Block by MOPS reveals a conformation change in the CFTR pore produced by ATP hydrolysis

Hiroshi Ishihara and Michael J. Welsh

Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ATP hydrolysis by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel predicts that energy from hydrolysis might cause asymmetric transitions in the gating cycle. We found that 3-(N-morpholino)propanesulfonic acid (MOPS) blocked the open channel by binding to a site 50% of the way through the electrical field. Block by MOPS revealed two distinct states, O1 and O2, which showed a strong asymmetry during bursts of activity; the first opening in a burst was in the O1 state and the last was in the O2 state. Addition of a nonhydrolyzable nucleoside triphosphate prevented the transition to the O2 state and prolonged the O1 state. These data indicate that ATP hydrolysis by the nucleotide-binding domains drives a series of asymmetric transitions in the gating cycle. They also indicate that ATP hydrolysis changes the conformation of the pore, thereby altering MOPS binding.

chloride channel; gating; cystic fibrosis transmembrane conductance regulator; buffer; anion; 3-(N-morpholino)propanesulfonic acid

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE CYSTIC FIBROSIS transmembrane conductance regulator (CFTR) is an epithelial Cl- channel with complex regulation (7, 22, 29). Amino acid sequence analysis, biochemical characterization, and functional studies indicate that CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain (RD). The MSDs, which are each composed of six transmembrane sequences, contribute to the formation of the Cl- conducting pore. The RD contains a number of consensus phosphorylation sites; phosphorylation of the RD by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) is required for the channel to open.

The NBDs in CFTR hydrolyze ATP to control channel activity. This conclusion is based on several findings: 1) hydrolyzable forms of MgATP are required to open the channel, 2) several nucleotide species and inorganic phosphate analogs alter channel gating in specific ways, 3) site-directed mutations in the NBDs produce specific alterations in channel gating, and 4) biochemical studies show that CFTR and an isolated NBD1 function as ATPases by hydrolyzing ATP (1-3, 6, 9, 14-16, 23, 30). This hydrolysis is required for the channel to open (1). As first suggested by Baukrowitz et al. (3), ATP hydrolysis is also required for the channel to close. On the basis of the effect of site-directed mutations in key residues in NBD1 and NBD2 and on the effect of agents such as 5'-adenylylimidodiphosphate (AMP-PNP), pyrophosphate, and orthovanadate, it has been proposed that ATP hydrolysis at NBD1 opens the channel into a burst of activity and that ATP hydrolysis at NBD2 terminates the burst and closes the channel (4, 5, 8).

The input of energy from ATP hydrolysis predicts that CFTR will have discrete conformations that do not exist at thermodynamic equilibrium. Moreover, the sequence of ATP hydrolysis at the two NBDs predicts that the channel will progress through an ordered, relatively irreversible series of distinct conformational states. Previous studies with the patch-clamp technique have identified two conformations of the protein, which are resolved as open and closed states. However, with only two recognizable gating conformations, open and closed, it is not possible to observe directly asymmetric transitions in the gating cycle. An indication that additional conformations exist comes from the recent observation of Gunderson and Kopito (9) that the open state may have two discrete conductances. We found that the buffer 3-(N-morpholino)propanesulfonic acid (MOPS) produced a flickery block of the channel that allowed us to discern additional conformations of the channel linked to ATP hydrolysis.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells and CFTR expression systems. We used two different expression systems to study wild-type CFTR: HeLa cells transiently expressing CFTR using the vaccinia virus-T7 RNA polymerase expression system and C127 mouse mammary epithelial cells stably expressing CFTR. We have previously described both systems (18, 21). Data obtained from both cell types were identical and were combined for analysis.

Chemicals and solutions. MOPS was obtained from Fisher Scientific (Fair Lawn, NJ). The catalytic subunit of PKA was from Promega (Madison, WI). All other reagents were from Sigma Chemical (St. Louis, MO). For excised, inside-out patch-clamp experiments, both sides of the membrane were bathed with a solution containing 140 mM N-methyl-D-glucamine chloride and 5 mM MgCl2 and buffered with the indicated concentrations of MOPS or 10 mM tricine (pH 7.3). To activate CFTR, 75 nM of PKA and 1 or 3 mM of Na2ATP were added to bath (cytoplasmic) solution; PKA was present for all conditions.

