| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
MS 0093 Received 10 September 1999; accepted after revision 24 January 2000.
 |
ABSTRACT |
|---|
 TopAbstract IntroductionMethodsResultsDiscussionReferences |
|---|
F508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of F508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and F508-CFTR channels expressed in NIH3T3 mouse fibroblasts.
F508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel.
F508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that F508-CFTR is not defective in ATP-dependent gating.
F508-CFTR cell membrane. Our data showed that the estimated channel density for F508-CFTR was 
10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by 200-fold. We thus conclude that the functional defect (a decrease in Po) of F508-CFTR is as important as the trafficking defect (a decrease in the number of functional channels in the plasma membrane) in cystic fibrosis pathogenesis. |
INTRODUCTION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
CFTR is an epithelial chloride channel, mutations in which cause cystic fibrosis (Riordan et al. 1989). The predicted topology of CFTR includes two repeats of six membrane-spanning segments, two nucleotide binding domains (NBD1 and NBD2) and a regulatory (R) domain. It is believed that protein kinase A (PKA) activates CFTR by phosphorylating multiple serine residues in the R domain. However, after phosphorylation by PKA, the opening and closing of phosphorylated CFTR channels is coupled to ATP hydrolysis at NBD1 and NBD2 (reviewed by Gadsby et al. 1995; Gadsby & Nairn, 1999; Sheppard & Welsh, 1999; cf. Zeltwanger et al. 1999). Since cellular protein phosphatases constantly counteract the activity of PKA in intact cells, in vivo CFTR activity should be determined by a balance between phosphorylation by kinases and dephosphorylation by phosphatases, as well as by ATP-dependent gating of the phosphorylated channels.
More than 800 disease-associated mutations in the CFTR gene have been identified (Cystic Fibrosis Genetic Analysis Consortium, accessible at http://www.genet.-sickkids.on.ca). Deletion of a single amino acid, phenylalanine 508 (F508), accounts for 70 % of these mutations and is present in 90 % of the patients with cystic fibrosis. Two physiological abnormalities have been demonstrated for the F508-CFTR channel. First, most of the mutant proteins are retained in the endoplasmic reticulum (i.e. trafficking defect) and subsequently degraded (Cheng et al. 1990; Ward et al. 1995). Second, the small fraction of the mutant channels that actually reach the plasma membrane respond poorly to the cAMP stimulation (i.e. functional defect). The molecular mechanisms and the relative role of these defects in the pathophysiology of cystic fibrosis are yet to be elucidated. However, each defect can be rectified experimentally. The defect of F508-CFTR in membrane trafficking can be partially corrected by incubating cells at a lower temperature (Denning et al. 1992) or in the presence of chemical chaperones (e.g. Sato et al. 1996). Although the reduced open probability (Po) of the plasma membrane F508-CFTR in the presence of cAMP stimulation (Dalemans et al. 1991; Haws et al. 1996; Hwang et al. 1997) is not corrected by lowering the culture temperature (Hwang et al. 1997), pharmacological reagents such as genistein can dramatically increase the Po of F508-CFTR (Hwang et al. 1997).
Since millimolar 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, can ameliorate the functional defect of the F508-CFTR channel expressed in Xenopus oocytes (Drumm et al. 1991), it has been hypothesized that at high cellular cAMP, F508-CFTR can behave like wild-type (WT) channels. This hypothesis was further supported by studies using more specific phosphodiesterase inhibitors such as milrinone (Kelley et al. 1996). However, millimolar concentrations of IBMX exceeds the amount required for inhibition of phosphodiesterases (Yamamoto et al. 1983). It has been shown that millimolar IBMX does not further increase cAMP levels generated by a maximally effective concentration of forskolin (He et al. 1998). Furthermore, the relationship between [cAMP] and CFTR activity follows a saturating function, and at a maximally effective concentration of forskolin, the cAMP-dependent CFTR activity is already saturated (Al-Nakkash & Hwang, 1999). Thus, millimolar IBMX is likely to act on CFTR through a cAMP-independent mechanism (He et al. 1998; Al-Nakkash & Hwang, 1999). This conclusion also suggests that the functional defect of F508-CFTR channels resides in a step(s) beyond the production of cAMP and subsequent activation of PKA. We therefore examined both PKA-dependent phosphorylation activation and ATP-dependent gating of F508-CFTR to understand the biophysical basis of the functional defect.
