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Journal of Physiology (2002), 540.1, pp. 29-38
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013234
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ABSTRACT |
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Lyotropic pseudohalide anions are potentially useful as high affinity probes of Cl- channel pores. However, the interaction between these pseudohalides and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel have not been described in detail. Here we show that Au(CN)2- ions applied to the intracellular face of membrane patches from stably transfected baby hamster kidney cells inhibit CFTR channel currents by at least two mechanisms, which can be distinguished at the single channel level or by inhibiting channel closure using 2 mM pyrophosphate. Low concentrations (< 10 µM) of Au(CN)2- significantly reduced CFTR channel open probability. This effect was apparently voltage insensitive, independent of extracellular Cl- concentration, and lost following exposure to pyrophosphate. Higher concentrations of intracellular Au(CN)2- caused an apparent reduction in unitary current amplitude, presumably due to a kinetically fast blocking reaction. This effect, isolated following exposure to pyrophosphate, was strongly voltage dependent (apparent Kd 61.6 µM at -100 mV and 913 µM at +60 mV). Both the affinity and voltage dependence of block were highly sensitive to extracellular Cl- concentration. We propose that Au(CN)2- has at least two inhibitory effects on CFTR currents: a high affinity effect on channel gating due to action on a cytoplasmically accessible aspect of the channel and a lower affinity block within the open channel pore. These results offer important caveats for the use of lyotropic pseudohalide anions such as Au(CN)2- as specific high affinity probes of Cl- channel pores.
(Received 4 September 2001; accepted after revision 10 January 2002)
Corresponding author P. Linsdell: Department of Physiology & Biophysics, Dalhousie University, Sir Charles Tupper Medical Building, Halifax, Nova Scotia, Canada B3H 4H7. Email: paul.linsdell{at}dal.ca
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INTRODUCTION |
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Studies of Cl- channel pore structure and function have been hampered by the lack of the kind of high-affinity toxin probes which target many classes of cation channels. In contrast, a wealth of different anions are usually permeant in Cl- channels and as a result permeant ions have been used extensively as probes of Cl- channel pores (e.g. Bormann et al. 1987; Halm & Frizzell, 1992; Rychkov et al. 1998; Smith et al. 1999; Qu & Hartzell, 2000; Linsdell, 2001a). Previously we have used permeant anions to identify regions contributing to anion selectivity (Linsdell et al. 1997b, 1998, 2000) and to intrapore anion binding sites (Gupta et al. 2001; Linsdell 2001a) in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel pore.
Permeant ions which bind within the pore with high affinity have been very useful in elucidating the permeation mechanism in cation channels. For example, Ba2+ ions have been used as high affinity probes of ion binding sites within K+ channel pores, both in functional (electrophysiological) studies (Neyton & Miller, 1988a,b; Hurst et al. 1996; Harris et al. 1998; Ogielska & Aldrich, 1998; Vergara et al. 1999) and in structural (X-ray crystallography) studies (Jiang & MacKinnon, 2000). The close agreement between the conclusions drawn from these two approaches (Jiang & MacKinnon, 2000) offers encouragement that functional studies of ion channel permeation are not yet outdated, particularly in cases where direct structural data is lacking.
Recently, pseudohalides have been described as novel high affinity permeant anions in CFTR (Smith et al. 1999) and Ca2+-activated Cl- channels (Qu & Hartzell, 2000). These small anions (N(CN)2-, SeCN-, Au(CN)2-, C(CN)3-) were described as showing both high permeability and high intrapore binding affinity (as judged by their ability to block Cl- permeation) in CFTR (Smith et al. 1999) and as such may prove useful in clarifying both the molecular determinants of anion binding within the pore (Gong et al. 2002) and the relationship between binding and permeability (Smith et al. 1999; Linsdell, 2001a; Gong et al. 2002). Furthermore, the high electron density conferred by the gold atom in Au(CN)2- makes it an ideal candidate for direct structural studies of Cl- channel pores, studies which are currently in their infancy (Mindell et al. 2001). However, the precise mechanism of action of Au(CN)2- and other pseudohalide anions on wild-type CFTR has not been fully examined.
In the present study we offer evidence that Au(CN)2- has multiple inhibitory effects on CFTR Cl- currents, some of which may reflect actions outside the channel pore. These effects offer some caveats for the future use of pseudohalides as functional and structural probes of Cl- channels.
