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REPORT
MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
School of Biomedical Sciences and Hunter Medical Research Institute, Faculty of Health, The University of Newcastle, Callaghan, New South Wales, Australia
Submitted 17 June 2005 ; accepted in final form 16 January 2006
ABSTRACT
Block of a sarcoplasmic reticulum anion channel (SCl channel) by disulfonic stilbene derivatives [DIDS, dibenzamidostilbene-2,2'-disulfonic acid (DBDS), and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)] was investigated in planar bilayers using SO
as the conducting ion. All molecules caused reversible voltage-dependent channel block when applied to either side of the membrane. DIDS also produced nonreversible channel block from both sides within 1–3 min. Reversible inhibition was associated with a decrease in channel open probability and mean open duration but not with any change in channel conductance. The half inhibitory concentration for cis- and trans-inhibition had voltage dependencies with minima of 190 nM and 33 µM for DBDS and 3.4 and 55 µM for DNDS. Our data supports a permeant blocker mechanism, in which stilbenes block SCl channels by lodging in the permeation pathway, where they may dissociate to either side of the membrane and thus permeate the channel. The stilbenes acted as open channel blockers where the binding of a single molecule occludes the channel. DBDS and DNDS, from opposite sides of the membrane, competed for common sites on the channel. Dissociation rates exhibited biphasic voltage dependence, indicative of two dissociation processes associated with ion movement in opposite directions within the trans-membrane electric field. The kinetics of DNDS and DBDS inhibition predict that there are two stilbene sites in the channel that are separated by 14–24 Å and that the pore constriction is 
10 Å in diameter.
4,4'-dinitrostilbene-2,2'-disulfonic acid; dibenzamidostilbene-2,2'-disulfonic acid; permeant blocker
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Stilbenes have been used to study anion fluxes across the sarcoplasmic reticulum (SR) of muscle. At micromolar concentrations they inhibited the transport of Cl–, Pi, oxalate, and SO across the SR membrane (4, 11, 12). With the use of the bilayer method, single channel recordings have identified an anion channel (SCl channel) that was strongly inhibited by the stilbene DIDS (20). It was highly regulated, being activated by µM cytoplasmic Ca2+ (17) and inhibited by cytoplasmic acidification (<7 pH), millimolar adenine nucleotides (1, 14), and inositol phosphates (16). It was activated and inhibited by oxidation and reduction, respectively (15). SCl channels have a bell-shaped voltage dependence with maximal activation between 0 and –80 mV (with respect to the lumen; Ref. 18).
Although the SCl channel was first identified as a Cl– channel (75 pS in 250/50 mM Cl–) (18), it has since been found to be a divalent anion channel, passing SO(60 pS in 250 mM SO) and the divalent form of Pi (maximum conductance = 22 pS) (20). The physiological ion for this channel is not clear. The SCl channel is unlikely to serve as a Cl– channel in muscle because its permeability to Cl– is three to six times lower than to divalent anions such as Pi and SO(20) and intracellular Cl– concentration (<10 mM) is relatively low. The SCl channel is the only SR channel known to pass SO and thus is likely to be the basis for the stilbene-sensitive fluxes reported in early studies (see above). In this study, the SO current through the SCl channel was used to examine the inhibition kinetics of DIDS, DBDS, and DNDS. This study aims to elucidate their mechanism of action and establish whether the molecules block the pore and permeate the channel. As yet, nothing is known about the structure of the SCl channel. A comparison of the block by stilbenes with different molecular sizes (DBDS and DNDS; see Fig. 1) has produced our first insights into the architecture of the SCl pore.
MATERIALS AND METHODS
Preparations of SR microsomes.
SR vesicles were prepared from the back and leg muscles of New Zealand White rabbits. The rabbits (4 kg wt) were euthanized with a barbiturate (Lethobarb) overdose (7.5 ml iv) before the muscle was removed for biochemical processing. SR vesicles were prepared as described by Laver et al. (20).
Lipid bilayers, chemicals, and solutions.
