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J Physiol (2003), 549.2, pp. 375-385
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.038216
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ABSTRACT |
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The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel pore is blocked in a voltage-dependent manner by a broad range of anionic substances added to the cytoplasmic side of the membrane. Here we investigate the origin of the voltage dependence of block by intracellular Au(CN)2-, a highly permeant lyotropic anion which also acts as a high-affinity blocker of Cl- permeation. Not only the affinity, but also the voltage dependence of block by intracellular Au(CN)2- ions is strongly dependent on extracellular Cl- concentration; following replacement of most extracellular Cl- by glucose or by impermeant anions, block by Au(CN)2- shows greatly weakened voltage dependence. This suggests that coupled movement of Au(CN)2- and Cl- ions within the pore contributes to the voltage dependence of block. This explanation requires that interactions between different anions take place within the pore, implying simultaneous binding of multiple anions to intrapore sites. Other anions are able to substitute for extracellular Cl- and interact with intracellular Au(CN)2- ions. Analysis of the effects of different extracellular anions on the apparent affinity and voltage dependence of block by intracellular Au(CN)2- ions suggests that extracellular anions do not need to permeate through the channel in order to destabilize Au(CN)2- binding within the pore, implying that this destabilizing effect results from binding to an externally accessible site in the permeation pathway. We propose that multiple anions can bind simultaneously within the CFTR channel pore, and that repulsive interactions between bound anions speeds anion exit from the pore.
(Received 19 December 2002; accepted after revision 14 March 2003; first published online 4 April 2003)
Corresponding author P. Linsdell: Department of Physiology and Biophysics, Dalhousie University, Sir Charles Tupper Medical Building, Halifax, Nova Scotia B3H 4H7, Canada. Email: paul.linsdell{at}dal.ca
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INTRODUCTION |
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Many classes of ion channels have pores which are capable of holding more than one permeant ion simultaneously (Hodgkin & Keynes, 1955; Neyton & Miller, 1988a,b; Doyle et al. 1998; Morais-Cabral et al. 2001; Hille, 2001). As ions pass through channel pores in single file, they interact electrostatically with each other, leading to a number of so-called multi-ion pore effects (Hille, 2001). Electrostatic repulsion between ions bound simultaneously within the multi-ion pores of voltage-gated cation channels is thought to be the physical basis of high ionic flux rates through these pores (Baukrowitz & Yellen, 1996; Doyle et al. 1998; McCleskey, 1999; Miller, 2000; Morais-Cabral et al. 2001).
Functional evidence for multi-ion pore behaviour has also been observed in many anion channels (Bormann et al. 1987; Halm & Frizzell, 1992; Levitan & Garber, 1998; Rychkov et al. 1998; Qu & Hartzell, 2000; Fahlke, 2001), including the cystic fibrosis transmembrane conductance regulator (CFTR) (Tabcharani et al. 1993; Linsdell, 2001a; Zhou et al. 2001). However, in contrast to the situation described above for cation channels, the relevance of multiple anion occupancy to the normal permeation mechanism of Cl- channels is not known. Recently, the first crystal structure of a Cl- channel, a prokaryotic ClC-type channel, was reported (Dutzler et al. 2002). Although this structure revealed only one Cl- ion bound within the pore, it was hypothesized that a second Cl- ion binding nearby would result in electrostatic destabilization and consequent Cl- permeation (Dutzler et al. 2002).
Since multi-ion pore behaviour requires simultaneous binding of multiple ions to intrapore binding sites, it is often revealed using high-affinity channel blocking ions, surrogates of the physiological permeant ion with greatly increased residency times within the pore (Neyton & Miller, 1988a,b; Harris et al. 1998; Spassova & Lu, 1998; Antonov & Johnson, 1999; Jiang & MacKinnon, 2000; Zhu & Auerbach, 2001a,b). Previously, we have shown that the permeant Au(CN)2- ion is a high-affinity probe of anion binding sites and interacts with multiple sites within the CFTR pore (Gong et al. 2002a; Linsdell & Gong, 2002). In order to investigate ion-ion interactions within the CFTR pore, we have now examined the effects of extracellular permeant anions on block by intracellular Au(CN)2-.