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METHODS |
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Excised, inside-out patch clamp recordings were carried out on baby hamster kidney (BHK) cells stably transfected with human CFTR in the pNUT vector (Linsdell & Hanrahan, 1996; Chang et al. 1998), kindly provided by Dr John Hanrahan (McGill University, Montréal, PQ, Canada). Cells were grown at 37 °C in 5 % CO2 in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12, supplemented with 5 % fetal bovine serum, 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 0.25 µg ml-1 fungizone and 500 µM methotrexate (all from Life Technologies, Burlington, ON, Canada, except for methotrexate: Faulding, Vaudreuil, PQ, Canada). For patch clamp recording, cells were seeded onto 22 mm glass coverslips and used after 2-4 days.
For inside-out patch recordings, CFTR channels were activated following patch excision by exposure to 30-140 nM protein kinase A catalytic subunit (PKA; prepared in the laboratory of Dr M. P. Walsh, University of Calgary, AB, Canada; Hanrahan et al. 1998) plus 1 mM MgATP, as described previously (Linsdell & Hanrahan, 1996, 1998; Hanrahan et al. 1998). Both pipette (extracellular) and bath (intracellular) solutions contained (mM): 150 NaCl, 2 MgCl2, 10 Tes, except in Fig. 5, where extracellular NaCl was replaced by 150 mM sodium gluconate. Solutions were adjusted to pH 7.4 using NaOH. Potassium dicyanoaurate (KAu(CN)2) was added to the bath solution as required from a stock solution made up in normal patch clamp buffer. Unless stated otherwise, all chemicals were from Sigma-Aldrich (Oakville, ON, Canada), except KAu(CN)2 (Strem Chemicals, Newburyport, MA, USA). Pipette resistances were 1-2 M
for recording macroscopic currents from inside-out patches, and 3-6 M for single channel recordings. Currents were filtered at 50 Hz (single channel) or 100 Hz (macroscopic current) using an 8-pole Bessel filter (Frequency Devices, Haverhill, MA, USA or Warner Instruments, Hamden, CT, USA), amplified using an Axopatch 1D or 200B amplifier (Axon Instruments, Union City, CA, USA), digitised at 250 Hz using a DigiData 1200 or 1322A interface (Axon Instruments) and analysed using pCLAMP6 or pCLAMP8 (Axon Instruments) or DRSCAN (Hanrahan et al. 1998) software. Data fitting was carried out using SigmaPlot version 5.0 (SPSS, Chicago, IL, USA) or Excel (Microsoft, Redmond, WA, USA) software.
Macroscopic current-voltage (I-V) relationships in inside-out patches were constructed using depolarising ramp protocols with a rate of change of voltage of 100 mV s-1 (Linsdell & Hanrahan, 1996, 1998). Background (leak) currents recorded before the addition of PKA were subtracted digitally, leaving uncontaminated CFTR currents (Linsdell & Hanrahan, 1996, 1998; Hanrahan et al. 1998). Blocker concentration-inhibition relationships were commonly fitted by a Hill equation of the form:
Fractional unblocked current = 1/(1 + (B/Kd)nH), (1)
where B is the blocker concentration, Kd the apparent blocker dissociation constant, and nH the slope factor or Hill coefficient.
During single channel recordings, relative channel activity before and after treatment was compared by measuring the number of channels (N) multiplied by open probability (PO), estimated using a 50 % threshold crossing method in DRSCAN software, as described previously (Hanrahan et al. 1998).
All recordings were carried out at room temperature, 21-24 °C. Mean values are given ± S.E.M. For graphical presentation of mean values, error bars represent ± S.E.M., where this is larger than the size of the symbol.
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RESULTS |
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Effects of intracellular Au(CN)2-
Previously we have characterised the effects of a number of different CFTR channel blockers by applying them to the intracellular face of inside-out patches excised from BHK cells (Linsdell & Hanrahan, 1996, 1999; Gupta et al. 2001; Linsdell, 2001b). This protocol offers the advantage that CFTR channel activity, which is controlled by intracellular factors (Gadsby & Nairn, 1999) can easily be manipulated. We therefore wished to examine the effects of intracellular Au(CN)2- under similar conditions. Previously, only extracellular effects of the pseudohalides have been reported (Smith et al. 1999).