Unless otherwise stated, lipid bilayers were produced using an 8:2 mixture of phosphatidylethanolamine and phosphatidylcholine dissolved in 20 µl n-decane to a final concentration of 50 mg/ml. Bilayers containing 30% phosphatidylserine also contained 50% phosphatidylethanolamine and 20% phosphatidylcholine. Lipids were obtained from Avanti Polar (Alabaster, AL). Bilayers were formed across a hole in a Delrin cup (aperture 100–200 µm diameter) separating the cis- and trans-solutions. SCl channels were mostly recorded using cis-solutions containing 250 mM MgSO4, 10 mM MgCl2, 10 TES (pH 7.4), and trans-solutions containing 50 mM MgSO4, 10 mM MgCl2, and 10 TES (pH 7.4). These solutions permitted measurement of SCl activity via their SO permeability. This obviated the effects of interfering signals from the BCl channels which are impermeable to SO and K+ channels, which are blocked by Mg2+. Ruthenium red (5 µM) was added to the cis-solution to inhibit ryanodine receptor activity.
Vesicles were added to the cis-solution where incorporation into the lipid bilayer was facilitated by the osmotic gradient (higher osmotic potential in the cis-bath) and the presence of 1–5 mM cis-Ca2+. Channels oriented in the bilayer such that their cytoplasmic side faced the cis-solution.
DNDS (Molecular Probes), DIDS, and DBDS (MP Biomedicals) were prepared as concentrated stocks in dimethyl sulfoxide (DMSO) in a range of 100 µM to 150 mM and added via aliquot additions to the cis- and trans-solutions. A large range of stock concentrations was prepared to keep the bath concentrations of DMSO to a minimum, preventing potential DMSO/SCl interactions. This precaution proved unnecessary because, unlike the ryanodine receptor (25), DMSO has no effect on the activity of the SCl (see RESULTS). In some experiments, DIDS was applied by perfusion tubes placed near the bilayer (25). This arrangement permitted both application and washout of DIDS.
Data acquisition and analysis. Bilayer holding potentials were controlled using an Axopatch 200B amplifier (Axon Instruments). All electrical potentials are expressed as cytoplasmic side relative to luminal side at virtual ground. During the experiments, the bilayer current was recorded after low-pass filtering at 5 kHz, sampled at 50 kHz, and simultaneously stored on computer disk using data interfaces (Data Translation DT301 or DT3001) under the control of in-house software written in Visual Basic. Before analysis, the single channel recordings were filtered at 200 Hz and resampled at 1 kHz.
Analyses of channel conductance and open and closed durations were made using the Hidden Markov Model algorithm (5). The Hidden Markov Model finds the maximum likelihood estimate of the channel current transitions present in the record (i.e., an idealized representation of the recording) and provides for the determination of channel amplitudes, transition probabilities, and transition rates. It operates on the assumption that the channel current signal is the sum of a first-order, finite-state, Markov process, and white, uncorrelated, Gaussian noise of a known variance. Analysis was carried out on single-channel recordings, with steady baseline, varying in duration from 10 to 60 s.
Dwell-time frequency histograms of channel open and closed dwell times were compiled from idealized representations of the recordings containing between 102 and 103 events. The distributions are displayed either as probabilities or frequencies (the number of events of a particular duration per second of recording) and plotted using the "log-bin" method suggested by Sigworth and Sine (27). Sampling bins were equally spaced on a log scale with 7 bins per decade. Putative gating models were evaluated by fitting theoretical probability distribution functions to the dwell-time histograms using the Q-matrix method (6, 7). The algorithm of Blatz and Magleby (2) was used to calculate the effects of current steps that were too short to be detected. For current records, this detection limit (dead time) of channel events ranged from 0.5 to 1 ms, depending on their signal-to-noise ratio. Optimum model fits to the data were obtained using Newton’s method and the quality of fit was determined from the root-mean-square of the differences between the theory and data.
RESULTS
DIDS: reversible and nonreversible inhibition.
