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J. Biol. Chem., Vol. 280, Issue 51, 41997-42003, December 23, 2005

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State-dependent Chemical Reactivity of an Engineered Cysteine Reveals Conformational Changes in the Outer Vestibule of the Cystic Fibrosis Transmembrane Conductance Regulator^*

From the School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230

Received for publication, September 19, 2005 , and in revised form, October 12, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are gated by binding and hydrolysis of ATP at the nucleotide-binding domains (NBDs). We used covalent modification of CFTR channels bearing a cysteine engineered at position 334 to investigate changes in pore conformation that might accompany channel gating. In single R334C-CFTR channels studied in excised patches, modification by [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET⁺), which increases conductance, occurred only during channel closed states. This suggests that the rate of reaction of the cysteine was greater in closed channels than in open channels. R334C-CFTR channels in outside-out macropatches activated by ATP alone were modified with first order kinetics upon rapid exposure to MTSET⁺. Modification was much slower when channels were locked open by the addition of nonhydrolyzable nucleotide or when the R334C mutation was coupled to a second mutation, K1250A, which greatly decreases channel closing rate. In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. These data indicate that the reactivity of the engineered cysteine in R334C-CFTR is state-dependent, providing evidence of changes in pore conformation coupled to ATP binding and hydrolysis at the NBDs. The data also show that maneuvers that lock open R334C-CFTR do so by locking channels into the prominent s2 subconductance state, suggesting that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The cystic fibrosis transmembrane conductance regulator (CFTR)² is a chloride channel with a predicted domain architecture including two membrane-spanning domains, each with six transmembrane domains, two nucleotide-binding domains (NBDs), and a regulatory domain (1). CFTR channel gating is controlled by binding and hydrolysis of ATP plus PKA-dependent phosphorylation at the regulatory domain; binding of ATP leads to dimerization of the two NBDs, which is linked to channel opening, whereas hydrolysis of ATP causes disassociation of the dimers and subsequent channel closure (2). Hence, these events in the NBDs induce conformational changes in the pore domain, which leads to opening and closing of the channel pore by an unknown mechanism.

Several lines of evidence suggest that the CFTR pore experiences more than two conformations (i.e. the closed and open states), including evidence that changes in anion selectivity and susceptibility to blockade are associated with the ATP-dependent gating cycle (3–14) (for a review, see Refs. 15 and 16). We recently showed that single wild type CFTR (WT-CFTR) channels exhibit two subconductance states as well as the full-conductance state (17); the stability and frequency of these substates are enhanced in some channels bearing mutations in the putative pore-lining regions. Using covalent labeling of channels bearing an engineered cysteine, we demonstrated that the subconductance and full conductance states represent different conducting states of a single chloride permeation pathway, which may reflect different conformations of the pore-lining helices (17). In that study, real time modification of single R334C-CFTR channels was observed during patch clamp experiments by the sulfhydryl-modifying agent, MTSET⁺, diffusing to the tip of the electrode; the resulting deposition of positive charge increased the open channel conductance. Strikingly, we never observed MTSET⁺-induced modification during an open burst. Therefore, we hypothesized that the accessibility or reactivity of the engineered cysteine in R334C-CFTR for modification by MTSET⁺ may be favored by the closed state. To test this hypothesis, we performed a series of experiments to measure the rate coefficients for modification by MTSET⁺ and MTSES^– under a variety of conditions that alter the channel open probability (P_o) of R334C-CFTR. Here we report for the first time in the CFTR pore that the rate coefficient for modification of an engineered cysteine by thiol-modifying reagents is significantly lower when channels are open compared with when channels are allowed to close. The results provide direct evidence that conformational changes in the outer vestibule of CFTR are linked to the ATP-dependent gating cycle.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Preparation of Oocytes and cRNA—For mutant R334C, site-directed mutagenesis used a nested PCR strategy in which the mutation was designed into antiparallel oligomers (18). The rest of the mutants used in this study were prepared with the QuikChange protocol (Stratagene; La Jolla, CA) using oligonucleotide-mediated mutagenesis. All mutant constructs were verified by sequencing across the entire open reading frame before use. For macropatch recordings, cRNAs were prepared from a high expression construct, which was kindly provided by Dr. D. Gadsby (Rockefeller University). Oocytes were injected in a range of 5–100 ng of CFTR cRNAs. Oocytes were incubated at 18 °C in modified Liebovitz's L-15 medium with the addition of HEPES (pH 7.5), gentamicin, and penicillin/streptomycin. Recordings were made 24–72 h after the injection of cRNAs.