As shown in Fig. 1A, addition of low concentrations of Au(CN)2- to the intracellular solution caused a reduction in CFTR Cl- current amplitude. However, the apparent affinity of block was surprisingly high compared to previous results; Smith et al. (1999) reported an apparent Ki of 1.06 mM for block of CFTR expressed in Xenopus oocytes by extracellular Au(CN)2-, although no dose-response data were offered. Several other unusual effects not previously reported are also apparent in Fig. 1. Block by lower concentrations (e.g. 1 µM, Fig. 1A) appeared less voltage dependent than block by higher concentrations. This is also reflected in an altered steepness of the concentration-inhibition relationship measured at different membrane potentials (Fig. 1B). These data, fitted as described in Methods, also clearly demonstrate the unusually shallow concentration dependence of block observed. Fits of the data shown in Fig. 1B by eqn (1) suggest a Kd of 6.94 µM and a Hill coefficient of 0.63 at -100 mV and a Kd of 18.5 µM and a Hill coefficient of 0.45 at +60 mV. Similar analyses at other voltages revealed a weak voltage dependence of Kd and a Hill coefficient consistently less than unity, especially at depolarised membrane potentials (Fig. 1C). This low Hill coefficient is inconsistent with a simple bimolecular blocking mechanism by intracellular Au(CN)2- ions.
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Figure 1. Inhibition of macroscopic CFTR Cl- currents by intracellular Au(CN)2- A, example CFTR I-V relationships recorded from two different inside-out membrane patches before (control) and following addition of 1 µM or 100 µM Au(CN)2- to the intracellular solution. B, mean fraction of control current remaining (I/I0) following addition of different concentrations of Au(CN)2-, at a membrane potential of -100 mV ( | ||
Separating the multiple effects of intracellular Au(CN)2-
Intracellular Au(CN)2- ions may potentially inhibit CFTR macroscopic currents by interfering with channel gating or by binding within and physically occluding the open channel pore. In an attempt to separate these two types of effects, we studied the effects of intracellular Au(CN)2- on CFTR channels in which the normal gating cycle had been interrupted (Lansdell et al. 2000; Linsdell, 2000; Zhou et al. 2001). This was achieved by applying 2 mM sodium pyrophosphate (PPi) to the intracellular solution following CFTR channel activation (Fig. 2A). Consistent with the ability of PPi to 'lock' CFTR channels in the open state under these conditions (Gunderson & Kopito, 1994; Carson et al. 1995), addition of 2 mM PPi caused a 2.49 ± 0.29-fold increase in CFTR macroscopic current conductance (n = 20) without altering the shape of the I-V relationship (Fig. 2A). After locking the channels open with PPi, addition of Au(CN)2- to the intracellular solution still inhibited CFTR macroscopic current (Fig. 2A), although the inhibition was both weaker and more strongly voltage dependent than in the absence of PPi (Figs 2, 3 and 4). Furthermore, in the presence of PPi, the concentration dependence of block was more consistent with a simple bimolecular blocking reaction (Fig. 2B and C). Fits of the data shown in Fig. 2B by eqn (1) suggest a Kd of 61.6 µM and a Hill coefficient of 1.01 at -100 mV and a Kd of 913 µM and a Hill coefficient of 0.88 at +60 mV. Only at the most positive voltages studied was the Hill coefficient substantially less than unity (Fig. 2C). Figure 2C also demonstrates the strong voltage dependence of block by Au(CN)2- observed in the presence of PPi, although the relationship between apparent Kd and membrane potential was clearly steeper at positive than at negative potentials, such that this relationship appears to level off at very negative voltages.
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Figure 2. Inhibition of macroscopic 'locked open' CFTR Cl- channel currents by intracellular Au(CN)2- A, following maximal stimulation with PKA plus ATP, addition of 2 mM sodium pyrophosphate (PPi ) to the intracellular solution causes a further voltage-independent stimulation of current amplitude (left panel), presumably due to channel 'locking' in the open state. Right panel, subsequent to stimulation with PPi (in a different patch), addition of 100 µM Au(CN)2- to the intracellular solution causes a strongly voltage-dependent inhibition. B, fractional current remaining following addition of Au(CN)2- in the presence of PPi at -100 mV ( | ||
The blocking effects of Au(CN)2- in the absence and presence of PPi are directly compared in Fig. 3 and Fig. 4. The altered concentration dependence of block is shown in Fig. 3. Au(CN)2- is clearly a more potent blocker in the absence of PPi than in its presence (Fig. 3A); PPi causes an 8.9-fold increase in Kd at -100 mV (Fig. 3B and Fig. 4A), and a 49-fold increase at +60 mV (Fig. 3C and Fig. 4A). The stronger voltage dependence of block in the presence of PPi is illustrated in Fig. 4A. Note that PPi also abolishes the concentration-dependence of voltage dependence (Fig. 4B), that is to say, in the absence of PPi block by higher concentrations of Au(CN)2- is clearly more voltage dependent (as judged by the ratio of the apparent Kd at -100 mV to that at +60 mV) than block by lower concentrations. In contrast, after locking the channels open with PPi, block is strongly and consistently voltage dependent at all blocker concentrations (Fig. 4B). All of these observed changes in the blocking effects of intracellular Au(CN)2- after locking the channels in the open state are consistent with the isolation of a lower affinity, voltage dependent component of the block. This presumably reflects the loss of a higher affinity, weakly- or non-voltage-dependent component which is only seen in channels in which the gating cycle is uninterrupted.