DIDS inhibited SCl channels by both reversible and nonreversible mechanisms (Fig. 2). Figure 2A shows an example of cis application of 40 µM DIDS to an SCl channel (cis- and trans-baths correspond to cytoplasm and SR lumen, respectively). SO current through the channel was irreversibly inhibited within seconds, just as reported in a previous study (18) using Cl– as the permeant ion. In addition, it was found that DIDS added to the trans-bath permanently blocked the channel within 1–3 min (N = 3 not shown). Before permanent closure, DIDS produced a flicker block (Fig. 2A, dashed arrow), which could be reversed by removing DIDS from the solution (Fig. 2B). Reversible inhibition was characterized by a marked increase in the number of brief channel closures, which was more pronounced at negative membrane potentials. The unitary current was not sensitive to DIDS, being 2.9 ± 0.2 pA (–40 mV, N = 3) in the presence of 0–60 µM DIDS. In control experiments, DMSO was added to the cis- and trans-baths and at concentrations of up to 2% had no observable effect on SCl channel conductance and open and closed times (not shown).
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conductance of SCl channels was 54 ± 1 pS in the presence of 250/50 mM MgSO4 (cis/trans) as determined from a linear fit to current-voltage data in Fig. 5B. This compares with a conductance of 75 pS in 250 mM Cl– (18), in which a large proportion of channel openings were conductance substrates (40% substate probability). In the presence of SO, substate probabilities were >10-fold lower than in Cl–. Maximum likelihood amplitude histograms revealed a 20 pS substate (indicated by the arrow in Fig. 5A). This substate accounted for <1% of the channel activity and so the channel gating analysis could be simplified by grouping substates and channel closures. Histograms in Fig. 5A show that DBDS reduced the proportion of the 54 pS channel openings (2 pA) and 20 pS substates while increasing the proportion of the baseline. Figure 5B shows that DBDS applied from either the cis- or trans-sides did not alter channel conductance. In the presence of DBDS (5 µM cis or 50 µM trans) the conductance changed by <2% (each from N = 3 paired experiments). Similarly, cis- or trans-DNDS produced <2% change in channel conductance (each from N = 4 paired experiments, not shown).
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The voltage dependencies of DBDS blocking kinetics are summarized in Fig. 8, E and F. Because open times, 
o, were inversely related to concentration, the DBDS binding kinetics were expressed by o x [DBDS] 
binding rate–1. The binding rates have an exponential dependence on bilayer voltage. Binding from cis- and trans-sides have opposite voltage dependencies with respect to the effect on o. Both cis- and trans-DBDS produce very similar values of c (c unbinding rate–1), which follow biphasic voltage dependencies with maxima at around –20 mV. This similarity is highlighted by the example shown in Fig. 8C (+40 mV), which shows means ± SE of closed histograms from several experiments. The voltage dependencies of DBDS binding and unbinding can be broadly understood in terms of the Woodhull (29) model for voltage-dependent ion block of channels, where the binding and unbinding rates have an exponential voltage dependence determined by the location of the blocker binding sites and energy barriers within the trans-membrane electric field. The strong voltage dependencies of DBDS binding suggest a binding site within the pore. Curiously, c, and hence unbinding rates, could be described by the sum of two dissociation mechanisms with opposite voltage dependencies, indicating molecular movements in opposite directions within the trans-membrane electric field. This is consistent with an unbinding mechanism that can release DBDS to either the cis- and trans-sides of the membrane. The similar values of c for cis- and trans-block indicate that the DBDS unbinding rate is independent of the side from which DBDS first enters the channel. This is consistent with a DBDS binding site within the pore that is accessible from both sides of the membrane. The possibility that DBDS permeates the channel is explored in Combined cis and trans inhibition.
DNDS: analysis of flicker block. The dose responses of DNDS-induced inhibition are shown in Fig. 9A and Ki are given in Fig. 9B and Table 1. Like DBDS, the Ki for cis- and trans-block by DNDS had opposite voltage dependencies, which were biphasic. The most potent affinities for cis- and trans-block were 3.4 and 5.5 µM, respectively. Thus DNDS is a less potent blocker than DBDS.