Electrophysiology—The electrophysiological recording methods used were similar to those described previously (17). For single channel recording, CFTR channels were studied in excised, inside-out patches at room temperature (22–23 °C). All single channel recordings for R334C- and R34C/K1250A-CFTR used asymmetrical [Cl^–] in order to increase the single channel amplitude at V_M = –100 mV, where the pipettes were filled with a low [Cl^–]-containing solution (see below). Oocytes were prepared for study by shrinking in hypertonic solution (200 mM monopotassium aspartate, 20 mM KCl, 1 mM MgCl₂, 10 mM EGTA, and 10 mM HEPES-KOH, pH 7.2) followed by manual removal of the vitelline membrane. Pipettes were pulled in four stages from borosilicate glass (Sutter Instrument Co.; Novato, CA) and had resistances averaging 10 megaohms when filled with low [Cl^–] pipette solution containing 30 mM NMDG-Cl, 270 mM NMDG-aspartate, 5 mM MgCl₂, 10 mM TES (pH 7.4). The sulfhydryl-modifying reagents MTSET⁺ or MTSES^– (200 µM) were backfilled into the pipettes before seal formation and allowed to diffuse to the tip during and after seal formation; solution lacking MTS reagent was used to fill the very tip of the pipette. Typical seal resistances were 200 gigaohms or greater. R334C-, R334C/K464A-, and R334C/K1250A-CFTR channels were activated by excision into intracellular solution containing 300 mM NMDG-Cl, 1.1 mM MgCl_2, 2 mM Tris-EGTA, 1 mM MgATP, 10 mM TES (pH 7.4), 50 units/ml PKA. In experiments designed to increase P_o of R334C-CFTR channels, 2.75 mM AMP-PNP was added to intracellular solution containing 1 mM MgATP and 100 units/ml PKA. CFTR currents were measured with an Axopatch 200B amplifier (Axon Instruments; Union City, CA) and were recorded at 10 kHz to DAT tape. For subsequent analysis, records were filtered at a corner frequency of 100 Hz and acquired using a Digidata 1322A interface (Axon) and computer at 2.5 ms/point with pClamp 8.0.

For outside-out macropatch recordings, electrode tips were filled with a modified intracellular solution (150 mM NMDG-Cl, 1.1 mM MgCl_2, 2 mM Tris-EGTA, 10 mM TES, pH 7.4) and then backfilled with the same solution containing either 1 mM MgATP plus 100 units/ml PKA or 1 mM MgATP, 100 units/ml PKA, and 2.75 mM AMP-PNP. Through time, nucleotides and PKA diffused to the intracellular face of the outside-out patch; CFTR channels were fully activated in 50–75 min. The normal extracellular solution (150 mM NMDG-Cl, 5 mM MgCl₂, 10 mM TES, pH 7.4) served as bath solution, to which we added MTSET⁺ or MTSES^– to reach final concentrations of 10–100 µM, respectively. The pipette potential was held at 0 mV and then stepped to either +80 or –80 mV during exposure to MTSET⁺. In the case of modification by MTSES^–, the pipette potential was held at 0 mV and then stepped to +80 mV during perfusion of MTSES^–. A fast perfusion system (model SF-77B; Warner Instruments, Hamden, CT) controlled by pClamp software was employed for all outside-out macropatch experiments; the time constant for solution exchange using this system is <25 ms as judged by activation of endogenous calcium-activated chloride channels (17). Outside-out macropatch recordings were performed with an Axopatch 200B amplifier operated by pClamp 8.0 software, filtered at 200 Hz, and analyzed using Clampfit 9.0.

Analysis of Single Channel and Macropatch Experiments—For single channel recordings, transition analysis used a 50% cut-off between the open and closed current levels. Burst duration analysis was completed on records from patches containing 1–4 active CFTR channels; we used a minimum interburst duration cut-off of 100 ms to discriminate between interburst gating and fast intraburst closings (19). The mean open burst duration was estimated as previously described (19, 20). The dwell time histograms of mean burst duration were constructed with Igor software (Wavemetrics; Lake Oswego, OR) and then fit to an exponential function. The bin widths for R334C-CFTR recordings in the absence of AMP-PNP were 100 ms, and for R334C-CFTR in the presence of additional AMP-PNP or for a dual mutant R334C/K1250A, bin widths were 500 and 1000 ms, respectively.