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Figure 3. Altered concentration dependence of Au(CN)2- block in the presence of PPi A, inhibition of macroscopic current by a low concentration of Au(CN)2- (3 µM, left) is not observed in the presence of 2 mM PPi (right, different patch). B and C, direct comparison of the concentration-inhibition curves constructed as described in Figs 1 and 2, in the absence ( | ||
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Figure 4. Altered concentration dependence of block in the presence of PPi A, comparison of the voltage dependence of Kd, estimated as described in Figs 1 and 2, in the absence ( | ||
The inhibitory effects of Au(CN)2- observed in the absence or presence of PPi also differed in their sensitivity to extracellular Cl- concentration. As shown in Fig. 5A and B, reducing extracellular Cl- concentration from 154 mM to 4 mM (by replacement with gluconate) did not affect the voltage-independent block of macroscopic currents by a low concentration of Au(CN)2- (3 µM). In contrast, the strongly voltage-dependent block of PPi-treated channels by a higher concentration (100 µM) of Au(CN)2- was very sensitive to extracellular Cl- concentration (Fig. 5A and C). The increased apparent affinity of block, as well as the weakened (or even reversed) voltage dependence of block when extracellular Cl- concentration is reduced to 4 mM (Fig. 5C), strongly suggests that the blocking effects of Au(CN)2- observed after locking the channels open with PPi reflect Au(CN)2- binding within the open channel pore.
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Figure 5. Dependence of block by intracellular Au(CN)2- on extracellular Cl- concentration A, example I-V relationships recorded with 4 mM extracellular Cl-, in the absence (left) or presence (right) of PPi (2 mM), before (control) and following addition of the stated concentration of Au(CN)2- to the intracellular solution. B, mean fractional current remaining following addition of 3 µM Au(CN)2- to the intracellular solution in the absence of PPi, with extracellular Cl- concentrations of 154 mM ( | ||
Effects on CFTR single channel currents
The experiments described above suggest that Au(CN)2- inhibits CFTR by at least two mechanisms. Consistent with this, two distinct effects of intracellular Au(CN)2- were identified at the single channel level. First, a low concentration (3 µM) of Au(CN)2- caused a significant reduction in channel open probability (Fig. 6). Although this effect was only studied at a single voltage (+50 mV), it appears sufficient in magnitude to account for the voltage-independent reduction in macroscopic current amplitude caused by this concentration of Au(CN)2- (Fig. 1B). Secondly, higher concentrations of Au(CN)2- caused an apparent reduction in unitary current amplitude which presumably reflects a kinetically fast blocking reaction (Fig. 7). This effect was strongly voltage dependent (Fig. 7) and appears to be able to account for the block of macroscopic current after locking the channels open with PPi (Fig. 2). For example, fits to the data shown in Fig. 7C by eqn (1) suggest Kd values of 68.7 µM at -50 mV and 930 µM at +50 mV, similar to those estimated in Fig. 2C in the presence of PPi (for simplicity, the Hill coefficient was fixed to unity in Fig. 7C). The fact that single channel currents could readily be recorded with Au(CN)2- concentrations as high as 300 µM implies that the high affinity effect of Au(CN)2- on open probability does not completely prevent channel gating.
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Figure 6. Effect of intracellular Au(CN)2- on CFTR channel open probability A, continuous recording from an inside-out membrane patch containing at least seven CFTR channels at +50 mV. Addition of 3 µM Au(CN)2- to the intracellular solution causes a decrease in channel activity, as further shown in the expanded subtraces before (a) and after (b) addition of Au(CN)2-. B, mean effect of 3 µM Au(CN)2- on CFTR open probability. To obviate any effects caused by variability in PO between patches, and to avoid any ambiguity concerning the total number of channels (N) in each patch, NPO was measured in one min bins before and after addition of Au(CN)2-, and compared to the mean NPO (-NPO) averaged over three min immediately prior to addition of Au(CN)2-. Addition of Au(CN)2- ( | ||
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Figure 7. Effect of intracellular Au(CN)2- on unitary current amplitude A, example single channel activity recorded from inside-out membrane patches at +50 mV (top) and -50 mV (bottom), in the absence (left) and presence (right) of 100 µM Au(CN)2-. Two channels are active in each case; all channels closed is indicated by the line to the far left. This concentration of Au(CN)2- causes a clearly voltage dependent decrease in apparent unitary current amplitude, as also shown by the mean i-V relationships measured under these conditions (B) in the absence ( | ||
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DISCUSSION |
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Highly lyotropic anions (i.e. those with low free energies of hydration), such as the pseudohalide Au(CN)2-, hold great potential as functional and structural probes of Cl- channel pores. Lyotropic anions tend to show both high permeability and tight binding within Cl- channel pores (Bormann et al. 1987; Smith et al. 1999; Linsdell, 2001a,c), which is presumed to reflect a relationship between these permeation parameters and anion dehydration (Smith et al. 1999; Linsdell, 2001a). In CFTR, Au(CN)2- shows both high permeability (Smith et al. 1999; Gong et al. 2002) and tight intrapore binding (Smith et al. 1999; present results). However, the present results suggest that Au(CN)2- may also have non-pore-mediated effects on CFTR channel currents which complicate its use as a probe of the pore.