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Combined cis and trans inhibition.
To further explore the possibility that DBDS and DNDS permeate through the anion pore experiments were carried out to detect interactions between blocking ions from opposite sides of the membrane. If blocker permeation occurs, then one would expect inhibitors on opposite sides of the membrane to exhibit competitive blocking kinetics. Ki was measured for cis-DBDS at –40 mV in the presence and absence of DNDS (5 mM) in the trans-bath. DNDS was chosen for the trans-bath because it was more soluble than DBDS (200 µM max) and could be used at concentrations well above its Ki. The addition of 5 mM DNDS in the trans-bath caused 85% reduction in Po. After this, addition of cis-DBDS caused further inhibition. The dose-response of DBDS inhibition, normalized to Po in the presence of 5 mM DNDS and no DBDS, is shown in Fig. 7A and the values for Ki are given in Table 1. DNDS in the trans-bath increased the Ki for cis-DBDS by 20-fold, which is a clear indication of competition between DBDS and DNDS on opposite sides of the membrane.
To gain a more detailed understanding of cis/trans drug interactions the combined effects of cis- and trans-block on closed dwell-time distributions were analyzed. The addition of cis- (20 µM) or trans- (2,000 µM) DNDS to channels, that were already inhibited by DNDS on the opposite side, increased the average duration of DNDS-induced channel closures (Fig. 11, A and C). In the case of the trans-addition (Fig. 11C), the lengthening of channel closures appeared to be due to a shift in the proportion of events associated with short and long exponential components. The lengthening of channel closures indicates that DNDS from cis- and trans-sides can simultaneously interact with the channel at what must be at least two separate sites.
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300 ms (Fig. 11, B and D, arrow). Although the addition of cis (2 µM) or trans (200 µM) DBDS increased the inhibition in channels already partly inhibited from the opposite side, this inhibition was not associated with a lengthening of time constant for DBDS-induced channel closures. Therefore, in the case of DBDS, there is no indication that two molecules can simultaneously bind from opposite sides of the channel.
Effect of permeant ion, SO42–, on stilbene inhibition.
In three experiments, inhibition by cis-DNDS was measured at –40 mV in the presence of symmetric 50 mM SO(cis and trans). Under these conditions, open and closed dwell-time histograms differed markedly from similar experiments carried out in fivefold higher (250 mM) cis-SO(Fig. 12). In 50 mM SO, 5 µM DNDS reduced mean open times to 6.5 ± 1 ms, which is threefold shorter than 22 ± 3 ms observed in 250 mM SO(N = 4). This is consistent with competition between DNDS and SO for common sites on the channel. Reduced SO concentration also increased closed durations. In the presence of 50 mM SO, DNDS induced mean closed time of 80 ± 10 ms, which is twofold longer than 39 ± 4 ms observed in 250 mM SO(N = 6).
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| (1) |
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| (2) |
, which is the degree of voltage dependence (the voltage-dependent equations are shown in Fig. 13). The model includes provision for the C and T sites to be both occupied (BCT). The values of were assumed to be independent of the presence of a second ion in the channel (see Table 2). Furthermore, to satisfy conditions for ionic equilibrium, addition constraints were placed on the parameters (see Table 2). Initially, Scheme II was fitted separately to voltage- and concentration-dependent dwell-time histograms from each experiment (e.g., data in Figs. 8, A and B, 10, A and B, 11, A–D, and 12, A and B). Within each experiment, this amounted to fitting 10–25 pairs of dwell-time histograms. The final parameter set was obtained from fitting mean data from many experiments, where the same parameters were used to fit cis- and trans- and combined cis/trans-inhibition (e.g., Figs. 8, D–F and 10, D–F).
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DISCUSSION
Disulfonic stilbene derivatives are permeant blockers.