Patch-clamp methods and data analysis. An Axopatch 1C amplifier (Axon Instruments, Foster City, CA) was used for voltage clamping and current amplification. A microcomputer and the pCLAMP software package (version 6.0.1, Axon Instruments) were used for data acquisition and analysis. Currents were recorded on videotape following pulse-code modulation with a PCM-2 analog-to-digital videocassette recorder adapter (Medical System, Greenvale, NY) for later analysis. The methods used for the experimental setup and the excised, inside-out patch configuration were previously described (4, 6). Voltages are referenced to the extracellular side of the membrane. Bath temperature was maintained by a temperature-controlled microscope stage (Brook Industries, Lake Villa, IL).

Replayed data were filtered at a 1-kHz corner frequency with a variable eight-pole Bessel filter (902LPF, Frequency Devices, Haverhill, MA) and digitized at 10 kHz. For analysis, data were digitally filtered with pCLAMP software using a Gaussian filter at either 500 Hz ("lightly" filtered) or 10 Hz ("heavily" filtered). These records were used to make all-points histograms for determination of current amplitude and to make events lists for open- and closed-time analyses. Transitions of <1 ms in duration were excluded. In some cases, current amplitudes of the O1 and O2 state were measured manually in the heavily filtered records. Single-channel open- and closed-time histograms were plotted from the idealized records with a logarithmic x-axis with 10 bins/decade. Histograms were fit with a one or more component exponential function using the maximum likelihood method. Burst analysis was performed from the idealized records as described previously (4), using a discriminator of 20 ms to separate interburst closures from intraburst closures. This value was determined from the single-channel recording closed-time histogram of wild-type CFTR activated by 1 mM ATP and 75 nM PKA in 10 mM tricine solution at 25°C. Similar values were obtained with other conditions.

In experiments in which the membrane patch contained more than one channel, regions of data with no superimposed openings were used for burst analysis (4). There was no statistical difference between burst durations derived from patches with only one active channel compared with patches with more than one active channel nor was there any trend toward an increase or decrease of lifetimes by inclusion of data from patches with more than one channel.

Results are expressed as means ± SE of n observations. Statistical significance was assessed with a paired or unpaired Student's t-test and a log-likelihood ratio test, where appropriate. P values of <0.05 were considered statistically significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Two open-gating states in CFTR studied in solutions containing MOPS. Figure 1 shows recordings of several bursts of activity obtained from an excised, inside-out patch of membrane that contained a CFTR Cl- channel. Within a burst of activity, we often observed two different gating patterns. For example, in trace a in Fig. 1, during the first two-thirds of the burst the channel showed a flickery pattern of gating in which the open state was frequently interrupted by very short closings. In contrast, during the last one-third of the burst the open state was less frequently interrupted. For convenience, we refer to the time that the channel is in the flickery gating pattern as the O1-gating state and the time that it shows the less flickery pattern as the O2-gating state. In general, most bursts of activity contained both patterns of gating. However, traces a-e in Fig. 1 show the gating pattern was variable. Inspection of the tracings also indicates that rigorous discrimination between the O1 and O2 states was difficult for several reasons. First, the closures and openings in the O1 state were very short. The short duration plus the small single-channel current made accurate measurements difficult. Second, the O2 state also contained short closures, although they appeared to be less frequent than in the O1 state (e.g., note traces a-d). Third, the relative durations of the O1 and O2 states were variable; for example, the flickering O1 state was a small proportion of the burst shown in trace b but occupied most of the burst in trace e. These considerations made it difficult to be certain whether both states were always present within a burst; for example, trace e might have been composed only of the O1 state or it may have ended in the O2 state. Therefore, as a way of qualitatively evaluating the gating behavior, we examined the effect of filtering the data. At the bottom of Fig. 1, we show the recording from which traces a-e were obtained after they were filtered at 10 Hz. In this heavily filtered recording, the very rapid kinetics are not resolved and the observed current is a mean value, reflecting the proportion of time that the channel spent in the open and closed states. Thus the O1 state "appears" to have a lower conductance than the O2 state. The two gating states we observed likely explain the two conductance states reported by Gunderson and Kopito (10) in which they heavily filtered the data from CFTR studied in planar lipid bilayers.


