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Journal of Physiology (2001), 536.2, pp. 459-470
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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Cystic fibrosis (CF) is a recessive genetic disease that affects approximately 1 in 2500 Caucasians (Zielenski & Tsui, 1995). This disease is associated with defects in the synthesis or function of a PKA-regulated chloride channel called the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan et al. 1989). Epithelial cells from CF patients have reduced chloride conductance, which results in abnormal salt and water transport (Quinton, 1983). The major cause of death in CF patients is lung infection, although the functions of the gastrointestinal tract, pancreas, sweat duct and reproductive organs also can be affected (Abman et al. 1991). The most common CF-associated mutation is 
F508 (~70 % CF mutations), which causes defective trafficking of CFTR to the cell surface (Cheng et al. 1990). Other mutations (e.g. G551D) exhibit normal protein trafficking but lower channel activity; many of these mutations are associated with less severe disease (Sheppard et al. 1993; Illek et al. 1999). Studies of mutants with partial loss of channel activity may help us better understand the mechanisms that regulate CFTR channel permeation and gating.
What regulates the opening and closing of the CFTR channel (i.e. channel gating) is not well understood. CFTR is a member of the superfamily of ABC (ATP Binding Cassette) transporters with two nucleotide binding domains (NBD1 and NBD2) (Hyde et al. 1990). These two nucleotide-binding domains are major regulators of CFTR channel opening and closing (reviewed in Gadsby & Nairn, 1999). CFTR also has a large cytoplasmic regulatory domain (R domain) that is not present in other ABC transporters. Phosphorylation of the R domain by cyclic nucleotide-dependent protein kinases is required for CFTR activation (Cheng et al. 1991). How the NBDs and R domain interact to regulate CFTR channel activity, and whether other regions of CFTR contribute significantly to channel gating, are unanswered questions.
Several groups have proposed cyclic gating models for CFTR based on the effects of ATP, non-hydrolysable ATP analogues and NBD mutations on channel gating (Baukrowitz et al. 1994; Carson et al. 1995; Gunderson & Kopito, 1995). In several of these early models channel opening is activated by ATP binding and hydrolysis, primarily at NBD1. However, the openings achieved by NBD1 are brief unless ATP also binds to NBD2, which then stabilizes the channel open state (reviewed in Gadsby & Nairn, 1999). More recent studies indicate that ATP hydrolysis may not be necessary to activate the channel; namely, ATP binding alone may be sufficient to cause channel opening (Ramjeesingh et al. 1999; Aleksandrov et al. 2000; Ikuma & Welsh, 2000). Channel closing may then be caused by ATP dissociation from the NBDs either by hydrolysis or unbinding. It also has been argued that the NBDs may operate in parallel rather than in series to regulate channel gating (i.e. each NBD may be able to control both channel opening and closing), but that NBD2 may dominate at millimolar ATP concentrations (Ikuma & Welsh, 2000). Clearly, the NBDs are important regulators of CFTR gating, but their precise roles in this process are still a mystery.
The amino terminal tail of CFTR (N-tail) also appears to play a role in regulating CFTR channel activity. Previous studies have shown that a syntaxin isoform (1A) binds to the N-tail of CFTR and inhibits macroscopic CFTR currents (Naren et al. 1997, 1998). Blocking the syntaxin- CFTR interaction reverses the inhibitory effect of syntaxin 1A on macroscopic CFTR currents. Syntaxin 1A may regulate CFTR function in part by affecting the trafficking of CFTR channels to or from the cell surface (Peters et al. 1999). However, the direct binding of syntaxin 1A to CFTR raises the possibility that this protein could modulate channel activity via its interaction with the CFTR N-tail. Indeed, in a recent study from our laboratory we identified a region within the N-tail (residues 46-60) that contains a series of acidic residues (D47, E51, E54, D58) which positively regulate CFTR channel activity (Naren et al. 1999). These residues partition onto one surface of a putative helix in the CFTR N-tail. The mechanism by which these negatively charged residues modulate channel activity is unknown, although it may involve a physical interaction with a portion of the R domain (Naren et al. 1999).
Our earlier studies had indicated that alanine substitutions at these acidic residues in the N-tail of CFTR decrease channel activity (Naren et al. 1999). A disease-associated mutant, D58N, maps to one of these acidic residues (Cystic Fibrosis Genetic Analysis Consortium: www.genet.sickkids.on.ca./cftr). This mutation was found on a patient with CBAVD (Congenital Bilateral Absence of Vas Deferens), a disease that typically associates with mild CF alleles (De Braekeleer & Ferec, 1996). Aspartic acid (D) and asparagine (N) have similar structures, except for the lack of negative charge in asparagine. Analysing the single channel properties of this mutant could help define the importance of negative charge at this position, as well as help explain the effects of this mutation on CFTR function. Here we report that the D58N CFTR mutant exhibits reduced macroscopic currents and accelerated deactivation kinetics in intact oocytes, and briefer channel openings in excised membrane patches as compared with the wild-type channel. The reduced chloride channel activity of D58N CFTR may explain how this mutation causes disease.