This study presents a broad data set that supports a permeant blocker mechanism for inhibition of the SCl channel by stilbenes (i.e., stilbenes block channels by lodging within the anion permeation pathway and can also permeate through the channel to the opposite side of the membrane). The following data provide evidence for binding of stilbenes in the anion permeation pathway. 1) The stilbenes are open channel blockers. The concentration dependencies of open and blocked durations indicate that binding can only occur when the channel is open and that the binding of a single stilbene molecule occludes the channel (Fig. 8D). 2) Stilbene molecules bind in the transmembrane regions. This is indicated by the strongly voltage-dependent binding kinetics shown in Figs. 8, E and F, and 10, E and F. 3) Stilbenes and permeant anions compete for common sites. The model fit to the data in Fig. 12 indicates that the binding rate of DNDS increases threefold from 5.6 to 19 (Ms)–1 when [SO] is decreased from 250 mM to 50 mM. Furthermore, two lines of evidence indicate that stilbene molecules permeate through the channel. 4) The stilbenes can block the channel from both sides of the membrane and from opposite sides they compete for common sites within the channel (Fig. 7A). 5) Stilbene dissociation can occur in opposite directions within the trans-membrane electric field. Hence, upon dissociation, the blocking molecule can enter either the cis- or trans-baths. This is demonstrated by the biphasic voltage dependencies of DBDS and DNDS dissociation in Figs. 8F and 10F.
It is unknown whether DIDS can also permeate the pore. However, the fact that DIDS can permanently block the channel from each side of the membrane is consistent with DIDS permeating the pore and cross linking with amino acid residues in the permeation pathway of the channel.
Functional evidence linking stilbene inhibition with binding in the permeation pathway has been reported for DNDS inhibition of epithelial anion channels (3). It was reported that DNDS was an open channel blocker that competed with permeant ions. However, DNDS only blocked from the cis-side of the channel and its action showed no significant voltage dependence. Permeant blockade of an anion channel by stilbenes is a novel finding. A permeant blocker mechanism has been invoked previously to explain the biphasic voltage dependence of dissociation from BK channels of alkaline earth ions, such as Ca2+ and Ba2+ (19, 24). In those studies, interactions between permeant K+ and blocking ions were able to elucidate the detailed mechanism for K+ permeation long before crystal structures for K+ channels were available.
The stilbene permeation mechanism. The simplest permeant blocker mechanism would involve a single binding site in the channel that was accessible from both sides of the membrane. Our data rules against this in favor of a multisite mechanism. First, the blocked durations for DNDS showed two exponential components, which depended on concentration and voltage (Fig. 10, B and F). Second, the presence of DNDS on both sides of the membrane produced blocked periods with longer duration than could be produced by DNDS on one side alone (Fig. 11, A and C).
To gain more information about the permeation mechanism an attempt was made to quantitatively fit two site models to the data. A model that did not fit the data was one in which DNDS could bind at one site within the pore and another site on the cis-surface, where binding modulated permeation via an allosteric effect. This model could not account for the combined action of DNDS on both sides of the membrane. The simplest two-site model that fitted our data was one in which DNDS could bind at two sites in the permeation pathway as shown in Fig. 13 and Scheme II. Examination of the model parameters in Table 2 reveals that DNDS kinetics are not significantly affected by the presence of another DNDS molecule, except for dissociation from the trans-site, which was slowed almost fourfold (i.e., kct-c = 5.4 s–1 and kt-o = 19 s–1 in Table 2). This reduced exit rate was responsible for the long exponential seen in the blocked time distributions. This stabilizing of the dual binding conformation is counter intuitive because one would expect proximity of two anions in the channel to mutually destabilize each other. This problem can be resolved by explicitly considering the effects of SO on stilbene permeation. The model fits to the data in Fig. 12 show that decreasing [SO] in the cis-bath from 250 mM to 50 mM causes a fivefold reduction in the rate of DNDS dissociation to the trans-bath. Thus it appears that the presence of SO in the pore destabilizes DNDS binding. In light of this finding, it can be seen that two adjacent DNDS molecules could be more stable than one DNDS molecule with SO in the pore. DNDS at the cis-site might displace SO and hence protect DNDS at the trans-site from the destabilizing effects of SO.