In the present study, the PKA-dependent activation time course of WT- or F508-CFTR was studied in inside-out membrane patches excised from NIH3T3 cells expressing either WT- or F508-CFTR. A comparison of ATP-dependent gating behaviour between WT- and F508-CFTR after channels were maximally activated with PKA was also performed. For WT-CFTR, the rate of macroscopic CFTR channel current activation increased as the PKA concentration was elevated. At an equal concentration of PKA, the activation of F508-CFTR was slower than that for WT-CFTR by approximately one order of magnitude. However, the steady-state Po values and single-channel kinetics for PKA-phosphorylated WT- and F508-CFTR in the presence of 2·75 mM ATP were not different. Furthermore, the ATP dose-response relationships (macroscopic current or single-channel Po) were not different for WT- and F508-CFTR, suggesting that the F508 mutation does not cause abnormalities in ATP-dependent gating. Our data were consistent with the hypothesis that the functional defect of F508-CFTR is due to a slower PKA phosphorylation rate rather than a defect in gating. Since F508 is physically located in NBD1 whereas PKA phosphorylation is believed to occur mostly in the R domain, our conclusion also supported a recent structural model (Armstrong et al. 1998) that places F508 in a region of NBD1 involved in interdomain interaction.
|
METHODS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cell culture and electrophysiology
NIH3T3 mouse fibroblasts stably expressing WT- or F508-CFTR channels (NIH3T3-CFTR and NIH3T3-F508, respectively) were described previously (Berger et al. 1991). Cells were grown at 37°C and in 5 % CO2 in Dulbecco's modified Eagle's medium-H21 supplemented with 2 mM glutamine and 10 % fetal bovine serum. For some whole-cell experiments, NIH3T3-F508 cells were incubated at 27°C for 2-3 days before use.
Single-channel patch-clamp recording
Cells were passaged and grown on small, sterile glass chips in 35 mm tissue culture dishes. Prior to recording, glass chips were transferred to a continuously perfused chamber located on the stage of an inverted microscope (Olympus Corp., Tokyo, Japan). CFTR channel currents were recorded at room temperature (22°C) with a patch-clamp amplifier (EPC9, Heka Electronic, Lambrecht, Germany), filtered at 100 Hz with a built-in three-pole Bessel filter, and stored on videotapes. Data were subsequently refiltered at 25 Hz with an eight-pole Bessel filter (Frequency Devices Inc., Havervill, MA, USA) and captured onto a hard disk at a sampling rate of 50 Hz. Patch-clamp electrodes were made from Corning 7052 glass capillaries (Warner Instruments, Hammed, CT, USA). The pipette resistance was usually 3-5 M
, and the seal resistance was >20 G. The pipette solution contained (mM): 140 N-methyl-D-glucamine chloride (NMDG-Cl), 2 MgCl2, 5 CaCl2 and 10 Hepes (pH 7·4 with NMDG). The superfusion solution contained (mM): 150 NaCl, 2 MgCl2, 1 EGTA, 5 glucose and 5 Hepes (pH 7·4 with NaOH). For experiments using excised inside-out patches, the bath solution contained (mM): 150 NMDG-Cl, 10 EGTA, 10 Hepes, 8 Tris and 2 MgCl2 (pH 7·4 with NMDG). Pipette potential was held at 50 mV relative to the bath. Downward deflections represent channel opening.