For outside-out macropatch experiments, we used pClamp 9.0 to fit the time course of covalent modification of CFTR currents using an exponential function to obtain the time constant, , which was converted to a rate coefficient with units of M^–1 s^–1 by dividing by [MTSET⁺] or [MTSES^–]. In all experiments, we compared the quality of the fit of the data by a single exponential function and the fit by the sum of two exponentials by assessing the values of the correlation coefficient and S.D., as well as by visual inspection of the goodness of fit to the data trace itself. For those experiments where the data were described best by a single exponential function (Figs. 2A, 5B, and 7A), fitting the data with the sum of two exponentials led to 1) a smaller correlation coefficient, 2) a larger S.D. value, and 3) poor superimposition of the fit line on top of the data trace.

Source of Reagents—Unless otherwise noted, all reagents were obtained from Sigma. MTSET⁺ and MTSES^– were purchased from Toronto Research Chemicals Inc. MTSET⁺ and MTSES^– were first suspended in deionized water at a concentration of 100 mM, frozen in aliquots at –20 °C, and thawed and diluted into recording solution immediately before use. L-15 medium was from Invitrogen. PKA was from Promega (Madison, WI).

Statistics—Unless otherwise noted, values given are mean ± S.E. for n observations. Statistical analysis was performed using the t test for unpaired or paired measurements by Sigma Stat 2.03 (Jandel Scientific; San Rafael, CA), with p < 0.05 considered indicative of significance.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
R334C Channels Are Modified by MTSET⁺ Only in the Closed State—We have shown previously that the MTSET⁺-induced covalent modification of a cysteine engineered at CFTR position 334 (in transmembrane domain 6) increased single channel conductance without altering gating properties (17, 18). In contrast, WT-CFTR does not respond to either MTSET⁺ or MTSES^– when these reagents are applied to the outside surface of the membrane (data not shown; see Ref. 18). Excised, inside-out patch clamp recordings in which the pipette was backfilled with MTSET⁺ allowed us to watch the process of modification in real time as the MTSET⁺ diffused to the electrode tip and deposited positive charge at the engineered cysteine (Fig. 1A). Despite hours of recording of R334C-CFTR channels, the process of modification was observed to occur only during the closed interval (i.e. sometime between the last opening with lower conductance and the first opening with higher conductance). This observation led us to hypothesize that modification of R334C-CFTR might be favored by the closed state. To test this hypothesis, we coupled the R334C mutation with a mutation at the Walker lysine of NBD2, K1250A, which prolongs the open burst duration of CFTR channels (21–23). Fig. 1B shows a recording of a single R334C/K1250A-CFTR channel, where the electrode was backfilled with 200 µM MTSET⁺. Modification was delayed until the channel transitioned to a brief closed state. Upon reopening, channel amplitude was increased, reflecting covalent modification by MTSET⁺. Hence, even when P_o was increased by the K1250A mutation, modification at R334C did not take place in the open state. These observations strongly suggest that modification of R334C-CFTR by MTSET⁺ is favored by the closed state.

As shown in Fig. 1A and reported previously (17), R334C-CFTR channels exhibit stable subconductance behavior, including transitions to s1, s2, and f conductance states. In contrast, all of the open bursts of R334C/K1250A-CFTR before modification lacked transitions between conductance states and were "locked" in the s2 state (Fig. 1B). Following MTSET⁺-induced modification, R334C/K1250A-CFTR channels opened to, and remained locked in, the s2 state. Amplitudes for the s2 state in R334C/K1250A-CFTR were not significantly different from the amplitudes of the s2 state of R334C-CFTR before and after modification (p = 0.85) (17). These data suggest that interruption of the ATP-dependent gating cycle leads to stabilization of the pore conformation, resulting in fewer transitions between the three open conductance levels characteristic of R334C-CFTR; this provides further support for the notion that transitions between open conductance levels in CFTR channels are linked to NBD-mediated gating events.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Real time modification of R334C-CFTR and R334C/K1250A-CFTR channels by MTSET+. A, a representative trace for R334C-CFTR in the excised, inside-out patch configuration during real time modification by MTSET+ backfilled in the pipette. Over the course of 10–15 min, the channel was modified by MTSET+ diffusing into the tip of the patch pipette, as reflected by the increase in the single channel amplitude. Modification occurred between the end of the last unmodified opening with lower amplitude and the beginning of the first modified opening with higher amplitude (i.e. while the channel was closed). Current levels for unmodified R334C-CFTR are indicated by the four dashed lines (in order from top to bottom) c, s1, s2, and f, representing the closed, subconductance 1, subconductance 2, and full conductance states. B, representative trace for R334C/K1250A-CFTR under identical conditions. Current levels are indicated by dashed lines; c, o-pre, and o-post represent closed, unmodified open, and modified open, respectively.