To better define the functional role of the N-tail in CFTR channel gating, we examined the effects of mutating this cluster of acidic residues on the regulation of channel activity by PKA, ATP and by manoeuvres that block ATP hydrolysis (e.g. the poorly hydrolysable analogue, AMP-PNP). AMP-PNP, when used in combination with ATP, can induce prolonged openings or even lock open the wild-type CFTR channel, presumably through its binding to NBD2 (Hwang et al. 1994: Mathews et al. 1998b); thus, it is a useful reagent to examine the involvement of NBDs in CFTR regulation. We observed that wild-type CFTR and N-tail mutant channels were similarly phosphorylated by PKA and exhibited half-maximal activation at nearly identical ATP concentrations. However, the N-tail mutations prevented the prolonged channel openings that are normally induced by AMP-PNP or by an NBD2 mutation that inhibits ATP hydrolysis (K1250A). Based on these results, we propose a functional model in which the amino terminal tail of CFTR regulates channel gating by facilitating entry into a long open state and/or by stabilizing this longer open state. The gating transition that is modulated by the N-tail appears to be downstream of ATP binding and upstream of ATP hydrolysis, apparently at NBD2.
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METHODS |
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Mutagenesis
The cDNA-encoding wild-type CFTR was a kind gift of Dr Eric Sorscher of the University of Alabama at Birmingham. CFTR mutants were generated in the pcDNA3 plasmid (Invitrogen). The N-tail triple mutant (D47A, E54A, D58A) was prepared by using the Stratagene site-directed mutagenesis kit. D58N CFTR was constructed by PCR mutagenesis. A mutagenic oligonucleotide was included in a PCR reaction using a 1 kb fragment of pcDNA3-wild-type CFTR as template, which contained the coding region for the CFTR N-tail (amino acids 1-332). The upstream primer included an Asp 718 site; the downstream primer included a Kpn 21 site. After digestion the PCR product was ligated back into pcDNA3-wild-type CFTR pre-digested with Asp 718 and Kpn 21. Mutations were confirmed by sequencing the entire 1 kb fragment. For oocyte injections wild-type and mutant CFTR cRNAs were prepared by using the T7 Megascript transcription kit from Ambion Inc.
Phosphorylation experiments
For in vitro phosphorylation experiments, wild-type and mutant CFTR proteins were expressed in COS-7 monkey fibroblast cells using the vaccinia expression system and immunoprecipitated using a COOH terminal monoclonal antibody (Genzyme Corp.) as described previously (Naren et al. 1998). The assay was performed at 30 °C for 12 min in 50 mM Tris-HCl (pH 7.5), 1 mM MgCl2 and 0.1 % bovine serum albumin (BSA) using varying concentrations of PKA catalytic subunit (Promega) with 10 µCi [32P]ATP. The samples were resolved by SDS-PAGE (5 % gels) and analysed by autoradiography. For in vivo phosphorylation experiments, COS-7 cells expressing wild-type or mutant CFTR were washed with PO4- -free buffer (10 mM Hepes (pH 7.4), 141 mM NaCl, 3 mM KCl, 0.9 mM MgCl2, 1.7 mM CaCl2 and 10 mM glucose) and treated with 0.4 mCi ml-1 [32P]orthophosphate for 90 min in this buffer. The cells were washed in PO4- -free buffer minus label and treated with CFTR activation cocktail (10 µM forskolin, 100 µM 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) and 100 µM 3-isobutyl-1-methylxanthine (IBMX) for 10 min. The cells were then lysed and CFTR was immunoprecipitated, resolved by SDS-PAGE and analysed by autoradiography as described above. CFTR protein amounts in the cell lysates were analysed in parallel by performing immunoprecipitations from equal numbers of unlabelled cells at the same seeding and transfection. CFTR protein in the immunoprecipitates was detected by Western blotting as described previously (Naren et al. 2000).