Whole-cell patch-clamp recording
Whole-cell currents were recorded with an Axopatch-1D patch-clamp amplifier (Axon Instruments, Foster City, CA, USA). Voltage pulses were generated using Igor software (Wavemetrics, Lake Oswego, OR, USA) and XOP (developed by Dr Richard Bookman at the University of Miami, Miami, FL, USA). Current traces, filtered at 1 kHz with a built-in four-pole Bessel filter, in response to voltage pulses (-100 to +100 mV at 20 mV increments) were digitized (at 2 kHz) directly into the computer hard disk through an ITC-16 interface (Instrutech Corp., Greatneck, NY, USA). The pipette solution contained (mM): 85 aspartic acid, 5 pyruvic acid, 10 EGTA, 10 Hepes, 20 tetraethylammonium-Cl, 5 Tris, 10 MgATP, 2 MgCl2 and 5·5 glucose (pH 7·4 with CsOH). The bath solution contained (mM): 145 NaCl, 5 glucose, 5 Hepes, 2 MgCl2, 1 CaCl2, 5 KCl and 20 sucrose (pH 7·4 with NaOH). A chloride gradient of external concentration 156 mM and internal concentration 24 mM was used to generate an outward CFTR current at a holding potential of 0 mV. Sucrose (20 mM) was added to the bath to avoid activation of swelling-induced chloride conductance. This holding current at 0 mV was normalized with the membrane capacitance to yield a current density. Since in native NIH3T3 cells that are not transfected with CFTR neither forskolin nor forskolin plus genistein induced any current (n = 14), it is concluded that the currents activated in NIH3T3-CFTR or NIH3T3-F508 cells were carried by CFTR.
Reagents
Forskolin, purchased from Calbiochem, was stored as a 20 mM stock solution in dimethyl sulfoxide (DMSO) at 4°C. Genistein, purchased from LC laboratories (Woburn, MA, USA), was stored as a 100 mM stock solution in DMSO at -20°C. MgATP and all salts and buffers were purchased from Sigma, and MgATP was stored as a 250 mM stock solution. The catalytic subunit of protein kinase A (PKA) was purchased from Promega. All [PKA] are reported as assayed by Promega (Flockhart & Corbin, 1984). The same lot of PKA was used to avoid variations in specific activity.
Kinetic analysis
PKA activation time course analysis. After excision of the membrane patch into an inside-out mode, the pipette was moved to a position a few micrometres away from the solution outlet to ensure that the pipette tip was completely immersed in the stream of the perfusate. ATP (2 mM) was perfused to the patch to ensure an absence of channel activity due to basal phosphorylation. Freshly made PKA solution was then continuously perfused together with ATP until a steady-state macroscopic current was reached. The fast solution change perfusion system (< 10 µl dead-space volume and complete solution change within 1 s in the chamber) ensures that the diffusive process of these compounds is not the rate-limiting step of our experiments. The mean current amplitude was measured, using Igor software as described previously (Wang et al. 1998), over every 5 s for the first 2 min of PKA addition and subsequently over every 30 s until a steady state was obtained. These mean current values were then normalized to the maximal steady-state mean current amplitude to yield percentage activation. The PKA activation time course was fitted with a sigmoidal function or an exponential function using Igor software. This sigmoidal function is a product of multiple single exponential functions with the same time constant (see Fig. 2 legend).
ATP dose-response relationship of macroscopic F508-CFTR channel current. After a steady-state F508-CFTR channel activity was reached in the presence of PKA and ATP, PKA was removed from the bath and different concentrations of ATP were applied. Macroscopic F508-CFTR channel current at tested concentrations of ATP was bracketed with the current responses to 2·75 mM ATP (see Zeltwanger et al. 1999). To create the dose-response relationship between macroscopic F508-CFTR current and [ATP], the ratio of the mean current in the presence of tested [ATP] and that with 2·75 mM ATP was plotted vs. [ATP].
Single-channel kinetic analysis. We calculated single-channel Po from recordings that showed fewer than three simultaneous levels of open channel current over >3 min of continuous traces. All-point amplitude histograms were first generated with the Igor software. The area under individual peaks of the all-point amplitude histogram was then used to calculate Po using the following formula:
where A0, A1, . . . , AN are: the area under the closing peak, the first channel opening peak, . . . , the Nth channel opening peak, respectively. Dwell time analysis was performed for recordings containing a single channel using Igor software as described previously (Wang et al. 1998).