View larger version (12K): [in this window] [in a new window]   FIGURE 2. Modification of R334C-CFTR is slowed in the presence of AMP-PNP. A, a representative recording of macroscopic current of R334C-CFTR. The pipette potential was held at 0 mV and then stepped to +80 mV. The arrow indicates the rapid application of 10 µM MTSET+ to the outside surface of the patch. The dashed line indicates a fit of the data to a first-order exponential function with = 5.3 s in this record. B, a representative recording of macroscopic current from R334C-CFTR in the presence of ATP + AMP-PNP. The kinetics of modification under this experimental condition were described best by the sum of two exponential functions, with values of 1 = 5.6 s and 2 = 104 s in this record.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Real time modification of R334C-CFTR in the presence of ATP + AMP-PNP. Three traces from one single channel patch recording in excised, inside-out mode, with real time modification by diffusion of MTSET+ to the patch tip. This individual patch contained at least three R334C-CFTR channels, recorded in the presence of ATP plus AMP-PNP. Experimental conditions were otherwise identical to those in Fig. 1. A, the first trace, from early in the experiment, shows two channels locked open (the upward arrow indicates an example of a locked open burst) and at least one undergoing normal gating (arrowhead). Neither is modified. B, in the second trace, from near the middle of the experiment, only the channel that was not locked open has undergone modification. C, in the third trace, later in the record, both channels have been modified, but only one is locked open. The arrowheads indicate the s2-to-f transition in unlocked channels, for an unmodified channel in the upper trace and for modified channels in the middle and lower traces. The lower dashed line in the bottom trace shows that channels locked open by AMP-PNP were locked into the s2 open state (compare with the unlocked opening at the left). The upper dashed lines represent the closed current level.

Kinetics of Macroscopic Modification Were Altered in NBD Mutants—As described above, CFTR channel P_o can be altered by mutations at the Walker A lysines that are involved in catalysis of ATP (21–23). Mutation K1250A reduces the channel closing rate (Fig. 1B). Mutation K464A in NBD1 leads to a great reduction in the channel opening rate. We studied outside-out macropatches from oocytes expressing R334C/K1250A- or R334C/K464A-CFTR to determine the effects of these gating domain mutations on the kinetics of modification, using experimental procedures similar to those described above. Upon exposure to MTSET⁺, the macroscopic current for R334C/K1250A-CFTR increased rapidly at first, followed by a slower increase in current, reflecting a complicated modification process (Fig. 5A); the kinetics of modification were described best by the sum of two exponential functions. Hence, the consequences of introducing the K1250A mutation were similar to the consequences of the addition of nonhydrolyzable nucleotide; the time-course of macroscopic modification was biphasic, with a component that is much slower than that seen in the single mutant with ATP alone. In five experiments (TABLE ONE), ₁ averaged 12.5 ± 0.94 s (fractional amplitude 74.7 ± 1.3%), which was significantly larger than the value of for R334C-CFTR in the presence of ATP alone and the value of ₁ for R334C-CFTR in the presence of ATP + AMP-PNP (p < 0.001). For those five experiments, ₂ averaged 225 ± 29 s (fractional amplitude 25.3 ± 1.3%), which was somewhat larger than the value of ₂ for R334C-CFTR in the presence of ATP + AMP-PNP (p = 0.049). The modification rate coefficients for MTSET⁺ in R334C/K1250A-CFTR were 9,840 ± 626 M^–1s^–1 and 482 ± 65 M^–1 s^–1, respectively.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Effects of AMP-PNP on mean burst duration of unmodified R334C-CFTR channels. A, R334C-CFTR channels recorded with ATP and PKA. The patch contained at least three channels. B, a dwell time histogram constructed from mean burst durations in 519 bursts from 12 patches in experiments such as in A; under normal conditions, the mean burst duration histogram was fit best with a first-order exponential function (solid line), having a time constant B = 0.46 s. C, after R334C-CFTR channels were activated by ATP and PKA, 2.75 mM AMP-PNP was added to the solution. The patch contained at least two active channels. Under these conditions, some openings exhibited "normal" gating behavior (brief openings), and there were also some prolonged bursts reflecting channels locked open. D, a dwell time histogram was constructed from mean burst durations in 1266 bursts from six experiments such as in C; the histogram was fit best with the sum of two exponential functions, with B1 = 0.52 s and B2 = 10.3 s. Values in parentheses indicate fractional amplitudes for each component of the second-order fit.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. MTSET+-induced modification of R334C/K1250A-CFTR and R334C/K464A-CFTR. Shown are outside-out macropatches from oocytes expressing either R334C/K1250A-CFTR (A and C) or R334C/K464A-CFTR (B). Experimental conditions were identical to those in Fig. 2. The pipette potential was held at 0 mV and then stepped to either +80 mV (A and B) or –80 mV (C). The arrows indicate the rapid application of 10 µM MTSET+. The process of modification of R334C/K1250A-CFTR by MTSET+ at Vm =+80 mV was fit best with the sum of two exponential functions, with 1 = 14.8 s and 2 = 158 s in this experiment. The kinetics of modification of R334C/K1250A-CFTR by MTSET+ at Vm = –80 mV also were described best by the sum of two exponential functions, having values of 1 = 12.9 s and 2 = 126 s in this experiment. In contrast, the kinetics of modification of R334C/K464A-CFTR by MTSET+ were fit best with a first-order exponential function, with = 1.92 s in this experiment.