Two mechanisms of inhibition
Application of Au(CN)2- to the intracellular face of the membrane strongly inhibits macroscopic CFTR currents (Fig. 1). However, this inhibition clearly reflects at least two distinct effects, which can be distinguished at the single channel level or by locking channels in the open state with 2 mM PPi. We will refer to the relatively high affinity effect of Au(CN)2- on single channel open probability (Fig. 6) as the 'gating effect' and the lower affinity effect on unitary current amplitude (Fig. 7) as the 'conduction effect'.
The gating effect is evident at very low concentrations of Au(CN)2- (3 µM, Fig. 6) and is presumably responsible for the voltage-independent inhibition of macroscopic current observed with concentrations of 0.3-3 µM (Fig. 1B), concentrations at which Au(CN)2- has no apparent effect on unitary current amplitude (Fig. 7C). Isolation of the gating effect using 3 µM Au(CN)2- suggest that this effect is independent of extracellular Cl- concentration (Fig. 5A and B). The gating effect is apparently lost following addition of 2 mM PPi; under these conditions, macroscopic current is unaffected by Au(CN)2- concentrations below 10 µM and the effect of higher concentrations is well fitted by a simple bimolecular blocking reaction with a Hill coefficient close to unity (Fig. 2). Such a high affinity component of block is not observed when Au(CN)2- is added to the extracellular solution during whole cell recording (X. Gong & P. Linsdell, unpublished observations), consistent with previous work (Smith et al. 1999).
The conduction effect can be isolated by measuring unitary current amplitude (Fig. 7) or from the effect on macroscopic currents following addition of PPi to the intracellular solution (Fig. 2). Under these two sets of conditions, the effects of Au(CN)2- are comparable and inhibition can be well fitted by a simple bimolecular blocking reaction (Fig. 2 and Fig. 7). The isolated conduction effect is strongly voltage dependent (Figs 2, 4 and 7), and both the apparent affinity and voltage dependence of this component of block are highly sensitive to the extracellular Cl- concentration (Fig. 5A and C).
Potential molecular mechanisms of Au(CN)2- action
The properties of the conduction effect are highly suggestive of a kinetically fast, open-channel block mechanism. Individual blocking and unblocking events are too fast to be resolved in the filtered single channel current record (Fig. 7A). The block is strongly voltage dependent (Figs 2, 4 and 7), consistent with block within the transmembrane electric field; however, this voltage dependence appears to result from electrostatic interactions between Au(CN)2- and Cl- ions within the pore. Thus, replacement of almost all extracellular Cl- with the impermeant anion gluconate not only increases the apparent affinity of block, but also reverses the voltage dependence (Fig. 5). This mechanism of blocker voltage dependence is similar to that proposed for the unknown cytosolic factor responsible for flickery block of CFTR channels in cell-attached patches (Zhou et al. 2001).
The origin of the gating effect is less certain. While it is possible that this high affinity effect on channel gating is also mediated by Au(CN)2- binding within the pore, the lack of high affinity current inhibition by extracellular Au(CN)2- (Smith et al. 1999; X. Gong & P. Linsdell, unpublished observations) implies that the site(s) responsible for the gating effect are more accessible from the intracellular solution. Numerous cytoplasmic aspects of the CFTR molecule, such as the regulatory (R) domain, nucleotide binding domains, cytoplasmic loops, and amino and carboxy terminal tails (Gadsby & Nairn, 1999; Sheppard & Welsh, 1999; Kirk, 2000; Ostedgaard et al. 2001; Zou & Hwang, 2001) influence channel gating and are potential targets of intracellular Au(CN)2--induced changes in channel open probability. From the present results it is not even certain that the gating effect represents a direct effect on the CFTR molecule. CFTR is closely associated with numerous other proteins which can affect its gating (Zhu et al. 1999; Kirk, 2000). Clearly more work is required to establish the mechanism of the gating effect induced by low concentrations of intracellular Au(CN)2- ions. Ultimately Au(CN)2- may prove useful as a high-affinity probe of non-pore regions of CFTR, and also of the mechanism of channel gating. However, in terms of the use of Au(CN)2- in investigations of pore structure and function, the gating effect is an unwanted complication.