Data are expressed as arithmetic means ± S.E.M. Statistical comparison was made using Student's t test. A value of P < 0·05 was considered to be statistically significant.
|
RESULTS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
PKA-dependent phosphorylation activation of WT- and F508-CFTR
It has been shown previously that F508-CFTR channels respond poorly to cAMP stimulation with characteristically prolonged closed times in cell-attached patches (Dalemans et al. 1991; Haws et al. 1996; Hwang et al. 1997). Figure 1 shows representative current traces in cell-attached patches from NIH3T3 cells stably expressing F508-CFTR. In the presence of 10 µM forskolin only a few opening events were observed over a 3 min time span. Further addition of a maximally effective concentration of CPT-cAMP (200 µM, see Al-Nakkash & Hwang, 1999), a membrane-permeant cAMP analogue, failed to increase the channel activity. In four experiments the mean macroscopic current amplitude in the presence of forskolin and CPT-cAMP was 1·2 ± 0·3 times that in the presence of forskolin alone, suggesting that 10 µM forskolin already maximally activates the cAMP-dependent CFTR activity (He et al. 1998; Al-Nakkash & Hwang, 1999). Contrary to previous reports (Drumm et al. 1991; Kelley et al. 1996), these data suggest that supersaturating concentrations of cAMP fail to rectify the functional defect of the plasma membrane F508-CFTR channels. Thus, it is likely that the functional defect of F508-CFTR resides in the step(s) beyond the production of cAMP and the activation of PKA. Although F508-CFTR channels do not respond to increasing [cAMP], addition of 20 µM genistein in the continued presence of forskolin and CPT-cAMP caused a 28·5 (± 5·6)-fold (n = 12) increase in the mean macroscopic current amplitude. In patches containing one single F508-CFTR channel (Fig. 1, inset), it can be seen that genistein drastically increases the Po of F508-CFTR. By visual inspection, it appears that genistein prolongs opening bursts and shortens closings as reported previously (Hwang et al. 1997; Wang et al. 1998).
 |
View larger version [in this window] [in a new window] |
|
|
F508-CFTR and effect of genistein
A continuous current trace in a cell-attached patch shows activation of | ||
Since CFTR channel activity in an intact cell is determined by both PKA-dependent phosphorylation and ATP-dependent gating, defects in either process could account for the low Po of F508-CFTR observed in cell-attached patches. We first examined the PKA-dependent activation in inside-out membrane patches excised from NIH3T3 cells expressing either WT-CFTR or F508-CFTR by monitoring the time course for macroscopic current activation upon addition of purified PKA and ATP. After excision of the membrane patch into an inside-out configuration, 2 mM ATP was applied for 3-5 min to ensure that there was no channel activity before addition of PKA. Absence of channel opening prior to application of PKA in most of our patches suggests a negligible basal phosphorylation of the CFTR protein when expressed in NIH3T3 cells.
Figure 2A shows representative traces of the PKA-dependent activation of macroscopic WT-CFTR channel current in excised inside-out patches. As the [PKA] was increased from 6·25 to 62·5 U ml-1, the time taken to reach a steady state was shortened. Activation time courses (Fig. 2B) were quantified by plotting the normalized activation vs. time (see Methods). At lower [PKA] (6·25 or 12·5 U ml-1), the activation time courses show an initial delay. Fitting the data (smooth curves in Fig. 2B) with a sigmoidal function yielded time constants of 310·3 ± 23·4 s (n = 6) and 163·9 ± 10·4 s (n = 7), respectively. At higher [PKA] the activation time courses approximate a single exponential function with smaller time constants of 70·4 ± 6·5 s (n = 6) for 50 U ml-1 PKA and 35·9 ± 4·4 s (n = 6) for 62·5 U ml-1 PKA. Note that even at the highest [PKA] tested, the time constant is two orders of magnitude slower than the time constants for ATP-dependent gating of strongly phosphorylated CFTR (see below, and also Zeltwanger et al. 1999). Figure 3 shows the relationship between PKA concentration and the apparent activation rate (1/time constant). A curvilinear relationship between [PKA] and the apparent activation rate suggests that phosphorylation at multiple sites is required for maximal activation of CFTR channels.