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METHODS |
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Experiments were carried out on two mammalian cell lines, baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells stably transfected with wild-type human CFTR, prepared as described previously (Gong et al. 2002b; Linsdell & Gong, 2002). BHK cells were used for macroscopic current experiments and CHO cells for single channel experiments. Both single channel and macroscopic CFTR current recordings were made using the excised, inside-out configuration of the patch clamp technique. Channel activity was induced following patch excision by exposure to 40-130 nM protein kinase A catalytic subunit (PKA) plus 1 mM MgATP in the bath. Both pipette (extracellular) and bath (intracellular) solutions were based on one containing (mM): 150 NaCl, 2 MgCl2, 10 TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonate). Chloride concentration on either side of the membrane was then varied by replacing NaCl by glucose. To maintain osmolarity, glucose concentrations of up to 300 mM (to replace 150 mM NaCl) were used. For experiments involving different anions (see Fig. 4), the extracellular solution contained (mM): 150 NaX, 2 MgCl2, 10 TES, where X is the anion of interest; a list of all extracellular anions tested is given in Fig. 4 and Fig. 6. All solutions were adjusted to pH 7.4 with NaOH. Given voltages have been corrected for liquid junction potentials existing between dissimilar pipette and bath solutions; the magnitude of these junction potentials was either calculated using pCLAMP8 software (Axon Instruments, Union City, CA, USA) or measured as described previously (Hanrahan et al. 1998). All chemicals were from Sigma-Aldrich (Oakville, ON, Canada), except KAu(CN)2 (Strem Chemicals, Newburyport, MA, USA) and PKA (prepared in the laboratory of Dr M. P. Walsh, University of Calgary, AB, Canada, as described previously; Hanrahan et al. 1998).
Current traces were filtered at 50 Hz (for single channel currents) or 100 Hz (for macroscopic currents) using an eight-pole Bessel filter, digitized at 250 Hz, and analysed using pCLAMP8 software (Axon Instruments). Macroscopic current-voltage (I-V) relationships were constructed using depolarizing voltage ramp protocols with a rate of change of voltage of 100 mV s-1 (Linsdell & Hanrahan, 1996; Linsdell & Gong, 2002). Preliminary spectral analysis suggests that, at 100 µM, rates of Au(CN)2- block and unblock are of the order of 3-5 kHz (data not shown), consistent with effects on apparent unitary current amplitude (Fig. 2C and D; see also Linsdell & Gong, 2002). These rates are very much faster than the rate of change of voltage, ensuring that steady-state block is measured using this voltage ramp protocol. Background (leak) currents recorded before addition of PKA were subtracted digitally, leaving uncontaminated CFTR currents (Linsdell & Hanrahan, 1996, 1998). To isolate the pore-blocking effects of Au(CN)2- on CFTR macroscopic currents, channels were 'locked' in the open state by addition of 2 mM sodium pyrophosphate (PPi) to the bath solution following attainment of full PKA-stimulated current amplitude (Linsdell & Gong, 2002; Gong et al. 2002a; see Fig. 2A).
The relationship between the fraction of the control current remaining following addition of Au(CN)2- (I/I0) and membrane voltage was analysed using two different versions of the Woodhull (1973) model of voltage-dependent channel block. Initially (Fig. 3A), the simplest version of this model was applied:
I/I0 = Kd(V)/(Kd(V) + [Au(CN)2-]), (1)
where I is the current in the presence of blocker, I0 the control, unblocked current, and Kd(V) is the voltage-dependent dissociation constant, the voltage dependence of which is given by:
Kd(V) = Kd(0) exp (-z
VF/RT), (2)
where z is the valence of Au(CN)2- (-1), is the fraction of the transmembrane electric field traversed by Au(CN)2- ions in reaching their binding site within the pore from the intracellular solution, and F, R and T have their normal thermodynamic meanings.
Subsequently (eg. Fig. 3B) a modified model which allows unblock by blocker permeation was used (see Woodhull, 1973):
Kd(V) = (k-1/k+1) + (k+2/k+1), (3)
where k+1 represents the rate constant for Au(CN)2- entry into the pore from the intracellular solution, k-1 the rate constant for exit into the intracellular solution, and k+2 the rate constant for exit to the extracellular solution. The voltage dependence of the resulting dissociation constants is given by:
(k-1/k+1) = K1(0) exp (-zVF/RT), (4)
(k+2/k+1) = K2(0) exp(-z (2 - 1) VF/2RT). (5)
Because rates of block and unblock were not routinely estimated experimentally, we have made no attempt to analyse the effects of different conditions on k+1, k+2 and k-1, and have instead focused on the overall Kd(V), which appears to be well described by the fits (e.g. Fig. 3B and Fig. 5).