View larger version (37K): [in this window] [in a new window]   FIGURE 6. Kinetics of modification of single R334C/K1250A-CFTR channels. A, a real time modification experiment, where this patch contained at least three channels. Experimental conditions were identical to those in Fig. 3. Three traces are shown from one single channel patch recording. The dashed lines indicate the closed current level. The first trace shows two prolonged openings from channels locked open and at least one channel not locked open (arrowhead). Neither of the channels have yet been modified. In the second trace, only the channel that was not locked has undergone modification (arrowhead). In the third trace, from near the end of the experiment, both remaining channels have been modified. All openings of R334C/K1250A-CFTR channels exhibited conductance equivalent to the s2 conductance state of R334C-CFTR. B, the mean burst duration histogram constructed from open bursts of unmodified channels from experiments such as in A was fit best with the sum of two exponential functions, with B1 = 4.1 s and B2 = 20.4 s (548 bursts from n = 7 patches).

View larger version (10K): [in this window] [in a new window]   FIGURE 7. MTSES–-induced modification of R334C-CFTR and R334C/K1250A-CFTR. Outside-out macropatches were pulled from oocytes expressing either R334C-CFTR or R334C/K1250A-CFTR. Experimental conditions were similar to those described in the legends to Figs. 2 and 5. A, R334C-CFTR channels were activated by ATP plus PKA. The amplitude of macroscopic current was decreased by 79% upon modification by 50 µM MTSES– in this experiment. The dashed line indicates a fit of the data to a first-order exponential function, having = 4.5 s in this experiment. B, a representative recording of macroscopic current of R334C/K1250A-CFTR under identical conditions. The process of modification in the double mutant was fit best by the sum of two exponential functions (dashed line), with values of 1 = 10.8 s and 2 = 144 s, respectively, in this experiment. The amplitude of macroscopic current was decreased by 85% upon modification by MTSES–.

Because one might suggest that the apparent state dependence of modification reflects interference from Cl^– in or near the mouth of the channel, we next asked whether the direction of Cl^– movement affected the kinetics of modification by extracellular MTSET⁺. Fig. 5C shows a representative experiment, where V_m was held at 0 mV and then stepped to –80 mV. Upon rapid exposure to MTSET⁺, macroscopic inward current was increased, reflecting modification of R334C/K1250A-CFTR channels. The kinetics were fit best with a sum of two exponential functions, providing time constants that were very similar to those measured from experiments at V_m =+80 mV (₁ = 13.4 ± 0.8 s and ₂ = 217 ± 52 s; n = 3; p > 0.5). These results indicate that the direction of anion movement does not affect the rate of modification by MTS reagent at R334C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we made use of covalent modification of engineered cysteine residues to investigate potential changes in the conformation of the outer vestibule of the CFTR channel pore between open and closed states. Single R334C-CFTR channels studied using real time modification only showed a reaction to MTSET⁺ during a closed state, even when channel P_o was increased dramatically by exposure to mixtures of ATP and AMP-PNP or by the addition of the Walker A mutation K1250A. Macropatch currents recorded from oocytes expressing R334C-CFTR increased rapidly upon abrupt exposure to MTSET⁺ (or decreased rapidly upon abrupt exposure to MTSES^–). Under conditions that increase channel activity (i.e. R334C-CFTR with ATP + AMP-PNP, or R334C/K1250A-CFTR with ATP alone), the kinetics of modification were slowed. Under conditions that decrease channel activity (R334C/K464A-CFTR), the rate of modification was increased dramatically. These data are consistent with a difference in the reactivity of the engineered cysteine to MTS reagents between the open and closed channel states, which most likely reflects changes in the conformation of the pore, or at least the outer vestibule, driven by gating events at the NBDs. These results provide the first evidence of movement in the CFTR pore domain correlated with channel gating state.