The dual inhibitory effects of intracellular Au(CN)2- are somewhat reminiscent of the inhibitory effects of high concentrations (> 30 µM) of genistein, which causes both a voltage-dependent decrease in CFTR unitary current amplitude and a voltage-independent decrease in channel open probability (Lansdell et al. 2000). It was suggested that these dual effects of genistein resulted from binding within the pore and to the first nucleotide binding domain respectively (Lansdell et al. 2000). Interestingly, macroscopic current inhibition by genistein, but not by the open channel blocker glibenclamide, was abrogated by PPi (Lansdell et al. 2000). Previously we showed that PPi had no effect on the inhibition of macroscopic CFTR Cl- currents by intracellular arachidonic acid (Linsdell, 2000). In the present study, PPi was able to dissociate the putative pore-mediated versus non-pore-mediated effects of intracellular Au(CN)2- (Figs 3, 4 and 5). A similar strategy using the CFTR mutant K1250A, which shows prolonged openings similar to that of wild type CFTR after locking the channels open with PPi (Gunderson & Kopito, 1995), has been used to dissociate open channel blocking from gating events at the single channel level (Zhou et al. 2001).
Au(CN)2- as a probe of the CFTR channel pore
Permeant anions which bind within the pore with high affinity are potentially of great use as probes of intrapore ion binding sites (see Introduction). Previously, the lyotropic SCN- anion has been used as a probe of anion binding sites in CFTR (Tabcharani et al. 1993; Mansoura et al. 1998; Gupta et al. 2001; Linsdell, 2001a). Figure 2A indicates that Au(CN)2- binding within the CFTR channel pore shows a Kd of < 200 µM at 0 mV, consistent with it binding at least 30-fold more tightly than SCN- under similar conditions (Linsdell, 2001c).
The conduction effect, reflecting open channel block of the CFTR pore by Au(CN)2-, is highly sensitive to the extracellular Cl- concentration (Fig. 5), suggesting that Au(CN)2-, in common with other CFTR pore blocking anions (McDonough et al. 1994; Linsdell et al. 1997a; Sheppard & Robinson, 1997; Linsdell & Hanrahan, 1999; Zhou et al. 2001), experiences electrostatic interactions with other permeant anions bound within the pore. This suggests that, as with Ba2+ block of K+ channels (Neyton & Miller, 1988a,b), Au(CN)2- block may be used to probe multiple interacting permeant ion binding sites within the CFTR pore. In the following study, as a first attempt to identify such sites, we survey the effect of mutations at various sites within the sixth transmembrane region on Au(CN)2- block.
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REFERENCES |
|---|
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
BORMANN, J. , HAMILL, O. P. & SAKMANN, B. (1987). Mechanism of anion permeation through channels gated by glycine and  -aminobutyric acid in mouse cultured spinal neurones. Journal of Physiology 385, 243-286 |
[Abstract] |
| CARSON, M. R., WINTER, M. C., TRAVIS, S. M. & WELSH, M. J. (1995). Pyrophosphate stimulates wild-type and mutant cystic fibrosis transmembrane conductance regulator Cl- channels. Journal of Biological Chemistry 270, 20466-20472 | [Abstract/Full Text] |
| CHANG, X. -B., KARTNER, N., SEIBERT, F. S., ALEKSANDROV, A. A., KLOSER, A. W., KISER, G. L. & RIORDAN, J. R. (1998). Heterologous expression systems for study of cystic fibrosis transmembrane conductance regulator. Methods in Enzymology 292, 616-629 | [Medline] |
| GADSBY, D. C. & NAIRN, A. C. (1999). Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiological Reviews 79, S77-107 | [Medline] |
| GONG, X., BURBRIDGE, S. M., COWLEY, E. A. & LINSDELL, P. (2002). Molecular determinants of Au(CN)2- binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl- channel pore. Journal of Physiology 540, 39-47 | [Abstract/Full Text] |
| GUNDERSON, K. L. & KOPITO, R. R. (1994). Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane conductance regulator channel gating. Journal of Biological Chemistry 269, 19349-19353 | [Abstract] |
