which means only an OH group difference, does not have an effect on the permeability properties of the channel. This observation nicely fits with the lack of the effect of NORP on Kv1.1, which also is equipped with the signature sequence GYGD. We think that the small NORP induced shift of the voltage–current curve is because of the influence of other amino acid residues that are not necessarily in the signature sequence motif.

In conclusion, we have taken advantage of the specific manner of action of two drugs used in human medicine, to produce different phenotypes of the cloned two-pore cTBAK-1 K+ channel expressed in Xenopus oocytes. To date, and to our knowledge, no drugs have been reported that could modify the selectivity of this emerging family of leak K+ channels. Based on a pharmacological approach on the one hand and on a mutagenesis study on the other, this work also highlights the significance of the crucially important GYGD sequence in this family of 2-P channels.

Time for primary review 28 days.


   Acknowledgements
 
We thank Yoshihisa Kurachi (Osaka University, Osaka, Japan) for kindly providing the mouse cTBAK-1 clone. We are grateful to Joost Pil and Isabelle Huys for their help in molecular biology. This work was partly supported by grants FKFP 622/2000, OTKA F035251 and ETT 010/2001 to G.P.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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