Electrophysiology
Frogs were obtained from Xenopus One (Ann Arbor, MI, USA). Oocytes were collected under anaesthesia according to local ethical committee/national guidelines. The frogs were humanely killed after the final collection. Clumps of oocytes were defolliculated by being placed in a 10 ml plastic tube containing OR-2 solution (82 mM NaCl, 5 mM MgCl2, 2 mM KCl, 5 mM Hepes, pH 7.5) plus 2 mg ml-1 collagenase A. Oocytes were then harvested in OR-2 solution and transferred to half-strength Leibovitz medium (Life Technology) plus 15 mM Hepes (pH 7.5), 15 % heat-inactivated horse serum and 1 % penicillin/streptomycin overnight for recovery. Wild-type and mutant CFTR cRNAs were injected into oocytes 2-5 days before using for voltage or patch clamp recording. All recordings were done at 21-22 °C. For the two-electrode studies the cRNA amounts for wild-type CFTR and the mutants varied depending on the nature of the experiment and are indicated in the figures and legends. For the patch clamp studies 1 ng wild-type cRNA and 5-10 ng mutant cRNAs were injected. More mutant cRNAs were injected to provide sufficient channel activity for quantitative analysis of channel gating (see below).
Voltage clamp recordings were performed in a low-calcium ND-96 solution containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 0.2 mM CaCl2, 5 mM Hepes (pH 7.5). Macroscopic currents mediated by CFTR were activated by perfusion with the same low-calcium ND-96 solution containing 1 mM IBMX, 10 µM forskolin, and 200 µM dibutyrl-cAMP. Oocytes were clamped at -50 mV during the recording unless otherwise noted.
For the patch clamp studies oocytes were shrunk in the bath solution (see below) for 10-15 min before removing the vitelline membrane using fine tip forceps. All patch clamp recordings were done in an inside-out excised configuration in a bath solution containing 140 mM N-methyl-D-glucamine (NMDG), 0.5 mM MgCl2, 1 mM EGTA, 10 mM Hepes (pH 7.4 with HCl). The pipette solution contained 140 mM NMDG, 0.5 mM MgCl2, 0.2 mM CaCl2, 10 mM Hepes (pH 7.4 with HCl). Pipettes were pulled to a tip resistance of 15-20 M
from Corning 8161 glass (Warner Instruments). PKA catalytic subunit and Mg-ATP were added to the bath solution following the excision and were continuously present throughout the experiment. A final concentration of 80 units ml-1 PKA was used for all patch clamp experiments. The added Mg-ATP concentration was 1.5 mM unless otherwise noted. All patch clamp experiments were performed at 21-22 °C. The holding potential was -80 mV (pipette-side ground).
Data analysis
All curve fittings for the kinetic experiments were performed using Microcal Origin software. Single channel analysis was performed using pCLAMP6 (Axon Instruments). Signals from single channel recordings were filtered at 200 Hz. Only records with stable activity for greater than 10 min were analysed. Since CFTR channel openings are typically interrupted by very brief closures that are ATP-independent (intraburst flickerings), we ignored openings shorter than 20 ms to exclude these flickerings from our analysis. Significant differences in burst durations between wild-type and mutant channels were also observed when shorter cut-off times were used; however, the burst durations were obviously underestimated when using thresholds shorter than 20 ms (results not shown). Since most excised membrane patches contained multiple channels, we used the cycle time method described by Mathews et al. (1998a) to calculate the mean burst and interburst duration. Channel open probability (Po) was estimated as:
where ti is the time spent above a threshold set at 0.5, 1.5, 2.5 . . . times the single channel current amplitude, N is the number of channels in a patch and T is the duration of the record. Mean burst duration (
o) and interburst duration (c) were calculated by the cycle time method (Mathews et al. 1998a) as:
o = [(NPo)T]/(number of openings),
c = [(N-NPo)T]/(number of openings - 1).
In this method the calculation of burst duration is not dependent on knowing channel number, but the estimates of channel Po and interburst duration are dependent on the numbers of channels in a patch. Thus, this method provides reasonable estimates of burst duration, but may lead to underestimates of interburst duration (and overestimates of Po) for less active mutant channels for which the numbers of channels in the patch may be underestimated.
Data are presented as mean ± S.E.M. The numbers of oocytes (two-electrode voltage clamp studies) or patches (single channel studies) analysed are denoted as n. Student's unpaired t tests were performed to examine differences between wild-type CFTR and the mutants. Differences were considered to be statistically significant if P values were lower than 0.05 (indicated by asterisks in the figures).
Chemicals
The PKA catalytic subunit was purchased from Promega, and forskolin was from CalBiochem. All other chemicals were from Sigma Chemical Company.
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RESULTS |
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D58N CFTR exhibits reduced channel activity
The disease-associated D58N CFTR mutant exhibited lower macroscopic currents upon activation with a cocktail containing cAMP, forskolin and IBMX than wild-type CFTR when equivalent cRNA amounts were injected into Xenopus oocytes (Fig. 1). The currents mediated by D58N CFTR were somewhat greater than those observed for a triple mutant in which three acidic residues were replaced with alanines (D47A, E54A, D58A). The currents mediated by D58N CFTR and the N-tail triple mutant also deactivated faster than those of wild-type CFTR after removal of the cAMP-containing cocktail (Fig. 1C). The wild-type channel exhibited a complex time course of deactivation (i.e. a lag followed by a gradual decline), whereas the N-tail mutants exhibit a rapid decline with no lag that could be approximated as a single exponential decay (data not shown). Accelerated deactivation following washout of an activating cocktail, has been described for a number of CFTR gating mutants, notably those with mutations in NBD1 or the R domain (Wilkinson et al. 1996, 1997). This finding, coupled with our observation that each of these N-tail mutants can be expressed as the mature form of CFTR with no obvious defect in biosynthetic processing (Naren et al. 1999; Fig. 3 and, for D58N CFTR, unpublished data), suggested to us the possibility that the D58N mutation influences the gating properties of the CFTR channel.