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Fig. 1.   Examples of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel gating in the presence of MOPS. Top: tracings were filtered at 500 Hz and were taken at positions indicated by letters a-e in bottom trace (which was filtered at 10 Hz). Tracings were obtained from excised, inside-out patches. Holding potential was -80 mV, and temperature was 25°C. Scale bars are shown. Dashed lines indicate closed (C) state; O1 and O2 states are indicated in bottom trace. cAMP-dependent protein kinase was present in the bath solution of this and all subsequent experiments.

The flickery pattern of gating suggested the possibility of channel block by something in the solution. Our solutions contained 10 mM MOPS as a buffer, and previous reports have suggested that related buffers such as N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) can block other Cl- channels (11, 32). Therefore, we substituted tricine for MOPS because it did not block an outwardly rectifying Cl- channel (11). With 10 mM tricine as the buffer (Fig. 2A), the gating pattern resembled only the O2 state; we saw none of the fast flickery behavior observed in the O1 state with MOPS as the buffer (Fig. 1). Instead, gating resembled what we observed at positive voltages with MOPS (see Fig. 4).


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Fig. 2.   Examples of CFTR Cl- channel gating in the presence of 10 mM tricine at 25°C (A), 10 mM MOPS at 15°C (B), and 10 mM MOPS at 35°C (C). Tracings shown at top (of A-C) were filtered at 10 Hz, and data at bottom (of A-C) were filtered at 500 Hz. * Site of bottom traces. See legend of Fig. 1 for other details.

In the past, we had not observed two different gating patterns; however, we usually studied CFTR at 35-37°C. Therefore, we studied patches under conditions identical to those used in Fig. 1A, except that temperature was varied. When temperature was reduced to 15°C, both the O1 and O2 states were readily apparent and the duration of the O2 state appeared to be prolonged (Fig. 2B). In contrast, when temperature was increased to 35°C, we could not resolve a distinct O2 component (Fig. 2C); the records showed only fast flickery behavior similar to that observed in the O1 state at 25°C. It seems probable that channels studied in MOPS buffer at 35°C also have two open states. However, perhaps because the duration of the O2 state decreases as temperature increases, we could not distinguish O2 and O1. Of note, the conditions under which the two gating states were readily apparent, temperatures less than 35°C and solutions that contain MOPS, were the same as those employed by Gunderson and Kopito (10) when they reported two conductance states.

Previous studies performed under different conditions have shown that open times for CFTR Cl- channels were well fit by a single exponential function (30). Figure 3 shows that, when we used tricine as the buffer at 25°C, the open times were well fit with a single exponential function. In contrast, when we used 10 mM MOPS as the buffer, open-time histograms were fit better by two than by one exponential function. This suggests the presence of two populations of open times. Table 1 shows values for short and long open times within bursts of activity as well as closed times. The data suggest that there are two closed times within bursts of activity, but, because the short closed time is near the limits of resolution for our recordings, we may have underestimated their occurrence and, consequently, absolute values of open times. The average open time in tricine was longer than in MOPS, primarily because of a paucity of short closings. With both MOPS and tricine, a reduction in temperature prolonged the long open time (Table 1).


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Fig. 3.   Examples of open-time histograms obtained from single-channel recordings used to produce the tracings in Figs. 1 and 2, A and B. Tracings were obtained in 10 mM tricine at 25°C (A) or 10 mM MOPS buffer at 25°C (B) or 15°C (C). Data are open dwell-time analysis of 1 channel. Line shows the fit to a single exponential in the case of 10 mM tricine and a double exponential for 2 tracings shown with MOPS buffer. With tricine as the buffer (A), use of 2 exponential functions did not produce a significantly better fit than a single exponential function (n = 5). With MOPS as the buffer, in 6 of 6 cases at 25°C (B) and 4 of 4 cases at 15°C (C), open-time histograms were fit better by 2 than by 1 exponential function. Note the different scale of the x-axis for 10 mM tricine (A).