Our data indicate that the rate of covalent modification at R334C differs dramatically between closed and open channels. This result could be explained by physical hindrance of the interaction between the reagent and the cysteine if the side chain were buried in protein or lipid during the open state. However, we previously found that the macroscopic conductance of whole oocytes expressing R334C-CFTR channels was sensitive to bath pH, due to titration of the partial negative charge on the unmodified cysteine (17, 18). This observation suggests that R334C indeed faces the water-soluble pore while the channels are open, because protons can access this residue. Hence, the state-dependent ability of MTS reagents to interact with the engineered cysteine of R334C-CFTR most likely reflects a difference in the reactivity of that cysteine during channel closure rather than physical obstruction that reduces accessibility. The difference in reactivity may reflect the impact of another residue, which shifts the pK_a for MTSET⁺. Hence, we cannot say that R334C changes its position between open and closed states but rather must limit ourselves to saying that the orientation of R334C relative to the other residue or the distance between them changes as a function of channel gating. Nonetheless, these data suggest that changes in the rate coefficients for thiol-modifying reagents at R334C under different experimental conditions reflect conformational changes in the outer vestibule of the CFTR pore, which are associated with ATP-dependent gating.

Our results also provide further evidence that transitions between the three major open conductance states are linked to ATP-dependent gating events at the NBDs. As described previously (17), channels formed by WT-CFTR and many pore domain mutants, including R334C-CFTR, exhibit two subconductance states (s1 and s2) as well as the full conductance state (f). The subconductance states in some mutant channels differ markedly in their stability and probability of occurrence from that seen in WT-CFTR (17).

In R334C-CFTR channels, the most stable conducting state is the s2 state, whereas in WT-CFTR channels, the most stable conducting state is the f state (17). Results from the present study show that when R334C-CFTR channels are locked open by either AMP-PNP or the addition of the K1250A mutation, they are locked into the s2 state. In contrast, previous studies show that WT-CFTR channels locked open by the same maneuvers are locked in the f state (21–23). These observations suggest that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs. Consistent with this notion, we recently reported that WT-CFTR channels locked open by either AMP-PNP or vanadate (and K1250A-CFTR channels with ATP alone) exhibit a reduced frequency of flickery closures compared with WT-CFTR channels in the presence of ATP alone (27). R334C-CFTR channels almost always transition briefly to the f state before closure (see the arrowheads in Fig. 3) (17); the f state may represent an unstable conformation that serves as a transition intermediate between the stable s2 state and the stable c state. Hence, the stability of the open conductance states appears to be determined by the processes of binding and hydrolysis at the NBDs. The mechanism that couples conformational changes at the NBDs to conformational changes in the pore is an interesting subject for future study.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant DK-056481 and American Heart Association Established Investigator Grant 0140174N (to N. A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: School of Biology, Georgia Institute of Technology, 310 Ferst Dr., Atlanta, GA 30332-0230. Tel.: 404-385-2955; Fax: 404-894-0519; E-mail: Nael.McCarty{at}biology.gatech.edu.

² The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PKA, protein kinase A; MTSET⁺, [2-(trimethylammonium)ethyl] methanethiosulfonate; MTSES^–, 2-sulfonatoethyl methanethiosulfonate; TES, N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid; NMDG, N-methyl-D-glucamine; AMP-PNP, 5'-adenylyl-,-imidodiphosphate; NBD, nucleotide-binding domain; WT-CFTR, wild type CFTR.

	ACKNOWLEDGMENTS

 
We thank G. Cui, M. Fuller, C. Thompson, and D. Dawson for comments.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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