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Figure 1. D58N CFTR and N-tail triple mutant (D47A, E54A, D58A) exhibit lower macroscopic currents and faster deactivation than wild-type CFTR A, schematic diagram of CFTR topology (left) and helical wheel plot of N-tail region of interest (right) showing locations of the mutations analysed in this study. B, macroscopic currents for wild-type (WT) CFTR, D58N CFTR and the triple mutant. CFTR-mediated currents represent currents activated with a cAMP-containing cocktail (see Methods). Equal amounts of wild-type, triple mutant or D58N CFTR cRNAs (1 ng) were injected into oocytes. n values are indicated above each bar. Error bars represent S.E.M. values. Asterisks indicate significant differences from wild-type CFTR (P < 0.05) C, deactivation of the macroscopic currents following washout of the cAMP-containing cocktail. Shown are representative current traces and mean estimates of the time to half-maximal deactivation (t1/2; n = 4 for wild-type CFTR, n = 4 for triple mutant CFTR, n = 5 for D58N CFTR). Deactivation half-time was estimated as the time required for the current to decrease to 50 % of the peak current attained following the application of cocktail. Since smaller currents tend to have faster deactivation rates (authors' unpublished observations), higher amounts of mutant CFTR cRNAs were injected (1 ng, 20 ng and 10 ng for wild-type, N-tail triple and D58N mutant CFTR, respectively) in order to achieve comparable macroscopic currents. In pilot experiments we observed that wild-type CFTR deactivated slower than the N-tail mutants, even when the peak currents mediated by the wild-type channel were lower (i.e. when we injected much less wild-type CFTR cRNA). Thus, the faster deactivation exhibited by the N-tail mutants cannot be explained simply by lower peak currents for these mutants. | ||
To test the effects of the D58N mutation on CFTR gating we examined the properties of D58N CFTR channels in excised membrane patches. D58N CFTR exhibited a lower single channel open probability (Po) under conditions that maximally activate the wild-type channel (Fig. 2). Although the Po of D58N CFTR was substantially reduced as compared with the wild-type channel, it was not as markedly reduced as the macroscopic currents in intact oocytes (see Fig. 1B). We cannot rule out the possibility that this quantitative difference is due to some effect of the D58N mutation on channel density in the intact oocyte, although it is just as likely that this difference is due to the different activating conditions used in these two assays. The unitary currents (i.e. single channel conductances) were not obviously affected by this or other N-tail mutations that we have tested (see also Naren et al. 1999). The reduction in Po exhibited by D58N CFTR was due in large part to a reduction in the duration of channel openings (i.e. to briefer open channel bursts; see Methods for details). Thus, this disease-associated mutant exhibits a decrease in Po and channel open time that is qualitatively similar to that previously observed for synthetic mutants in which D58 and nearby acidic residues were replaced with alanine (Naren et al. 1999).
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Figure 2. D58N CFTR exhibits decreased channel activity in excised membrane patches due in part to shorter channel openings A, representative patch clamp records from inside-out excised patches that contained at least two active wild-type (WT) or D58N CFTR channels. B, C and D, mean single channel open probabilities, open channel burst durations and interburst durations, respectively, for wild-type CFTR and D58N CFTR. See Fig. 1 legend for definitions of error bars and asterisks. Burst durations and interburst durations were estimated from multichannel patches using cycle time analysis as described in the Methods. The principal effect of the D58N mutation was to reduce the open channel burst duration. | ||
N-terminal tail mutations appear to have little effect on PKA phosphorylation or ATP sensitivity
Phosphorylation of the R domain by PKA is required for CFTR channel activation (Cheng et al. 1991). Since the N-tail can physically interact with a peptide containing the R domain in vitro (Naren et al. 1999), it is possible that mutations that disrupt this interaction could affect phosphorylation of this domain. As an initial test of this possibility, we examined the phosphorylation of several N-tail mutants by PKA. Mutants in which multiple acidic residues in the N-tail were replaced with alanine were chosen for these and the following experiments, since these mutants exhibit a greater degree of chloride channel dysfunction than the D58N CFTR mutant (Fig. 1 and Naren et al. 1999). We observed no apparent differences between any of the N-tail mutants and wild-type CFTR when bulk PKA phosphorylation was measured either in vitro (Fig. 3A) or in vivo (Fig. 3B). At present we cannot rule out the possibility that the N-tail mutations have more subtle effects on the phosphorylation of individual residues, since the R domain has at least four PKA sites that can be phosphorylated in vivo (Cheng et al. 1991). However, it appears that these mutations have little effect on global phosphorylation of the R domain.