| GUNDERSON, K. L. & KOPITO, R. R. (1995). Conformational states of CFTR associated with channel gating: the role of ATP binding and hydrolysis. Cell 82, 231-239 | [Medline] |
| GUPTA, J., EVAGELIDIS, A., HANRAHAN, J. W. & LINSDELL, P. (2001). Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region. Biochemistry 40, 6620-6627 | [Medline] |
| HALM, D. R. & FRIZZELL, R. A. (1992). Anion permeation in an apical membrane chloride channel of a secretory epithelial cell. Journal of General Physiology 99, 339-366 | [Abstract] |
| HANRAHAN, J. W., KONE, Z., MATHEWS, C. J., LUO, J., JIA, Y. & LINSDELL, P. (1998). Patch-clamp studies of cystic fibrosis transmembrane conductance regulator chloride channel. Methods in Enzymology 293, 169-194 | [Medline] |
| HARRIS, R. E., LARSSON, H. P. & ISACOFF, E. Y. (1998). A permeant ion binding site located between two gates of the Shaker K+ channel. Biophysical Journal 74, 1808-1820 | [Abstract/Full Text] |
| HURST, R. S., TORO, L. & STEFANI, E. (1996). Molecular determinants of external barium block in Shaker potassium channels. FEBS Letters 388, 59-65 | [Medline] |
| JIANG, Y. & MACKINNON, R. (2000). The barium site in a potassium channel by X-ray crystallography. Journal of General Physiology 115, 269-272 | [Abstract/Full Text] |
| KIRK, K. L. (2000). New paradigms of CFTR chloride channel regulation. Cellular and Molecular Life Sciences 57, 623-634 | [Medline] |
| LANSDELL, K. A., CAI, Z., KIDD, J. F. & SHEPPARD, D. N. (2000). Two mechanisms of genistein inhibition of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in murine cell line. Journal of Physiology 524, 317-330 | [Abstract/Full Text] |
| LINSDELL, P. (2000). Inhibition of cystic fibrosis transmembrane conductance regulator chloride channel currents by arachidonic acid. Canadian Journal of Physiology and Pharmacology 78, 490-499 | [Medline] |
| LINSDELL, P. (2001a). Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore. Journal of Physiology 531, 51-66 | [Abstract/Full Text] |
| LINSDELL, P. (2001b). Direct block of the cystic fibrosis transmembrane conductance regulator Cl- channel by butyrate and phenylbutyrate. European Journal of Pharmacology 411, 255-260 | [Medline] |
| LINSDELL, P. (2001c). Thiocyanate as a probe of the cystic fibrosis transmembrane conductance regulator chloride channel pore. Canadian Journal of Physiology and Pharmacology 79, 573-579 | [Medline] |
| LINSDELL, P., EVAGELIDIS, A. & HANRAHAN, J. W. (2000). Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore. Biophysical Journal 78, 2973-2982 | [Abstract/Full Text] |
| LINSDELL, P. & HANRAHAN, J. W. (1996). Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in a mammalian cell line and its regulation by a critical pore residue. Journal of Physiology 496, 687-693 | [Abstract] |
| LINSDELL, P. & HANRAHAN, J. W. (1998). Adenosine triphosphate-dependent asymmetry of anion permeation in the cystic fibrosis transmembrane conductance regulator chloride channel. Journal of General Physiology 111, 601-614 | [Abstract/Full Text] |
| LINSDELL, P. & HANRAHAN, J. W. (1999). Substrates of multidrug resistance-associated proteins block the cystic fibrosis transmembrane conductance regulator chloride channel. British Journal of Pharmacology 126, 1471-1477 | [Abstract/Full Text] |
| LINSDELL, P., TABCHARANI, J. A. & HANRAHAN, J. W. (1997a). Multi-ion mechanism for ion permeation and block in the cystic fibrosis transmembrane conductance regulator chloride channel. Journal of General Physiology 110, 365-377 | [Abstract/Full Text] |
| LINSDELL, P., TABCHARANI, J. A., ROMMENS, J. M., HOU, Y. -X., CHANG, X. -B., TSUI, L. -C., RIORDAN, J. R. & HANRAHAN, J. W. (1997b). Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions. Journal of General Physiology 110, 355-364 | [Abstract/Full Text] |
| LINSDELL, P., ZHENG, S. -X. & HANRAHAN, J. W. (1998). Non-pore lining amino acid side chains influence anion selectivity of the human CFTR Cl- channel expressed in mammalian cell lines. Journal of Physiology 512, 1-16 | [Abstract/Full Text] |