                              
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Table 1.   Effect of MOPS and temperature on open and closed times within bursts of activity

MOPS block of CFTR Cl- channels. To evaluate further the possibility that MOPS blocks the channel, we examined the effect of voltage on single-channel currents. Figure 4 shows data filtered at 500 Hz (left) and data filtered at 10 Hz (right). At a voltage of +80 mV, the flickering pattern of gating with short closures was not observed. However, as the voltage became more negative, the short closings and flickering gating behavior of the O1 state became more and more prominent. This was readily apparent after the data were heavily filtered.


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Fig. 4.   Effect of voltage on gating in the presence of 10 mM MOPS at 25°C. Tracings on left were filtered at 500 Hz, and tracings on right show the same data filtered at 10 Hz.

Figure 5A shows current-voltage (I-V) relationships for channels studied in tricine at 35 and 25°C. The I-V relationship of lightly filtered (500 Hz) data was linear, and the slope conductance was greater at 35°C (11.0 ± 0.21 pS, n = 3) than at 25°C (7.9 ± 0.02 pS, n = 4, P < 0.0001). Figure 5B shows the I-V relationship of channels studied in 10 mM MOPS at 25°C. When the data were lightly filtered at 500 Hz, the I-V relationship was relatively linear with slight rectification at the most negative voltages. After the data were filtered at 10 Hz, there was a clear distinction between the O1 and O2 states, with the O1 level showing greater rectification. At positive voltages, the I-V relationship was linear with a slope conductance of 7.4 ± 0.1 pS (n = 4) at 25°C and 10.2 ± 0.5 pS (n = 4, not shown in Fig. 5B) at 37°C. These conductance values are similar to those obtained from channels studied in tricine.


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Fig. 5.   Current-voltage relationships of channels studied in tricine and MOPS. A: buffer was 10 mM tricine at 25°C (square ) and 35°C (open circle ). Data were filtered at 500 Hz; n = 3-5. B: channels were studied at 25°C with 10 mM MOPS buffer. Recordings of O1 and O2 current levels were filtered at 10 Hz (bullet  and black-square, respectively). triangle , Current-voltage relationship of data filtered at 500 Hz; n = 2-5 for each. In most cases, SE bars are hidden by symbols.

We also examined the effect of increasing concentrations of MOPS (Fig. 6). At 0.5 mM MOPS, there was only a small amount of flicker in the lightly filtered tracing. However, both O1 and O2 states were apparent. As the concentration of MOPS increased to 25 mM, the fast flickery behavior became more prominent. In addition, when the data were filtered at 10 Hz, the distinction between O1 and O2 states became more obvious as the concentration of MOPS increased. The single-channel I-V relationships for the heavily filtered data are shown in Fig. 7, A and B. As the MOPS concentration ([MOPS]) increased, the I-V relationships for both O1 (A) and O2 (B) states showed increasing rectification, with the effects more pronounced for the O1 level.


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Fig. 6.   Effect of concentration of MOPS. Left: records filtered at 500 Hz at the indicated concentration of MOPS. Right: records filtered at 10 Hz. Traces at left were obtained at the points indicated by asterisks. Temperature was 25°C.


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Fig. 7.   Effect of concentration of MOPS on current-voltage relationships (A and B) and dissociation constant (Kd; C and D). Data for the O1 state are in A and C, and data for the O2 state are in B and D. Temperature was 25°C, and voltage was -80 mV. square , 25 mM MOPS. open circle , 10 mM MOPS. triangle , 5 mM MOPS. Data are means ± SE of 4-5 experiments for each condition. Data were filtered at 10 Hz. In most cases, error bars are hidden by symbols. Kd at 0 mV [Kd(0)] for the O1 state was 71 mM, and electrical distance sensed by MOPS (delta ) was 0.50 ± 0.01. Kd(0) and delta  for the O2 state depended on the MOPS concentration: with 25 mM MOPS they were 89 mM and 0.25 ± 0.01, and with 10 mM MOPS they were 261 mM and 0.54 ± 0.02, respectively.

The flickery pattern of gating in the O1 state at hyperpolarized voltages is consistent with classic open channel block. We calculated the voltage-dependent dissociation constant (Kd) for MOPS inhibition using the single-channel current amplitude of heavily filtered data in the O1 state as follows
<IT>K</IT><SUB>d</SUB>(<IT>V</IT>) = ([MOPS] · <IT>i</IT><SUB>MOPS</SUB>)/(<IT>i</IT><SUB>0</SUB> − <IT>i</IT><SUB>MOPS</SUB>) (1)
where Kd(V) is the voltage-dependent Kd at voltage V, iMOPS is the single-channel current of the O1 state in the presence of MOPS, and i0 is the single-channel current in the absence of MOPS (with tricine as the buffer), respectively. Figure 7C shows that Kd was strongly voltage dependent and the predicted Kd at 0 mV was 71 mM.