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Figure 3. The N-tail mutations have little effect on bulk CFTR phosphorylation A, in vitro phosphorylation of wild-type (WT) CFTR and double (E54A, D58A) and triple N-tail mutants. CFTR constructs where expressed in COS-7 cells, immunoprecipitated using a C-terminal antibody and treated with | ||
We also tested the responses of wild-type CFTR and the N-tail triple alanine mutant to increasing concentrations of ATP in excised membrane patches. The N-tail mutations could inhibit CFTR activity by reducing the ATP sensitivity of channel activation, since ATP binding to one or both NBDs is required to open the channel (Anderson et al. 1991; Winter et al. 1994). However, the N-tail triple mutant exhibited markedly lower Po values than wild-type CFTR over a wide range of ATP concentrations (Fig. 4A). Fitting the data with the Hill equation yielded a K1/2 of 146 ± 43 µM for wild-type CFTR (Hill coefficient n = 0.77, R2 = 0.989) and a K1/2 of 170 ± 210 µM for the N-tail triple mutant (Hill coefficient n = 0.53, R2 = 0.952). Superimposing the ATP dose-response curves for the two channel constructs confirmed that their relative responses to ATP were similar (Fig. 4B). Thus, the reduced single channel Po of the N-tail triple mutant over a wide range of ATP concentrations cannot be explained simply by a change in ATP sensitivity. These results imply that the N-tail of CFTR regulates channel gating by influencing a step downstream of ATP binding to the site or sites (presumably the NBDs) that activate channel opening.
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Figure 4. ATP dependence of channel activity for wild-type CFTR and the N-tail triple mutant A, open probabilities of wild-type (WT) CFTR and the N-tail triple mutant in excised patches at different concentrations of Mg-ATP. The open probability at each concentration was estimated from at least three patches containing 1-5 channels each. The continuous lines were obtained by fitting the data to the Hill equation. B, data of A normalized to maximum Po showing little or no shift of ATP sensitivity for the N-tail mutant channel. C, histograms of burst durations for wild-type CFTR and the N-tail triple mutant observed in single channel records at 1.5 mM ATP. Shown are the distributions of open channel bursts detected in records containing only one detectable channel each. Data were collected from multiple single channel records of wild-type (n = 7) and N-tail mutant channels (n = 9). | ||
Figure 4C verifies that the reduction in Po exhibited by the N-tail triple mutant was due in part to a reduction in the duration of channel openings. Shown are the distributions of channel burst durations at 1.5 mM ATP that were observed in records that contained only one detectable wild-type or N-tail mutant channel. The N-tail mutant channel exhibited a narrower range of openings that overlapped with the briefer openings observed for the wild-type channel. The results of this analysis of single channel records are consistent with our previous estimates of mean open channel burst duration for this mutant (Naren et al. 1999) and D58N CFTR (Fig. 2) in which multichannel patches were analysed using the cycle time method (see Methods for details). The fact that the N-tail mutant failed to exhibit the longer openings that were observed for the wild-type channel is consistent with the notion that the CFTR channel can have multiple open states (Gadsby & Nairn, 1999), and implies that the N-tail mutations affect a transition between brief and long channel openings and/or the stability of these longer openings.
N-tail mutations inhibit the long open channel bursts that predominate under conditions of reduced ATP hydrolysis by NBD2
It has been argued that the hydrolysis of bound ATP at NBD2 and the subsequent release of hydrolysis product from this NBD terminates channel opening (Hwang et al. 1994; Carson et al. 1995). Thus, long channel openings have been interpreted as representing stable binding of ATP to NBD2. AMP-PNP is a non-hydrolysable analogue of ATP that traps the channel in a long open state at 22 °C (Hwang et al. 1994; Mathews et al. 1998b). If the N-tail mutations affect the hydrolysis of ATP or a step downstream of ATP hydrolysis, then these mutations should have no effect on the long bursts that are activated by AMP-PNP. Conversely, if these mutants affect the entry of the channel into this long open state or the stability of this state, then they should inhibit the activation by AMP-PNP. Figure 5 shows that the N-tail triple mutant activated much slower in response to AMP-PNP addition than wild-type CFTR (half-time of 140 ± 20 s for the triple mutant versus 21 ± 4 s for wild-type CFTR). Furthermore, the triple mutant exhibited a lower steady-state Po following AMP-PNP addition (Fig. 5C). These data imply that: (i) the N-tail of CFTR regulates gating at a step prior to ATP hydrolysis (possibly at NBD2) and (ii) the N-tail triple mutant channel appears to be defective both at entering into the long open state that is stabilized by AMP-PNP (as evidenced by the reduced rate of AMP-PNP activation) and at remaining within this long open state (as evidenced by the lower steady-state Po following AMP-PNP activation).
