©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutant (F508) Cystic Fibrosis Transmembrane Conductance Regulator Cl Channel Is Functional When Retained in Endoplasmic Reticulum of Mammalian Cells (*)

Eva A. Pasyk (§) , J. Kevin Foskett (1)(¶)

From the (1) Division of Cell Biology, Hospital for Sick Children and the Department of Physiology, University of Toronto, Toronto, Ontario M5G 1X8, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane-localized chloride channel. Some mutations in CFTR, including one which affects most patients (F508-CFTR), prevent CFTR from exiting the endoplasmic reticulum (ER) where it is synthesized. To examine whether normal and mutant CFTRs function as chloride channels when they reside in the ER, the patch clamp technique was used to measure currents in the outer membrane of nuclei isolated from mammalian cells expressing CFTR. Both F508-CFTR as well as CFTR were revealed to function as cAMP-regulated chloride channels in native ER membrane. These results represent the first demonstrations of functional activity of CFTR in the biosynthetic pathway and suggest that conformational changes in the mutant protein, although recognized by ER-retention mechanisms, do not necessarily affect CFTR chloride channel properties, which may have implications for pathophysiology and therapeutic interventions in cystic fibrosis.


INTRODUCTION

Cystic fibrosis (CF)() is characterized by abnormal epithelial ion and fluid transport due to mutations in the CF gene product, CFTR (1, 2, 3, 4, 5) , a cAMP-regulated, DIDS-insensitive, linear, 6-14 picosiemens (pS) Cl channel (6, 7) . The most common (affecting 90% of patients) of many mutations in CFTR is a deletion of phenylalanine, residue 508 (F508-CFTR) (4) . Mammalian cells expressing F508-CFTR lack Cl permeability because the mutant protein fails to exit from the endoplasmic reticulum (ER) (8, 9, 10, 11, 12, 13, 14, 15, 16, 17) and localize normally in the plasma membrane (2, 18) . Abnormal processing of F508-CFTR is believed to result from its structural misfolding, since the processing defect is temperature-sensitive (19, 20) ; functional revertants have been generated by inserting point mutations in the protein (21) , and synthetic peptides and full-length CFTR containing the 508 mutation have reduced stability (20, 22) . In insect cells and Xenopus oocytes in which CFTR does not undergo biosynthetic arrest, plasma membrane-localized F508-CFTR functions as a Cl channel (23, 24) , and purified F508-CFTR from insect cells retains Cl channel activity when reconstituted in bilayers (23) . Furthermore, gross overexpression (25) or growth of some cell types at low temperatures (19, 20, 24) enables some F508-CFTR to reach the plasma membrane, where it then has Cl channel activity. Nevertheless, these studies do not address whether F508-CFTR functions as a Cl channel when it is retained in the ER in CF patients. Because the molecular mechanisms underlying ER retention have not been determined, it is unknown whether the Cl channel activity of mutant CFTR is affected by those conformational changes that cause its ER retention or whether the mechanisms that enable F508-CFTR to escape from the ER, presumably by enabling it to attain its native conformation, also influence its Cl channel function. Moreover, it is also unknown whether the retention mechanisms themselves alter or abolish the Cl channel activity of CFTR. The consequences of F508-CFTR Cl channel activity, or lack thereof, in the ER cannot be predicted because it has also not been determined if wild-type CFTR functions there. It has been proposed that several unexplained biochemical abnormalities in CF result from defective CFTR Cl channel activity in the Golgi (26) . Lacking, however, are measurements of CFTR activity in the biosynthetic pathway, the cellular compartments including ER and Golgi through which proteins traverse during their synthesis, processing, and trafficking to the plasma membrane. Here we demonstrate that both CFTR as well as F508-CFTR are functional cAMP-regulated Cl channels in native ER membrane in mammalian cells. These channel activities may indicate functional roles of CFTR in the biosynthetic pathway and suggest that lack of such activities due to ER retention of mutant CFTRs could underlie some pathophysiology of CF.