Voltage dependence suggests that MOPS binds within the electric field of the membrane. The electrical distance sensed by MOPS (delta ) can be calculated with the relationship (31)
<IT>K</IT><SUB>d</SUB>(<IT>V</IT>) = <IT>K</IT><SUB>d</SUB>(0)exp[(−<IT>z</IT>&dgr;<IT>FV</IT>)/(<IT>RT</IT>)] (2)
where z is the valence of MOPS, which is assumed to be -1 (see Fig. 8), Kd(0) is the Kd at 0 mV, and F, R, and T represent Faraday's constant, the gas constant, and absolute temperature, respectively. Assuming a single binding site for MOPS, we calculated that delta  = 0.50 ± 0.01 (n = 5) measured over the voltage range of -120 to -40 mV.

Comparisons of the tracings in Fig. 1 with those in Fig. 2B and the I-V relationships in Fig. 7B with those in Fig. 5A indicate that MOPS also affected the channel in the O2 state. Therefore, we calculated the voltage-dependent Kd for O2. Figure 7D shows that the Kd for the effect of MOPS on the O2 state was voltage dependent. However, when the concentration of MOPS changed, delta varied. These results indicate that the effect of MOPS in the O2 state is not consistent with a simple voltage-dependent block of an open channel as described by Woodhull (31). Thus MOPS block of CFTR in the O2 state is different from the open channel block observed in the O1 state.

Channel block by the anionic form of MOPS. MOPS is a buffer that exists in solution as the zwitterion (MOPS0) or the anion (MOPS-), and the ratio depends on the pH. To determine whether MOPS0 or MOPS- produced the rapid flickery block in the O1 state, we used the equation pH = pKa + log([MOPS-]/[MOPS0]), where pKa, the dissociation constant of MOPS, is 7.2 at 25°C. We altered the pH and the total [MOPS] to vary the concentration of MOPS- and MOPS0. Figure 8A shows that an increase in the concentration of MOPS- had a greater effect on gating than did MOPS0. For example, increasing the [MOPS-] from 2.8 to 13.9 mM at a constant pH of 7.3 markedly increased the flickery block (compare the first and second trace in Fig. 8A). Likewise, increasing the [MOPS-] from 2.8 to 13.8 mM at a nearly constant total [MOPS] had a similar effect (compare the third and fourth trace in Fig. 8A). The effect of [MOPS-] may be more readily apparent from examination of current amplitude of data obtained at negative voltages and filtered at 10 Hz (Fig. 8B). When [MOPS-] increased, the current amplitude in the O1 state decreased, whereas changes in the concentration of MOPS0 or in the pH did not correlate with effects on current amplitude. We obtained similar results when we examined the effect of MOPS- on the O2 state. Our previous unpublished observations have suggested little effect on gating of pH changes in this range. These data suggest that MOPS-, rather than MOPS0, blocked the channel in both the O1 and the O2 states, results consistent with a voltage-dependent effect.


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Fig. 8.   Effect of MOPS- (anion) and MOPS0 (zwitterion). A: example of traces obtained at different MOPS- and MOPS0 concentrations produced by variation of total MOPS concentration and pH. Specific conditions are indicated above each tracing. All tracings were obtained at 25°C and -80 mV. B: current amplitude of heavily filtered data for O1 (solid bars) and O2 state (open bars) under same conditions as in A. Concentrations of MOPS-, MOPS0, total MOPS, and pH are indicated. Data are means ± SE of 3-5 experiments for each condition.

To learn whether MOPS was able to block the channel from one or both sides, we substituted tricine as the buffer in either the intracellular or extracellular solution. Figure 9 shows that the flickery block was evident when MOPS was in the internal (bath) solution alone. In contrast, when only the external solution contained MOPS, flickery block was not observed at either hyperpolarizing or depolarizing voltages. These results indicate that MOPS can access its binding site only from the internal surface of the channel.