All other methodological details were as described in detail recently (Linsdell & Gong, 2002; Gong et al. 2002a,b). Experiments 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. Statistical comparisons between groups were carried out using Student's two-tailed t test, with P < 0.05 being considered a significant difference.
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RESULTS |
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Extracellular Cl--dependence of block by intracellular Au(CN)2-
Intracellular Au(CN)2- has two inhibitory effects on CFTR, affecting both channel gating and Cl- permeation through the pore due to open channel block (Linsdell & Gong, 2002). Previously we have used channels that have been 'locked' in the open state using PPi to discriminate between these two effects (Gong et al. 2002a; Linsdell & Gong, 2002; see Methods). The effect of extracellular Cl- concentration on block of 'locked open' CFTR channels by 100 µM intracellular Au(CN)2- is shown in Fig. 1A and B. Reducing extracellular Cl- concentration (by replacement of NaCl with glucose) increased the apparent affinity of block at depolarized voltages, leading to a clear decrease in the apparent voltage dependence of block. In contrast, reducing intracellular Cl- concentration to 10 mM had no apparent effect on block by intracellular Au(CN)2- (Fig. 1A and C). The effects of changing extracellular Cl- concentration appear to support a correlation between Au(CN)2- block and the electrochemical driving force on Cl- ions (Fig. 1D). However, this apparent correlation does, in fact, seem to reflect a specific effect of extracellular Cl- concentration, since changing the driving force by altering intracellular Cl- concentration does not have the same effect (Fig. 1D).
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Figure 1. Extracellular Cl- concentration alters the apparent affinity and voltage dependence of block by intracellular Au(CN)2- A, example CFTR I-V relationships, leak-subtracted as described in Methods, recorded from three different inside-out patches with the transmembrane Cl- ion gradients indicated. Currents were recorded following maximal CFTR channel activation with PKA and PPi, before (control) and after (+Au(CN)2) addition of 100 µM KAu(CN)2 to the intracellular solution. B, mean fraction of control current remaining (I/I0) at different voltages following addition of 100 µM Au(CN)2- to the intracellular solution, at different extracellular Cl- concentrations: | ||
Since the two inhibitory effects we previously identified for intracellular Au(CN)2- show different voltage dependencies and affinities (Linsdell & Gong, 2002), one concern related to the results shown in Fig. 1B is that changing the extracellular Cl- concentration may be changing the efficacy with which PPi locks CFTR channels in the open state, thereby obviating the use of PPi to discriminate open channel block from effects on channel gating. However, PPi does appear to be equally effective at all extracellular Cl- concentrations, since (1) single channel recordings show almost complete locking open with 2 mM PPi under different ionic conditions, before and after addition of 100 µM Au(CN)2- (data not shown), (2) the fractional increase in macroscopic current amplitude seen on addition of 2 mM PPi is the same for all extracellular Cl- concentrations (Fig. 2A and B), and (3) very similar results to those seen in Fig. 1 were obtained with the CFTR mutant K1250A, which normally shows very long open burst durations, in the absence of PPi (data not shown). Furthermore, the differential blocking effects of Au(CN)2- when the extracellular solution contained 154 mM or 4 mM Cl- were also observed at the single channel level (Fig. 2C-F); since unitary current amplitude also reflects isolated pore-blocking effects of Au(CN)2- (Linsdell & Gong, 2002), this close agreement suggests that the inhibitory effects of Au(CN)2- shown in Fig. 1 are predominantly the result of open channel block.