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Figure 5. The N-tail mutations inhibit channel activation by AMP-PNP A and B, representative traces showing wild-type (WT) and triple mutant single channel behaviour before and 3 min after adding 3 mM AMP-PNP. C, the effect of AMP-PNP (3 mM) on wild-type and mutant CFTR single channel Po as a function of time. AMP-PNP was added at time zero. Each point is the mean ± S.E.M. of at least three patches with multiple channels each. Data were fitted with sigmoidal functions as shown by the smooth lines. All experiments were done with 1 mM ATP continuously present in the bath. | ||
If the N-tail mutations destabilize the long channel openings that predominate under conditions of reduced hydrolysis by NBD2, then they should also inhibit the very long bursts that are characteristic of K1250A. K1250A CFTR is a NBD2 mutant that lacks a lysine at the Walker A motif that is critical for ATPase activity. This mutant normally exhibits slow deactivation kinetics and very long open channel bursts (Carson et al. 1995; Gunderson & Kopito, 1995; Wilkinson et al. 1996). We introduced the N-tail triple mutations into K1250A CFTR as another test of whether the N-tail regulates gating by affecting a step prior to ATP hydrolysis (i.e. at NBD2). The triple/K1250A mutant exhibited lower macroscopic currents (Fig. 6A) and faster deactivation (Fig. 6B) compared with K1250A in voltage clamp studies of intact oocytes. In single channel studies we observed that the very long bursts of K1250A CFTR were disrupted by the N-tail mutations (Fig. 7). The triple/K1250A mutant exhibited a lower single channel open probability due to a marked reduction in open channel burst duration (Fig. 7D). These results support the hypothesis that the N-tail controls channel gating at a step upstream of ATP hydrolysis (apparently at NBD2), and that one of the consequences of the N-tail mutations is to destabilize the long open state that otherwise occurs when ATP hydrolysis at this NBD is inhibited.
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Figure 6. The N-tail mutations inhibit the macroscopic currents and accelerate the deactivation kinetics of the hydrolysis mutant, K1250A CFTR A, macroscopic currents mediated by K1250A CFTR and triple/K1250A CFTR were measured in intact oocytes as described in Fig. 1 legend. Equal amounts of K1250A and triple/K1250A cRNAs (1 ng) were injected into oocytes (n = 6 oocytes for K1250A, n = 8 for triple/K1250A). B, deactivation of K1250A (n = 3) and triple/K1250A (n = 4) currents were monitored following washout of cAMP cocktail as described. 1 ng cRNA was injected for each construct. | ||
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Figure 7. The N-tail mutations disrupt the long open channel bursts of K1250A CFTR A and B, representative records from excised inside-out patches for K1250 and triple/K1250A, respectively (1.5 mM Mg-ATP for each). C and D, mean single channel Po and burst duration for K1250A (n = 4 patches) and triple/K1250A (n = 5 patches). | ||
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DISCUSSION |
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To date most studies of CFTR chloride channel gating have focused on the two nucleotide binding domains and the regulatory domain. The involvement of other regions of CFTR in the regulation of channel activity is poorly understood. Our earlier studies indicated that the N-tail of CFTR regulates channel activity apparently through its interaction with the amino terminal half of the R domain (Naren et al. 1999). This activity maps to a cluster of acidic residues that localizes to one surface of a putative helix in the N-tail.
In this study we examined the single channel properties and macroscopic current kinetics of a disease-associated mutant that maps to this region of the N-tail (D58N CFTR). This structurally subtle substitution (with the exception of the loss of negative charge) resulted in shortened burst duration in single channel studies and faster deactivation in whole cell voltage clamp studies. These effects of the asparagine substitution at residue 58 are similar to that previously observed for alanine mutations at this and neighbouring acidic residues (Naren et al. 1999). Our results indicate that: (i) the negative charge at this position is likely to be an important determinant of the involvement of the N-tail in regulating the duration of channel openings and (ii) the mild disease that associates with the D58N mutation could well be due to the partial loss of channel activity that is exhibited by this mutant.