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Fig. 9.   Effect of addition of MOPS to the intracellular or extracellular surface of the patch. Examples were obtained at 25°C and -80 or +80 mV as indicated. MOPS was applied to the intracellular (bath) or extracellular (pipette) surface with the indicated concentrations of MOPS or tricine. Similar results were obtained in 3 other experiments. Records were filtered at 500 Hz.

Sequential passage of the channel through O1 and O2 states. As is evident from an examination of Figs. 1, 2B, 4, 6, 8A, and 9, bursts of activity usually started with the flickery O1 state and ended with the O2 state. To evaluate this pattern more objectively, we measured the duration of the first opening and the last opening within a burst using pCLAMP software. Table 2 shows that the first opening was significantly shorter than the last opening. Moreover, the average durations of the first (7.3 ms) and last (26.1 ms) openings were very close to the short (6.1 ms) and long (31.7 ms) open times obtained from the open-time histograms (Table 1). These data indicate that bursts of activity usually began with the O1-gating state and ended in the O2-gating state. In contrast, for channels studied in 10 mM tricine at 25°C, the average duration of the first opening was not significantly different from that of the last opening. We could not distinguish a difference between the first and last openings for channels studied in 10 mM MOPS at 35°C, but, as indicated above, our resolution is limited under those conditions.

                              
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Table 2.   Duration of first and last opening within bursts of activity

AMP-PNP inhibits the transition from the O1 to the O2 state. The finding that the O1 state usually preceded the O2 state indicates that the two states are not randomly distributed but rather have a specific order in their occurrence. This would require energy input that would probably come from ATP hydrolysis by the NBDs. To evaluate this possibility further, we examined the effect of AMP-PNP. Previous studies have shown that, in the presence of ATP and PKA, AMP-PNP increases the duration of a small fraction of the bursts of activity (4, 14). The fact that AMP-PNP affects only a fraction of bursts is consistent with the observation that AMP-PNP competes poorly compared with ATP in inhibition of 8-azidoadenosine 5'-triphosphate photolabeling of CFTR (28). Previous work with site-directed mutants has suggested that hydrolysis at NBD2 terminates a burst of activity. These results suggest that AMP-PNP binds to NBD2 and, because it cannot be hydrolyzed, prevents the hydrolysis that ultimately terminates a burst and closes the channel. When we added 1 mM AMP-PNP to a channel studied in a 10 mM MOPS solution at 25°C, we observed occasional long bursts of activity (Fig. 10), as we and others had previously reported (4, 14). The heavily filtered tracings in Fig. 10 (top) show that when the channel was in a long burst of activity it was in the O1 state, whereas bursts of normal duration exhibited both O1 and O2 states. The tracings in Fig. 10 (bottom) show fast flickery behavior during the long AMP-PNP-induced bursts. These data suggest that the transition from the O1 to O2 state requires ATP hydrolysis.


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Fig. 10.   Effect of 5'-adenylylimidodiphosphate (1 mM) on single-channel currents. Solution contained 10 mM MOPS and 1 mM ATP. Experiments were performed at 25°C and -80 mV. Top: records filtered at 10 Hz. Bottom: records were filtered at 500 Hz and were obtained at the position indicated by a and b. Similar results were obtained in 2 other experiments.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

MOPS blocks CFTR Cl- channels. Our data indicate that MOPS blocked CFTR Cl- channels by two different mechanisms, manifest as the O1- and the O2-gating states. In both cases, block was concentration dependent and was due to the anionic form of MOPS (MOPS-) rather than the protonated form (MOPS0). The data suggest that MOPS was not able to pass through the channel because it did not block when added to the extracellular surface. These findings are consistent with the voltage dependence: block was only observed at negative voltages that would drive MOPS- from the intracellular solution into the channel. In the O1 state, MOPS produced a flickering block with frequent short closures characteristic of classic open-channel block. When the data were heavily filtered, the O1 state appeared to have a reduced single-channel conductance that reflected the proportion of time the channel spent in the open and closed states. In the O2 state, block was not resolved as discrete closings, probably because the kinetics were too fast to be resolved with our recording system. However, as in the O1 state, block in O2 was voltage dependent and appeared as a decreased conductance at negative voltages. Thus block in the O1 state corresponds to block of "intermediate" speed and block in the O2 state may correspond to "very fast" block as described by Hille (13).