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Figure 2. Validation of the use of PPi to study Au(CN)2- block A, example CFTR I-V relationships recorded with extracellular Cl- concentrations of 154 mM (left) or 4 mM (right), following attainment of full PKA-stimulated amplitude (control) and following subsequent addition of 2 mM PPi (+PPi) to the intracellular solution. B, mean fractional increase in macroscopic current amplitude at -100 mV seen following addition of PPi, at different extracellular Cl- concentrations. C and D, unitary currents recorded at -60 mV, in the absence (control) and presence of 100 µM Au(CN)2-, when the extracellular solution contained 154 mM Cl- (C) or 4 mM Cl- (D). Under these conditions, the mean fraction of control current remaining following addition of Au(CN)2- (E and F) is similar for unitary currents recorded in the absence of PPi ( | ||
The results shown in Fig. 1B clearly indicate that the voltage dependence of Au(CN)2- block is dependent on the extracellular Cl- concentration; as the Cl- concentration increases, so the voltage dependence of block becomes stronger. However, quantification of this effect is not straightforward. At all Cl- concentrations studied, the macroscopic current blocking effects of Au(CN)2- were poorly fitted by a simple Woodhull (1973) model of voltage-dependent block of the kind given by eqn (2) (Fig. 3A). This appears to result to a large extent from partial relief of block at hyperpolarized voltages, as previously noted (see Fig. 2, Linsdell & Gong, 2002). Since Au(CN)2- is highly permeant in CFTR (PAu(CN)2 > PCl; Smith et al. 1999; Gong et al. 2002a) this most likely reflects unblock by Au(CN)2- permeation, as previously reported for other channel types (e.g. Lansman et al. 1986; French & Wells, 1997; Bähring et al. 1997; Huang et al. 2000; Nimigean & Miller, 2002). This potentially complex behaviour can most simply be fitted using a modified Woodhull (1973) model in which unblock can occur by Au(CN)2- movement either back into the intracellular solution or 'forward' into the extracellular solution (see eqns (3)-(5), Methods). As shown in Fig. 3B, such a model significantly improved the fits, and also allowed data recorded under a variety of different ionic conditions (see below) and in different channel variants (Gong & Linsdell, 2003) to be compared quantitatively.
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Figure 3. Quantification of the effects of extracellular Cl- on Au(CN)2- block A and B, fits of mean data from Fig. 1B with different forms of the Woodhull (1973) model, either without (A) or with (B) channel unblock by blocker permeation (see Methods). Mean values of Kd(0) (C) and | ||
Mean parameters from fits of eqns (1) and (3)-(5) to data from individual patches reveal an approximately linear relationship between the apparent Kd for Au(CN)2- block at 0 mV membrane potential and extracellular Cl- concentration (Fig. 3C), suggesting competition between intracellular Au(CN)2- and extracellular Cl- ions within the pore. The fractional electrical distance experienced by Au(CN)2- (, estimated across the entire voltage range studied according to eqns (3)-(5), showed an apparently saturating increase with increasing extracellular Cl- concentration (Fig. 3D). The strong Cl- dependence of suggests that the voltage dependence of Au(CN)2- block may have multiple components: an intrinsic component which remains at very low extracellular Cl-, which may reflect Au(CN)2- movement within the transmembrane electric field, and a Cl--dependent component which appears to reflect coupled movement of Au(CN)2- and Cl- ions within the transmembrane electric field. This coupled movement occuring within the pore is a clear manifestation of multi-ion pore behaviour (Oliver et al. 1998; Spassova & Lu, 1998). While different kinds of interaction between extracellular Cl- and an intracellular, permeant blocker such as Au(CN)2- are possible (French & Shoukimas, 1985; Neyton & Miller, 1988a,b; Spassova & Lu, 1988; Nimigean & Miller, 2002; see Discussion), the effects shown in Fig. 3C and D are most consistent with a competitive interaction between Cl- and Au(CN)2- inside the pore. Thus, Au(CN)2- movement into the pore from the intracellular solution is associated with movement of a bound Cl- ion from the pore into the extracellular solution, and so movement of these two anions within the transmembrane electric field occurs as a concerted step.