At present we do not know the extent to which the accelerated deactivation observed in intact oocytes and the shortened single channel bursts observed in excised membrane patches for the N-tail mutants are mechanistically related. These kinetic effects occur over different time domains (seconds versus minutes). However, there does appear to be a rough correlation between macroscopic deactivation kinetics and single channel burst duration for a number of CFTR mutants that have been assayed for both parameters (e.g. K1250A CFTR, which exhibits both prolonged channel bursts and slower macroscopic deactivation; Carson et al. 1995; Gunderson & Kopito, 1995; Wilkinson et al. 1996). It seems likely that the rate of deactivation following removal of the cAMP-containing cocktail reflects at least in part the rate of channel dephosphorylation. If so, perhaps mutations that destabilize (e.g. N-tail mutations) or stabilize (e.g. K1250A) channel openings also influence the stability of channel phosphorylation. We did not detect any effect of the N-tail mutations on the steady-state phosphorylation of CFTR (Fig. 3); however, it is conceivable that the rates of dephosphorylation following stimulus removal are altered by these mutations. Analysis of the dephosphorylation kinetics of these and other CFTR gating mutants will be required to clarify this point.
ATP binding and hydrolysis at the NBDs play important roles in CFTR channel gating (Gadsby & Nairn, 1999). ATP binding to one or both NBDs is required for channel opening (Anderson et al. 1991), although it is still controversial whether the hydrolysis of ATP is required to initiate channel opening and whether the two NBDs operate in series or in parallel to control gating (Ramjeesingh et al. 1999; Aleksandrov et al. 2000; Ikuma & Welsh, 2000). AMP-PNP can lock the channel into a more stable open state at 22 °C, supposedly by binding to NBD2 (Hwang et al. 1994; Mathews et al. 1998b). To further examine the mechanism by which the N-tail modulates ATP-dependent channel gating, we tested the responses of the wild-type and N-tail triple mutant (D47A, E54A, D58A) to ATP and AMP-PNP in single channel studies. There were no obvious differences in ATP sensitivity or bulk phosphorylation between wild-type CFTR and the N-tail triple mutant. However, the N-tail mutant activated much more slowly and achieved a lower steady-state Po following the addition of the poorly hydrolysable AMP-PNP. In addition, the N-tail mutations destabilized the long bursts that are a feature of an NBD2 mutant (K1250A) that is defective at ATP hydrolysis (Ramjeesingh et al. 1999). The lack of effect of the N-tail mutations on ATP sensitivity indicates that ATP binding to the site or sites that activate channel opening is probably not markedly altered by these alanine substitutions. Also, it is unlikely that these residues dramatically affect ATP hydrolysis at NBD2 or post-hydrolysis steps in gating, since the inhibitory effects of the N-tail mutations on channel activity persisted when ATP hydrolysis was inhibited by AMP-PNP or by mutating NBD2. In summary, these data suggest that the N-tail regulates a step downstream of ATP binding but upstream of ATP hydrolysis, probably at NBD2.
Figure 8 provides a simplified functional model of the involvement of the amino terminal tail of CFTR in channel gating. CFTR channel openings are activated by ATP binding to one of the NBDs in accordance with recent findings (Ramjeesingh et al. 1999; Ikuma & Welsh, 2000). In this model, the CFTR N-tail modulates the transition from a brief open state to a longer open state. Such a transition between brief and long openings is a feature of most other models of CFTR gating. Entry into the long open state has been argued to be driven by ATP binding to NBD2 (reviewed in Gadsby & Nairn, 1999), but other mechanisms are possible (e.g. Ikuma & Welsh, 2000). As argued above, we favour the notion that this N-tail-regulated transition occurs downstream of ATP binding to sites that activate channel opening (one or both NBDs) because the N-tail mutants appear to have little effect on the ATP sensitivity of channel activation (Fig. 4). We place this transition upstream of ATP hydrolysis because these mutants do inhibit the long bursts that are otherwise activated by manoeuvres that inhibit ATP hydrolysis (AMP-PNP and K1250A mutation). Presumably this hydrolytic event occurs at NBD2, although we cannot rule out the involvement of NBD1 in this step since NBD2 mutations may indirectly affect ATP binding or hydrolysis at NBD1 (Ramjeesingh et al. 1999). It appears from our data that the N-tail both facilitates the entry of the channel into the longer open state (as evidenced by a decreased rate of activation of N-tail mutants by AMP-PNP) and inhibits exit from this longer open state (as evidenced by destabilization of the long bursts of K1250A CFTR by introducing the N-tail mutations).