MATERIALS AND METHODS

Three CHO lines that stably expressed wild-type CFTR, F508-CFTR, or the vector only (controls; no CFTR) (16, 27) were used. Western blot analysis using a CFTR monoclonal antibody (Genzyme 24-1) revealed a 160-kDa band and a lesser 140-kDa band, reflecting mature extensively glycosylated and pre-Golgi core-glycosylated CFTR, respectively, in cells expressing wild-type CFTR; lower levels of only the 140-kDa band in the cells expressing F508-CFTR; and an absence of CFTR in control CHO cells, as previously reported (16) (data not shown). CHO cells were cultured in 75-cm flasks at 37 °C in -minimum essential medium supplemented with 7% fetal calf serum, 1% penicillin, and streptomycin and 20 or 200 µM methotrexate (vector- or CFTR-transfected cells, respectively). In most experiments (90%), cells were treated with sodium butyrate (5 mM) for 18 h prior to isolation (14) . Confluent cells were suspended by brief trypsinization, washed twice with the phosphate-buffered saline and once in an isolating solution containing (in mM): 120 KCl, 5 MgCl, 10 HEPES, and 1 phenylmethylsulfonyl fluoride, pH 7.0 with NaOH, resuspended in 1 ml of the isolating solution, and homogenized at 4 °C with a motor-driven Teflon pestle (Glas-Col) at 1700 rpm for 30 s (30 strokes). The integrity of nuclear membranes of isolated nuclei was verified by electron microscopy. Nuclei that attached to the glass bottom of a 1-ml chamber were perfused with solutions for patch clamp experiments. Heat-polished patch pipettes (tip, <0.5 µm in diameter) filled with the patch clamp buffer had resistances of 20-50 megaohms. Stable (10-60 min) seals (20-100 gigaohms) of the outer nuclear membrane were obtained in 70-80% of attempts. Thus, nuclear pores were closed and impermeable in our nuclear membrane patches, in agreement with patch clamp studies of isolated Xenopus oocyte nuclei (28) . Unless specified, in all experiments the excised patch configuration was used and the pipette and bath contained (in mM): 120 NMDG-Cl, 3 MgCl, 0.1 CaCl, 1.1 EGTA, 10 HEPES, 1 MgATP, 5 glucose, pH 7.1, with HCl, with PKA (90 nM) included in the pipette solution (cytoplasmic face of the membrane faces into the pipette). DIDS was added directly to the bath to achieve final desired concentration. All experiments were performed at room temperature. The applied potential was: V - V = V - V (in excised patches). Accordingly, positive current flowed from pipette to bath. Currents were amplified (Axopatch-1D, Axon Instruments, Inc.), digitized (Macintosh computer via ITC-16 interface, Instrutech Corp.), and written directly onto the hard disk (Pulse+PulseFit software, HEKA Electronik). Data were analyzed using Pulse+PulseFit and MacTac (Skalar Instruments). All data were filtered at 300 Hz unless indicated. Open probability (P) of single channels was determined by summing all bursts of openings with closed times of <3 s from single channel patches. Total burst times from all single channel patches (53 and 8 s for F508-CFTR and wild-type CFTRs, respectively) were combined to calculate kinetic parameters. Data, expressed as mean ± S.E., were analyzed using standard statistical tests.


RESULTS AND DISCUSSION

We examined the Cl channel activities of ER-localized CFTRs by patch clamping the outer membrane of the nuclear envelope of nuclei isolated from CFTR-expressing CHO cells. Because the outer membrane is continuous with ER (29) , we reasoned that ER-localized ion channels would also be present there, as confirmed in recent single channel measurements of the inositol trisphosphate receptor in isolated Xenopus oocyte nuclei (28) . With the catalytic subunit of PKA and ATP in the pipette, excised patches from nuclei isolated from F508-CFTR-expressing cells revealed linear, 6-10-pS channels in 39/49 patches (Figs. 1-3). The channels were Cl-selective since similar channels were observed with bath and pipette solutions containing Cs instead of NMDG, and they were observed at the Cs reversal potential with asymmetric Cs (120 mM CsCl in bath versus 20 mM CsCl, 100 NMDG-Cl in the pipette, or vice versa) (3 patches; data not shown). In the absence of PKA, either no channels (8/12 patches) or only an endogenous low conductance Cl channel (below) was observed (4/12 patches). The PKA requirement for channel activation was confirmed by alternately patching the same nucleus in the presence and absence of PKA (n = 5 nuclei) (Fig. 2). The PKA-stimulated Cl channel activities were blocked by external I (Fig. 1B; 4/4 patches) and were DIDS-insensitive (0.1-0.5 mM) (Fig. 1D; 7/7 patches). Thus, the properties of the Cl channels in the nuclear membrane in F508-CFTR-expressing cells are highly reminiscent of those described for wild-type CFTR in the plasma membrane (6, 7) .


Figure 2: PKA dependence of F508-CFTR Cl channels in the outer nuclear membrane. Currents were recorded alternately in the absence and presence of 90 nM PKA in the pipette at two pipette potentials as indicated. Each trace is from a different patch excised from the same nucleus. Similar results were obtained from 6 nuclei (17 patches).