It is clear from the data that DNDS and DBDS behave quite differently in the channel. The lower blocking potency of DNDS appears to be attributable to its considerably faster exit rates from the cis-site. Furthermore, the data shows no indication that more than one DBDS molecule can reside in the channel at any time. The blocked time distributions show only one exponential component and the combined effects of cis- and trans-DBDS produce blocked durations that were indistinguishable from the those caused by cis or trans alone (Fig. 11, B and D). The marked differences in the cis- and trans-blocking potencies of the stibenes stem mainly from the lower stilbene binding constants on the trans-side of the channel (Table 2).
It is worth noting that the voltage dependencies of the model parameters are larger than expected from the movement of a divalent ion through the transmembrane electric field. The Woodhull model predicts that the total magnitudes of s associated with translocation of DNDS across the membrane (o-c + c-o + c-t + t-c + t-o + o-t) should be 1, whereas in this study, it equals 2.47 for DBDS and 2.02 for DNDS. This underscores the fact that the model transition rates shown here do not depend solely on the energy profile for stilbenes within the pore. The model in Scheme II does not explicitly include the effects of SO permeation, which is shown to be an important modulator of the blocker kinetics. Voltage dependence of the model parameters will include contributions from the voltage dependence of SO occupancy of binding sites in the channel.
Scheme II was used to predict the rate of ion permeation through the SCl channel (Fig. 13). Although DNDS is a weaker channel blocker than DBDS, its maximal transport rate at each membrane potential is an order of magnitude higher than DBDS. Curiously, the concentration dependence of the DNDS transport rate shows a decrease at concentrations >10 µM, which is due to the lower permeability of DNDS when two molecules are in the pore.
Insights into channel architecture.
The presence of negative lipids has been shown to strongly attenuate the action of stilbenes on ryanodine receptor activity, indicating that the negative charges on the lipids are close enough to the binding site to repel anionic molecules (25). In contrast to this, negative lipids had no effect on DBDS inhibition of SCl channels (Fig. 7B). Hence the binding site is sufficiently far (>1 Debye length; 1 nm) from the lipid head groups so as not to be significantly affected by charge repulsion.
Because DBDS and DNDS pass through the pore, their molecular dimensions give clues to the size of the anion permeation pathway. The molecular cross section perpendicular to the long axis of the molecules fits into a rectangle 10 Å x 6 Å (Fig. 1). This places a lower limit on the pore diameter of 10 Å. Such a pore diameter is consistent with a channel that poorly discriminates between anion species but is selective among ions of different valency (9). The data suggest that the permeation pathway can only hold one DBDS molecule but can contain two DNDS molecules. If the binding sites are produced by interactions between the pore and the stilbene SO
groups and if the stilbene molecules are constrained to lay end-on-end within the pore then a separation of <24 Å between cis- and trans-sites would exclude a second DBDS from being in the pore. With the use of similar logic, two DNDS molecules could fit in the pore if the cis- and trans-sites are separated by more than 14 Å. This indicates that the cis- and trans-sites have a separation of 14–24 Å. The presence of permeant anions within the pore affects the dissociation kinetics of stilbene block. This property endows stilbene molecules with the potential to be powerful probes for the conduction pathway and the mechanisms for permeation of small anions.
GRANTS
This work was supported by National Health and Medical Research Council project grant 211030 and by an infrastructure grant from the Hunter Medical Research Institute. D. R. Laver was supported by an Australian Research Council Professorial Fellowship.
ACKNOWLEDGMENTS
We thank Melissa Dafo and Paul Johnson for assisting with the experiments and Dr. Renate Griffith for help in producing the molecular diagrams.
FOOTNOTES
Address for reprint requests and other correspondence: D. R. Laver, School of Biomedical Sciences and Hunter Medical Research Institute, Faculty of Health, The University of Newcastle, Callaghan, NSW 2308, Australia (e-mail: Derek.Laver{at}newcastle.edu.au)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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, 552 events over 26 s, 
, 193 events over 8 s, 
, 131 events over 2 s, and 
, 300 events >4 s.