The voltage-dependent Kd for block in the O1 state suggests that MOPS binds at a site ~50% of the way across the electrical field. Electrical distance does not necessarily indicate physical distance. However, in the two cases in which it has been determined, there has been excellent correlation between electrical distance and physical distance across the pore formed by the MSDs. Tabcharani et al. (26) found that SCN- bound within the CFTR channel at a site ~20% of the electrical distance through the membrane from the cytoplasmic side, in good agreement with the predicted location of Arg-347 with which it interacted (assuming an alpha -helix). McDonough et al. (20) found that diphenylamine-2-carboxylic acid bound at a site ~40% of the electrical distance through the membrane from the cytoplasmic side; this value is in good agreement with the predicted location of Ser-341 to which it bound. MOPS is predicted to have dimensions of 7 Å × 5.5 Å × 12 Å, and the pore of CFTR is predicted to have a diameter of ~5.5 Å (25). These considerations suggest that MOPS on the internal side can enter a pore that has a wide cytosolic mouth and progress at least 50% of the distance through the electrical field to its binding site. While it is bound, MOPS appears to occlude the pore, preventing the flow of Cl-. At some point external to the MOPS binding site, the pore presumably narrows to a diameter of ~5.5 Å. This model of the channel pore and its interaction with MOPS- in the O1 state are shown schematically in Fig. 11A.


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Fig. 11.   Model of MOPS interaction with CFTR in the O1 (A) and O2 (B) state. Membrane is represented by hatched area, and domains of CFTR are labeled. MSD, membrane-spanning domain. NBD, nucleotide-binding domain. Model shows the channel only during a burst of activity; long closed state between bursts, ATP hydrolysis at NBD1, and phosphorylation of the regulatory (R) domain are not represented. Model also does not show any interaction with MOPS during O2 state, although an interaction does occur that is different from that during the O1 state. B: ADP and Pi are shown dissociating from the channel for purposes of illustration; we have no data to determine if they remain bound or if one or the other dissociates. Tracing (middle) represents the gating of CFTR between the open (O) and closed (C) state.

The mechanism of MOPS interaction in the O2 state is more difficult to characterize. The concentration-dependent change in the proportion of the electrical distance sensed by MOPS indicates that the voltage dependence was not consistent with a simple open channel block. Therefore, for simplicity, in the model in Fig. 8 we do not show an interaction with MOPS during the O2 state.

Previous studies have shown that anionic buffers can block other Cl- channels. Hanrahan and Tabcharani (11) showed that an outwardly rectifying Cl- channel was blocked by HEPES and MOPS but not by tricine. However, in contrast to the MOPS block of CFTR, HEPES was able to block the outward rectifier from the extracellular surface and both the anionic and the zwiterionic forms of HEPES were effective. Yamamoto and Suzuki (32) also showed that a 35-pS Cl- channel from cultured Drosophila neurons was blocked by HEPES and MOPS. In CFTR, Sheppard et al. (24) and McCarty et al. (19) reported that CFTR showed brief flickery closings at hyperpolarizing voltages, an effect that may have been due to the use of 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.

Our observations also have practical implications for future studies of CFTR function. For studies designed to evaluate the conductive properties of the channel, the use of tricine as a buffer may be preferable to MOPS because the lack of the flickery kinetics would allow a more accurate determination of single-channel current. In addition, study of channels at 35°C will increase single-channel conductance. In contrast, for studies designed to evaluate channel regulation and gating, the use of MOPS as a buffer and temperatures <35°C may be useful because these conditions allow the identification of two different conformational states within bursts of activity. These findings also reconcile the report of Gunderson and Kopito (10) with previous studies from our and other laboratories that did not find evidence for two different "conductance" levels: those earlier studies did not use conditions that would make the two different gating states apparent. Finally, previous studies have shown that single CFTR Cl- channel currents can occasionally be observed to enter subconductance states (27, 30). The effect of MOPS we report here is different from those earlier studies in that the "appearance" of a subconductance is only evident with heavy filtering and there is a specific pattern of its occurrence during a burst of activity.