Extracellular anion selectivity of block by intracellular Au(CN)2-
The results shown in Figs 1-3 suggest that Cl- ions entering the channel from the extracellular solution and Au(CN)2- ions entering from the intracellular solution experience mutual repulsive effects. Since many monovalent anions besides Cl- are permeant in CFTR (Linsdell & Hanrahan, 1998), we tested the ability of other extracellular anions to substitute for Cl- in repelling intracellular Au(CN)2- ions from the pore. As shown in Fig. 4 and Fig. 5, block of locked-open CFTR channels by 100 µM intracellular Au(CN)2- was altered when NaCl in the pipette solution was replaced by other sodium salts. As described above (Fig. 3C), analysis of the blocking effects of 100 µM Au(CN)2- (or 1 mM Au(CN)2- in the case of SCN-, which greatly weakened Au(CN)2- block; Fig. 4 and Fig. 5) allowed comparison of the ability of different extracellular anions to destabilize Au(CN)2- binding within the pore. Estimates of Kd(0) and for intracellular Au(CN)2- with different extracellular anions are shown in Fig. 6. With the exception of acetate, all anions tested significantly weakened Au(CN)2- block relative to that seen with 300 mM glucose. The relative potency of different extracellular anions in repelling intracellular Au(CN)2- ions from the pore, judged from the Kd(0) values shown in Fig. 6, was SCN- >> Br- 
I- NO3- ClO4- Cl- > PF6- formate F- benzoate gluconate > acetate. This anion sequence does not appear to be closely related to either the anion permeability sequence (see Linsdell & Hanrahan, 1998) or anion conductance sequence (see Linsdell, 2001c). However, there does appear to be some relationship between the effect of different extracellular anions on Kd(0) and anion free energy of hydration (Fig. 7), since all weakly hydrated anions studied (lyotropes; Cl- , Br-, NO3-, SCN-, I-, ClO4-) show a greater ability to destabilize Au(CN)2- block than strongly hydrated anions (kosmotropes; benzoate, acetate, gluconate, formate, F-). Different extracellular anions also led to different voltage dependencies of block (as judged by effects on ; Fig. 6). There did appear to be some relationship between strongly voltage-dependent block by intracellular Au(CN)2- and permeability (Fig. 7); only anions showing a permeability equal or greater than that of I- (PI/PCl 0.24) gave a significantly greater value than that observed with glucose (Fig. 6), suggesting that the external anion-dependent component of observed in Fig. 3B only applies to relatively permeant anions. The biggest effect on was seen with those anions that combine high pemeability and high conductance (Cl-, NO3-, Br-; Fig. 6). Two anions which apparently destabilize Au(CN)2- block, ClO4- and PF6-, significantly reduced relative to that seen with glucose (Fig. 6B), suggesting a complex coupling of the movement of these anions in the pore.
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Figure 4. Block by intracellular Au(CN)2- is dependent on the extracellular anion Example I-V curves, constructed as described in Fig. 1, were recorded with 150 mM of the named anion present in the extracellular solution, before (control) and following addition of 100 µM Au(CN)2- to the intracellular solution. In each case, the scale is current in pA on the ordinate and membrane potential in mV on the abscissa. | ||
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Figure 5. Effect of different extracellular anions on block by intracellular Au(CN)2- Mean fraction of control current remaining (I/I0) at different voltages following addition of 100 µM Au(CN)2- to the intracellular solution with the named anion (150 mM) present in the extracellular solution. Mean of data from 4-5 patches. Glcn, gluconate; Benz, benzoate; Form, formate; Ace, acetate. Mean data have been fitted according to the Woodhull (1973) model including unblock by blocker permeation (see Methods). | ||
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Figure 6. Effect of different extracellular anions on the apparent affinity and voltage dependence of block by intracellular Au(CN)2- Kd(0) (A) and | ||
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Figure 7. Potential physical basis of extracellular anion effects The effects of different extracellular anions on both the apparent affinity (Kd(0)) and voltage dependence ( | ||
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DISCUSSION |
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The CFTR chloride channel pore is blocked in a voltage-dependent manner by a broad range of anionic molecules added to the cytoplasmic side of the membrane (Linsdell, 2001b). The conventional interpretation of the voltage dependence of block is that, because of their charge, these substances experience part of the transmembrane electric field as they move from the intracellular solution to their binding site within the channel pore. This view was supported by the finding that block by the negatively charged channel blockers SCN- (Tabcharani et al. 1993) and diphenylamine-2-carboxylate (DPC) (McDonough et al. 1994) were affected by mutations approximately the same predicted physical distance from the intracellular end of the sixth transmembrane region of the CFTR molecule as the fractional electrical distance sensed by these blockers. However, the present results suggest a different origin for at least part of the voltage dependence of block by intracellular Au(CN)2- ions. As shown in Fig. 1 and Fig. 3, the fractional electrical distance apparently experienced by Au(CN)2- was dependent on the concentration of extracellular Cl- ions, strongly suggesting that the voltage dependence of channel block results not only from Au(CN)2- movement within the transmembrane electric field but also in part from the coupled movement of Au(CN)2- and Cl- ions within this field. This apparent origin of the voltage dependence of Au(CN)2- block is reminiscent of that proposed for block of inward rectifier K+ channels by intracellular blocking cations (Oliver et al. 1998; Spassova & Lu, 1998, 1999) and is similar to that proposed for the voltage-dependent 'flickery' closures seen primarily during on-cell CFTR single channel recording, which appear to reflect open channel block by an unknown cytosolic anion(s) (Zhou et al. 2001). Interestingly, the voltage dependence of block of CFTR by other intracellular anions is reduced by lowering the extracellular Cl- concentration (McDonough et al. 1994; Sheppard & Robinson, 1997; Linsdell & Hanrahan, 1999; Gong et al. 2002b), suggesting that coupled movement of Cl- and blocking anions may be a common factor contributing to the apparent voltage dependence of CFTR channel block. CFTR channel currents appear to be subject to block by cytoplasmic anions (Zhou et al. 2001), and this process could potentially be modulated by physiological changes in the concentration and nature of the extracellular anion (Gray et al. 2002).