Figure 1: Properties of F508-CFTR Cl channels in excised patches of outer membranes of nuclei isolated from CHO cells. A, single PKA-activated F508-CFTR Cl channel. PKA (90 nM) was present in the pipette solution. Within bursts, P was 0.64, and analysis of open and closed time histograms revealed one open and one closed state ( = 42 ms and = 20 ms) (14). Pipette potentials were as indicated. The dashedline represents closed state (C). This figure is representative of 5 experiments. B, block of F508-CFTR single Cl channel activities by I. Shown is the excised patch with pipette solution containing (in mM): 120 KCl, 3 MgCl, 0.1 CaCl, 1.1 EGTA, 10 HEPES, 1 MgATP, 5 glucose, and 90 nM PKA, pH 7.1, with KOH. The bath (equivalent to extracellular side) contained a similar solution with 60 mM Cl replaced with I. Pipette potentials were as indicated. This figure is representative of 4 experiments. C, I-V relationships for A (opencircles) and B (closedcircles). The slope conductance under symmetrical conditions (A) was 8.8 ± 0.7 pS (3-5 measurements at each indicated voltage from 10 patches). In B with I on the lumenal side of patch, conductance was reduced to 3.5 ± 0.3 pS (n = 4 patches). The shift of extrapolated I-V curve (dashedline) toward positive potentials indicates the channel is selective for Cl over I, though currents at positive voltages could not be resolved. D, insensitivity of F508-CFTR Cl channels to DIDS. DIDS (0.3 mM) was without effect on multilevel F508-CFTR Cl channel activities. This figure is representative of 7 experiments.



No channels were detected in 24/56 patches of nuclei isolated from cells transfected with the vector only, whereas an endogenous linear 4-8-pS Cl channel was present in the rest (not shown). However, this channel could be distinguished from F508-CFTR because it (a) was observed only as a single channel, (b) displayed long closed states (several minutes) with only occasional single openings instead of bursts of openings seen with F508-CFTR, and (c) was blocked by DIDS (0.1-0.3 mM) (10/10 patches). This channel therefore more closely resembles a cAMP-independent Cl channel observed in plasma membranes of the CHO parental line (30) .

To evaluate the significance of F508-CFTR Cl channel activity, nuclei from cells expressing wild-type CFTR were also examined. Cl channels were observed that had essentially identical biophysical and pharmacological properties as ER F508-CFTR channels (Fig. 4). The frequency and kinetic properties of ER-localized F508-CFTR and wild-type CFTR were also similar. True single channels were rarely observed (5/39 and 2/14 active patches from F508-CFTR (Fig. 1A) and wild-type CFTR nuclei (not shown), respectively) whereas the remaining patches contained up to 20 channels (Fig. 1D and 2-4). In single channel patches, activities appeared as bursts of openings lasting 1.3-30 s (Fig. 1) separated by long (several minutes) closings. In contrast, multichannel patches remained continuously active (Fig. 1D and 2-4), and channels were often observed to ``lock'' into the open state, with no channel gating for long periods (mean, 8.8 ± 1.3 s; 16 observations from 5 patches) (Fig. 3A). Channel activities were often observed as simultaneous gating of two channels (Figs. 3B and 4B; 11/39 and 4/14 patches from F508-CFTR and wild-type CFTR nuclei, respectively). Such pronounced cooperative gating appears to be characteristic of CFTR in the ER and may suggest that CFTR functions as a dimer that favors pairs of channels being simultaneously open. Cooperative gating of plasma membrane-localized CFTR has also been observed (31) . The very long closed times in single channel patches and prevalence of simultaneous gating of channel pairs have not been reported for plasma membrane-localized CFTR, but the similar properties of wild-type and F508-CFTR Cl channels we observed suggest that these different kinetics are due to ER localization rather than altered conformation induced by the F508 mutation. Since processing of proteins in the ER is likely mediated by protein interactions, the apparent strong cooperative behavior of ER-localized CFTR, by suggesting CFTR-CFTR interactions, raises the possibility that such interactions may participate in retention or stability of normal and mutant CFTRs in the ER.


Figure 4: Properties of wild-type CFTR Cl channels in the outer nuclear membrane. A and B, PKA-dependent activation. The channels were observed with PKA in the pipette (14/18 patches) but not in its absence (9/10 patches). In the remaining patch, the endogenous Cl channel was present. In A and B recordings obtained alternately from the same nucleus in the presence and absence of pipette PKA confirmed the PKA requirement for channel activation (6/6 patches). This figure is representative of 10 experiments. A, multichannel patch with at least 4 channels. In single channel patches, P = 0.42, = 37 ms, and = 40 ms (80% of closures) and 80 ms (20% of closures) within bursts. B, simultaneous gating of pairs of wild-type CFTR Cl channels. The binomial probability of one channel open was 0.46 ± 0.01, whereas the observed probability was 0.07 ± 0.03 (p < 0.003). This figure is representative of 4 experiments. Current records in A and B are from different patches at indicated pipette potentials. Data in A were filtered at 200 Hz. C, linear I-V relationship for currents shown in A and B. The slope conductance under symmetrical solutions was 7.9 ± 0.7 pS (3 determinations at each indicated voltage from 6 patches). D, insensitivity of multilevel wild-type CFTR Cl channels to DIDS (0.3 mM). This figure is representative of 5 experiments.