The block we observed with MOPS reminds us of the behavior of CFTR studied in cell-attached patches. For example, Haws et al. (12) and McCarty et al. (19) showed that, in cell-attached patches, CFTR had a pronounced flickery behavior at hyperpolarizing but not depolarizing voltages. This gating is similar to the voltage-dependent behavior we observed with MOPS on the cytosolic surface of excised patches of membrane. Thus it is interesting to speculate that an intracellular anion might be a physiological blocker of CFTR. By analogy, intracellular polyamines are proposed to block and thereby regulate inwardly rectifying K+ channels (17).

Conformational changes in CFTR during an asymmetric gating cycle. The results show that CFTR can exist in three distinct protein conformations that can be distinguished as three different gating states. 1) The C state is a long closed state between bursts of activity. 2) The O1 state is an open state within bursts during which the channel conformation allows MOPS to bind a site in the pore and intermittently occlude Cl- flow. 3) The O2 state is an open state during which MOPS has a different, less well-defined interaction with the channel, perhaps producing a very fast block.

We found a strong asymmetry or directionality in the movement between these conformational states; they did not exist at thermodynamic equilibrium. The channel first moves from C to O1. This transition probably represents a series of steps that include binding of ATP to NBD1 and NBD2, but it appears to be hydrolysis of ATP at NBD1 that ultimately provides the energy for the entry into O1 and a burst of activity (4, 5, 8, 14). Then the protein is driven from the O1 conformation to the O2 state (Fig. 11) by ATP hydrolysis at NBD2. Consistent with this conclusion, AMP-PNP (which is thought to bind at NBD2) prevented the transition to O2 and prolonged the O1 state. This is also consistent with observations on the effect of pyrophosphate (10). Finally, the channel progresses from O2 back to the C state, a transition that appears to be irreversible because the channel will not open without ATP. In several respects, this model is similar to the one proposed by Gunderson and Kopito (10); an important difference is that we propose that ATP hydrolysis is required for the channel to open, based on previous work (1, 4, 8, 14, 16). Although the mechanisms involved in the O2-to-C transition are not known, it is interesting that it is temperature sensitive.

In Figs. 1, 2, 4, 6, and 8-10 we showed examples of these gating patterns and their variations. The most common pattern was a burst of activity that began with the O1 state and ended with the O2 state. This is consistent with our kinetic analysis that showed that, within a burst of activity, the first opening was shorter than the last opening, and the durations of the first and last openings were similar to the short and long open times identified by dwell-time analyses. However, other patterns were occasionally observed. For example, channels may have rarely closed from the O1 state (trace e in Fig. 1), especially in the presence of AMP-PNP (trace b in Fig. 10). Perhaps in these cases, ATP dissociated from CFTR (presumably NBD2) without undergoing hydrolysis. Nevertheless, our kinetic analysis suggests that the O1-to-C transition is uncommon.

Our results link the activity of the NBDs to alterations in a MOPS binding site. They indicate that input of external energy from ATP hydrolysis causes a conformational change in the pore. Thus they suggest that there may be a direct physical link between the NBDs and the MSDs in CFTR, a conclusion that provides a new insight into how ATP may gate the channel.

    ACKNOWLEDGEMENTS

We thank Pary Weber, Phil Karp, Theresa Mayhew, Virginia Song, and Amanda Niehaus for excellent assistance and our laboratory colleagues for helpful discussions. We especially appreciate the thoughtful comments and suggestions of Dr. David Sheppard. We thank Drs. John Marshall and Seng Cheng for the gift of C127 cells. We thank Boyd Knosp in the Image Analysis Facility for modeling MOPS.

    FOOTNOTES

This work was supported by the National Heart, Lung, and Blood Institute and the Howard Hughes Medical Institute (HHMI). H. Ishihara was an associate and M. J. Welsh is an investigator at the HHMI.

Present address of H. Ishihara: Second Dept. Med., Yamanashi Medical Univ., 1110, Shimogato Tamaho-cho, Nakakoma-gun, Yamanashi-ken 409-38, Japan.

Address for reprint requests: M. J. Welsh, Howard Hughes Medical Institute, Univ. of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242.

Received 10 March 1997; accepted in final form 14 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(4):C1278-C1289
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