The physical meaning of the electrical distance apparently traversed by Au(CN)2- ions is limited by the analytical model used and by the different potential interactions between ions inside the pore. The high permeability of Au(CN)2-, which probably results in the relief of block at hyperpolarized voltages, necessitates use of a modified Woodhull (1973) model (see Methods). However, this model assumes a single blocker binding site at a fixed electrical distance through the pore, whereas the apparent multi-ion pore effects observed in the present study suggest Au(CN)2- may interact with multiple sites, and further imply that only part of the voltage dependence of block reflects blocker movement within the transmembrane electric field. As presented, the model also assumes that Au(CN)2- itself does not carry a significant fraction of the current, only that it permeates to a sufficient extent to affect the voltage dependence of occupancy of the pore. In spite of these caveats, the model does provide an adequate description of the data under a range of different conditions (Fig. 3 and Fig. 5), allowing a reliable quantification of the effect of different ionic conditions on blocker affinity and relative voltage dependence.
The salient features revealed by this analysis of block are that extracellular Cl- ions destabilize Au(CN)2- binding within the pore (Fig. 3C) and also contribute to the voltage dependence of block, leading to a Cl--dependent component of (Fig. 3D). Together, these effects are most consistent with a competitive interaction between Cl- and Au(CN)2- ions inside the pore, which strongly implies that both ionic species can bind in the pore simultaneously. Movement of Au(CN)2- from the intracellular solution into the pore shows some intrinsic voltage dependence (Fig. 3D); in addition, Au(CN)2- block acquires additional voltage dependence due to its interaction with Cl- ions already bound in the pore. This results from the fact that, when Au(CN)2- enters the pore while it is occupied by a Cl- ion, this entry step is associated with the movement of the bound Cl- ion across part of the transmembrane electric field and into the extracellular solution. Other interactions between permeant ions may be envisioned; for example, Cl- binding in the pore may prevent unblock of the channel by Au(CN)2- permeation to the extracellular solution. Nevertheless, coupling of Au(CN)2- movement into the pore with Cl- exit from the pore is sufficient to explain the observed dependence of block on extracellular Cl- concentration.
Different mechanisms may allow movement of Au(CN)2- and Cl- ions within the pore to be coupled. If the two anions are bound closely together within the pore, they may experience mutual electrostatic destabilization (Baukrowitz & Yellen, 1996; Doyle et al. 1998; McCleskey, 1999; Miller, 2000; Morais-Cabral et al. 2001), as previously proposed for CFTR (Zhou et al. 2001). However, Spassova & Lu (1999) have proposed that movement of ions within channel pores may be coupled by a non-electrostatic mechanism, whereby binding of an ion to one site results in a subtle conformational change in the pore leading to unbinding of an ion bound to a separate site.
Other anions beside Cl- can destabilize Au(CN)2- binding within the pore (Figs 4-6), indicating that they have access to an anion binding site in the pore. The simplest interpretation of these results is that the ability of different extracellular anions to affect the Kd for Au(CN)2- block reflects their relative affinity for an intrapore binding site (see Spassova & Lu, 1999), although the observed Kd(0) will also be influenced by the presence of other anions within the pore. In this simple case, the affinity of extracellular anion binding appears only weakly related to anion permeability (Fig. 7). For example, ClO4- is only sparingly permeant but increases the Kd(0) for Au(CN)2- block more effectively than does Cl-, whereas formate is quite permeant but apparently unable to destabilize Au(CN)2- block (Fig. 6). This suggests the presence of an 'outer' binding site in the pore that is readily accessible to anions present in the extracellular solution. There is also no correlation with the previously identified anion conductance sequence (Linsdell, 2001c). Instead, the results shown in Fig. 7 suggest that anion binding to the external site is influenced by anion free energy of hydration; anions which are more readily dehydrated than Cl- (lyotropes) also destabilize Au(CN)2- binding more effectively than does Cl- itself, whereas all anions with greater free energy of hydration than Cl- (kosmotropes) are much less effective. In fact, since it has previously been shown that anion permeability in CFTR is related to the lyotropic sequence (Linsdell & Hanrahan, 1998; Linsdell et al. 2000), lyotropic anion binding to an extracellular site may indirectly appear to be related to permeability. Nevertheless, all anions tested with the exception of acetate significantly increased Kd(0) relative to that seen with extracellular glucose, suggesting a non-specific anion-dependent component of Au(CN)2- destabilization. These experiments may therefore isolate the effects of anion binding to an extracellularly accessible, lyotropic-selective anion binding site. Previous evidence has also suggested that lyotropic anions bind relatively tightly within the CFTR pore (Dawson et al. 1999; Smith et al. 1999; Linsdell, 2001a, 2001c; Gong et al. 2002a).