Figure 3: Kinetic features of F508-CFTR Cl channels in the outer nuclear membrane. A, long open state frequently observed in F508-CFTR Cl multichannel patches. Channels became ``locked'' in the open state during the interval indicated by the double-arrowed line. This figure is representative of 16 observations. B, simultaneous gating of pairs of F508-CFTR Cl channels. The probability of observing both channels open simultaneously (0.49) was used to calculate P (39). The binomial probability of observing one channel open was 0.42 ± 0.11, whereas the observed probability was 0.16 ± 0.05. Thus, the probability of each current level did not follow a binomial distribution for two independent channels (p < 0.04). C, O1, and O2 represent closed and two open levels, respectively. This figure is representative of 11 patches.



CFTR Cl channel conductance has been detected in plasma membranes and endosomes (32, 33) , but our evidence that wild-type CFTR can function as a regulated Cl channel in the ER represents the first measurements of CFTR activity in the biosynthetic pathway. Because both wild-type as well as F508-CFTR function in the ER and their half-lives there are similar (17, 34) , ER retention of the mutant CFTR will not be expected to affect the ionic composition of the ER lumen. In contrast, function of wild-type CFTR in the ER suggests that retention of F508-CFTR will result in the absence of normal Cl channel activity in post-ER compartments. Abnormal trans-Golgi pH and sialylation and mucoprotein sulfation observed in CF have been proposed to be due to aberrant Cl channel activity in Golgi compartments (26) , which may also account for altered activities of other ion channels in CF airway cells (35, 36, 37) . By demonstrating that CFTR may normally function en route to the plasma membrane, our results provide experimental evidence consistent with such hypotheses. Lack of CFTR function in the post-ER biosynthetic pathway may therefore contribute to some symptoms and abnormal cellular physiology in CF. Because our results demonstrate that CFTR can function as a regulated Cl channel in the biosynthetic pathway, it will now be important to determine if it functions in this pathway in vivo. Many other mutations in CFTR also cause its intracellular retention (18, 38) . The methods we have described will now enable examination of those mutant CFTRs to determine whether they also retain Cl channel activities, as well as to determine whether other plasma membrane ion channels are functional immediately after their biosynthesis in the ER, which may have implications for cell-specific regulation of organellar ion composition and function.

The demonstration that the major disease-causing mutant CFTR retains Cl channel activity when it is localized in the ER in mammalian cells suggests that conformational changes caused by the F508 mutation, although recognized by ER-retention mechanisms (8, 13, 14, 15, 16) , do not necessarily affect CFTR Cl channel properties or activation by cAMP-dependent mechanisms. The F508 mutation causes some loss of structure in synthetic peptides (22), but our data indicate that F508-CFTR is unlikely to be grossly misfolded when it is retained in the ER in vivo. By suggesting that CFTR domains involved in ER retention may not necessarily contribute to CFTR Cl channel function, our results may have implications for therapeutic interventions based on delivery of mutant CFTR to the plasma membrane, since they suggest that pharmacological reagents that could promote escape of CFTR from the ER might do so without adversely affecting its Cl channel activity, a prerequisite for restoring normal function.


FOOTNOTES

*
This work was supported by grants from the Canadian Cystic Fibrosis Foundation and National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Canadian Cystic Fibrosis Foundation Fellow.

To whom correspondence and reprint requests should be addressed: Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6889; Fax: 416-813-5028; E-mail: foskett@resunix.ri.sickkids.on.ca.

The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid; pS, picosiemens; ER, endoplasmic reticulum; CHO, Chinese hamster ovary; PKA, catalytic subunit of protein kinase A; NMDG, N-methyl-D-glucamine.


ACKNOWLEDGEMENTS

We thank J. Riordan and X.-B. Chang for providing transfected CHO cells, S. Cheng for providing the CFTR antibody, D. Mak and Y. Marunaka for valuable help in single channel analysis, L. J. Huan and D. Wong for technical assistance, and C. Bear, M. Buchwald, D. Mak, and S. Grinstein for comments on the manuscript.


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