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Figure 8. Functional model of the involvement of the N-tail in regulating CFTR channel gating According to this model the N-tail mutants are defective at entering into or remaining within a long open state that is normally terminated by ATP hydrolysis. AMP-PNP and the K1250A mutation normally prolong CFTR openings by blocking ATP hydrolysis, presumably at NBD2. The N-tail mutations appear not to affect ATP binding per se (Fig. 4), although we cannot rule out the possibility that these mutations also have effects on channel opening (see Discussion). More complicated gating models have been proposed to account for the involvement of the multiple NBDs in CFTR gating; however, we show this simplified model because it accounts for the present data with a minimum number of closed and open states. | ||
The results of our K1250A and AMP-PNP experiments appear to link functionally NBD2 and the N-tail in the regulation of CFTR channel gating. However, we cannot exclude additional interactions among the N-tail, NBD1 and the R domain. For example, the N-tail binds in vitro to a recombinant peptide (amino acids 595-740 ) that includes the linker region between NBD1 and the R domain (Naren et al. 1999). The results of recent functional studies of severed CFTR molecules (Chan et al. 2000) and structural studies of the NBD of bacterial histidine permease (Hung et al. 1998) indicate that the domain boundary for the CFTR NBD1 probably extends beyond residue 595. Thus, the R domain peptide that binds to the N-tail probably also includes the distal portion of NBD1. The R domain and both NBDs have been reported to physically as well as functionally interact (Neville et al. 1998; Lu & Pedersen, 2000); these domains may act co-operatively to regulate CFTR channel gating. Conceivably, the N-tail stabilizes CFTR channel activity by modulating interactions among these various domains. In this regard, it may be relevant that severed CFTR molecules that lack the R domain also exhibit shortened open channel bursts (Csanady et al. 2000). Perhaps the N-tail influences interactions between the phosphorylated R domain and one or both NBDs to help link channel activity to phosphorylation state. More biochemical and structural studies will be required to distinguish between this and other possible mechanisms by which the N-tail participates in channel gating.
Our data do not rule out the possibility that the N-tail may also play a role in regulating the opening of the channel. In this regard, the longer interburst duration exhibited by D58N CFTR (Fig. 2D) implies that this mutation may also inhibit channel opening. In addition, we have observed that chemical modification of these residues can inhibit channel opening rate in excised membrane patches (J. Fu & K. L. Kirk, unpublished data). Thus, although it seems clear that the N-tail plays a role in controlling how long the CFTR channel remains open, this region of the polypeptide may also be important for modulating how often the channel opens.
In summary, we have observed that a cluster of acidic residues in the amino terminal tail positively regulates CFTR activity by stabilizing channel openings. The present functional data indicate that this modulatory site plays a role in controlling the accessibility or stability of a long open state at a step that is downstream of ATP binding and upstream of ATP hydrolysis, presumably at NBD2. In addition, our analysis of the D58N CFTR mutant has revealed that a disease-associated mutation that maps to this cluster of acidic residues also inhibits channel activity. This defect in channel gating presumably contributes to the disease pathology that is a feature of this mutation. The involvement of the amino terminal tail of CFTR in channel gating may provide a physiological mechanism for fine tuning CFTR activity by proteins that bind to this cytoplasmic tail (e.g. syntaxin 1A). In addition, the amino terminal tail of CFTR could be a potential target for drugs to treat some forms of cystic fibrosis or disorders that are caused by hyperactivation of this chloride channel (i.e. secretory diarrhoea; Gabriel et al. 1994).
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Acknowledgements
The authors thank Saroja Reddy, Ge Li and Holly Gentry for excellent technical assistance and Glenda Tawbush for secretarial support. They also thank Drs Dale Benos and Michael Quick for their support and guidance, and Dr David Dawson for the K1250A mutant. This study was supported by grants from the NIH (DK53090 and DK56796) and Cystic Fibrosis Foundation (Naren 99G0).
Corresponding author
K. L. Kirk: Department of Physiology and Biophysics, 982 B MCLM, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
Email: kirk{at}physiology.uab.edu
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[32P]ATP at varying concentrations of PKA (see Methods for details). Inputs correspond to aliquots of cell lysates analysed in parallel by immunoprecipitation followed by immunoblotting with a CFTR antibody (repeated two times with similar results; see Methods for details). Bands B and C represent immature and mature CFTR, respectively. B, in vivo phosphorylation of wild-type CFTR and double and quadruple (E54, D58, D47, E51) mutants expressed in COS-7 cells. Cells were preloaded with [32P]orthophosphate and CFTR was activated using a cocktail (100 µM cpt-cAMP, 10 µM forskolin and 100 µM IBMX) for 10 min at 37 °C. Cells were lysed and immunoprecipitated as described in Methods. Inputs represent the amounts of the indicated CFTR constructs detected by immunoprecipitating CFTR from equal numbers of unlabelled cells followed by immunoblotting using a CFTR antibody (see Methods for details). The phosphorylation signals correlated well with the input signals; e.g. in this particular experiment the densitometric ratios for the band C forms of wild-type CFTR, double mutant and quadruple mutant were 100:85:50 (inputs) and 100:88:54 (32P signal), respectively (repeated three times with similar results). The triple mutant behaved like the double and quadruple mutants (data not shown).