Thiocyanate is far more potent than any other extracellular anion tested in destabilizing Au(CN)2- block (Figs 4-6) implying that it may bind much more tightly to extracellularly accessible binding sites than other anions. Thiocyanate is well-known as a high-affinity probe of many types of Cl- channel pores (reviewed in Dawson et al. 1999; Linsdell, 2001a) and has previously been shown to bind tightly within the CFTR pore (Tabcharani et al. 1993; Dawson et al. 1999; Linsdell, 2001a,c), but other permeant anions such as I- and ClO4- that appear to bind with similar affinity in CFTR (Linsdell, 2001c) do not destabilize Au(CN)2- block to an equivalent extent. While we do not know what makes SCN- so effective, it is interesting to note that SCN- is the only anion tested which combines strong block of CFTR Cl- currents (Linsdell, 2001c) with high permeability (PX/PCl > 1; Fig. 7). This combination of tight binding and high permeability also applies to Au(CN)2- itself (Smith et al. 1999; Linsdell & Gong, 2002).
Although the ability of extracellular anions to affect the Kd(0) of Au(CN)2- block appears to show some preference for lyotropic over kosmotropic anions, the effect of different anions on the voltage dependence of block (quantified as ; Fig. 6) instead appears more dependent on anion permeability (Fig. 7). In fact, the only anions which increased significantly above the value seen with extracellular glucose were those for which PX/PCl 
0.2 (SCN-, I-, Br-, NO3-, Cl- ; Fig. 6 and Fig. 7). This discrepancy may in part reflect the fact that while effects on Kd(0) result from anion occupancy of an external anion binding site, effects on require coupled movement of Au(CN)2- and other anions within the pore.
Au(CN)2- blocks Cl- permeation in CFTR by interacting with multiple amino acid residues in the pore (Gong et al. 2002a; Gong & Linsdell, 2003). The present results suggest that Au(CN)2- ions bound within the pore experience strong interactions with other anions entering from the extracellular solution to bind to other sites, and that these interactions serve to repel bound Au(CN)2- ions from the pore. Mutual electrostatic destabilization between permeant ions bound simultaneously within the pore is thought to be the physical basis of high ionic throughput in voltage-gated cation channels (Baukrowitz & Yellen, 1996; Doyle et al. 1998; McCleskey, 1999; Miller, 2000; Morais-Cabral et al. 2001) and has been proposed on structural grounds in a ClC channel pore (Dutzler et al. 2002). Our functional results suggest that a similar mechanism may act to maximize the conductance of the CFTR Cl- channel.
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Acknowledgements
We thank Susan Burbridge and Angie Lewis for technical assistance. This work was supported by the Canadian Institutes of Health Research and the Canadian Cystic Fibrosis Foundation (CCFF). X.G. is a CCFF postdoctoral fellow, P.L. is a CCFF scholar.
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154 mM, 
75 mM, 
50 mM, 
20 mM, 
10 mM, 
4 mM; intracellular Cl- concentration is 154 mM in each case. C, results of the same analysis at intracellular Cl- concentrations of 154 mM (
) and an extracellular Cl- concentration of 154 mM. In A, B and C, Cl- concentration was altered by substitution of glucose for NaCl. D, data from B and C replotted as a function of the calculated electrochemical driving force on Cl- ions, estimated as the difference between the membrane potential and the Cl- ion equilibrium potential (ECl) calculated from the Nernst equation. Mean of data from 4-7 patches in each case.