| MANSOURA, M. K., SMITH, S. S., CHOI, A. D., RICHARDS, N. W., STRONG, T. V., DRUMM, M. L., COLLINS, F. S. & DAWSON, D. C. (1998). Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore. Biophysical Journal 74, 1320-1332 | [Abstract/Full Text] |
| MCDONOUGH, S., DAVIDSON, N., LESTER, H. A. & MCCARTY, N. A. (1994). Novel pore-lining residues in CFTR that govern permeation and open-channel block. Neuron 13, 623-634 | [Medline] |
| MINDELL, J. A., MADUKE, M., MILLER, C. & GRIGORIEFF, N. (2001). Projection structure of a ClC-type chloride channel at 6. 5 Å resolution. Nature 409, 219-223 | [Medline] |
| NEYTON, J. & MILLER, C. (1988a). Potassium blocks barium permeation through a calcium-activated potassium channel. Journal of General Physiology 92, 549-567 | [Abstract] |
| NEYTON, J. & MILLER, C. (1988b). Discrete Ba2+ block as a probe of ion occupancy and pore structure in the high-conductance Ca2+-activated K+ channel. Journal of General Physiology 92, 569-586 | [Abstract] |
| OGIELSKA, E. M. & ALDRICH, R. W. (1998). A mutation in S6 of Shaker potassium channels decreases the K+ affinity of an ion binding site revealing ion-ion interactions in the pore. Journal of General Physiology 112, 243-257 | [Abstract/Full Text] |
| OSTEDGAARD, L. S., BALDURSSON, O. & WELSH, M. J. (2001). Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by its R domain. Journal of Biological Chemistry 276, 7689-7692 | [Full Text] |
| QU, Z. & HARTZELL, H. C. (2000). Anion permeation in Ca2+-activated Cl- channels. Journal of General Physiology 116, 825-844 | [Abstract/Full Text] |
| RYCHKOV, G. Y., PUSCH, M., ROBERTS, M. L., JENTSCH, T. J. & BRETAG, A. H. (1998). Permeation and block of the skeletal muscle chloride channel, ClC-1, by foreign anions. Journal of General Physiology 111, 653-665 | [Abstract/Full Text] |
| SHEPPARD, D. N. & ROBINSON, K. A. (1997). Mechanism of glibenclamide inhibition of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in a murine cell line. Journal of Physiology 503, 333-346 | [Abstract] |
| SHEPPARD, D. N. & WELSH, M. J. (1999). Structure and function of the CFTR chloride channel. Physiological Reviews 79, S23-45 | [Medline] |
| SMITH, S. S., STEINLE, E. D., MEYERHOFF, M. E. & DAWSON, D. C. (1999). Cystic fibrosis transmembrane conductance regulator. Physical basis for lyotropic anion selectivity patterns. Journal of General Physiology 114, 799-818 | [Abstract/Full Text] |
| TABCHARANI, J. A., ROMMENS, J. M., HOU, Y.-X., CHANG, X.-B., TSUI, L.-C., RIORDAN, J. R. & HANRAHAN, J. W. (1993). Multi-ion pore behaviour in the CFTR chloride channel. Nature 366, 79-82 | [Medline] |
| VERGARA, C., ALVAREZ, O. & LATORRE, R. (1999). Localization of the K+ lock-in and the Ba2+ binding sites in a voltage-gated calcium-modulated channel. Implications for survival of K+ permeability. Journal of General Physiology 114, 365-376 | [Abstract/Full Text] |
| ZHOU, Z., HU, S. & HWANG, T. -C. (2001). Voltage-dependent flickery block of an open cystic fibrosis transmembrane conductance regulator (CFTR) channel pore. Journal of Physiology 532, 435-448 | [Abstract/Full Text] |
| ZHU, T., DAHAN, D., EVAGELIDIS, A., ZHENG, S. -X., LUO, J. & HANRAHAN, J. W. (1999). Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C. Journal of Biological Chemistry 274, 29102-29107 | [Abstract/Full Text] |
| ZOU, X. & HWANG, T. -C. (2001). ATP hydrolysis-coupled gating of CFTR chloride channels: structure and function. Biochemistry 40, 5579-5586 | [Medline] |
Acknowledgements
We thank Susan Burbridge for technical assistance. This work was supported by the Canadian Institutes of Health Research and the Canadian Cystic Fibrosis Foundation (CCFF). P.L. is a CCFF scholar. X.G. is a CCFF postdoctoral fellow.
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) or +60 mV (
). Mean of data from 3-9 patches. The data have been fitted by eqn (1) as described in the text. C, voltage dependence of Kd (