 |
View larger version [in this window] [in a new window] |
|
|
Membrane patches were excised from NIH3T3-CFTR cells into an inside-out configuration. Pipette and bath contained equal [Cl-]. The membrane potential was held at -50 mV. A, PKA concentration-dependent activation of WT-CFTR. B, the time course of the PKA activation was generated by normalizing macroscopic CFTR current to the maximal steady-state level. The smooth lines were the result of fitting the data with a sigmoidal function, [1 - exp(-t/ | ||
 |
View larger version [in this window] [in a new window] |
|
|
 ) of macroscopic CFTR channel current
| ||
To compare the activation time course of F508-CFTR with WT-CFTR, 62·5 U ml-1 PKA was used to activate F508-CFTR (Fig. 4A). Figure 4B plots the percentage activation of F508-CFTR current vs. time. Compared to WT-CFTR in the presence of the same concentration of PKA, it took a much longer time to reach a steady-state activation for the mutant channel. The smooth curve in Fig. 4B represents the fit of the data with a sigmoidal function (time constant 252·0 ± 17·2 s, n = 7). Corresponding results for WT-CFTR are presented for comparison. Note that the shape and the time constant of the activation time course for F508-CFTR in the presence of 62·5 U ml-1 PKA are similar to those for WT-CFTR in the presence of a 10-fold lower concentration of PKA (Fig. 3). The mean current amplitude of F508-CFTR upon maximal activation was 50 % of that for WT-CFTR (Fig. 4C) presumably due to a lower channel density in NIH3T3-F508 cells (see below). These results suggest a 7-fold difference in the PKA-dependent activation rate between WT- and F508-CFTR.
 |
View larger version [in this window] [in a new window] |
|
|
F508-CFTR
Experimental conditions and methods of analysis were similar to those in Fig. 2. A, a representative recording of PKA-dependent activation of | ||
ATP-dependent gating of fully phosphorylated WT-CFTR and F508-CFTR
We next compared the ATP-dependent gating of F508-CFTR and WT-CFTR. F508-CFTR channels were first activated with 62·5 U ml-1 PKA and 2 mM ATP until a steady state was achieved. Different concentrations of ATP were subsequently applied. Figure 5A demonstrates the relationship between [ATP] and macroscopic F508-CFTR channel current. The macroscopic current amplitudes at different concentrations of ATP were normalized to the current level at 2·75 mM ATP. The dose-response relationship for WT-CFTR, previously published (Zeltwanger et al. 1999), was plotted here for comparison. There are no statistically significant differences between the ATP dose-response for WT-CFTR and that for F508-CFTR (Student's t test, P = 0·45, 0·20 and 0·19 for 0·1, 0·5 and 1·0 mM ATP, respectively). Curve fitting of the data with Hill's equation yields K½ of 128 ± 24 µM and 136 ± 68 µM for WT- and F508-CFTR, respectively. Figure 5B shows the relationship between single-channel Po and [ATP] for WT- and F508-CFTR. Again, no statistically significant differences in Po were observed at any [ATP] tested (Student's t test, P = 0·05, 0·31 and 0·22 for 0·1, 1·0 and 2·75 mM ATP, respectively). Curve fitting of the Po values for F508-CFTR with Hill's equation yields a K½ of 213 ± 62 µM and a maximal Po of 0·50 ± 0·07 (186 ± 38 µM and 0·43 ± 0·02 for WT-CFTR). To further examine the ATP-dependent gating of F508-CFTR, dwell time analysis was performed. Figure 6A shows representative single-channel traces of PKA-phosphorylated WT- or F508-CFTR in the presence of 2·75 mM ATP. Events from three patches containing one single F508-CFTR channel were pooled to generate cumulative open and closed time histograms (Fig. 6B). Comparing the open time and closed time constants between WT- and F508-CFTR (o = 0·45 ± 0·03 s and c = 0·52 ± 0·01 s for WT-CFTR, from Zeltwanger et al. 1999) in the presence of 2·75 mM ATP, we found no evidence for different single-channel kinetics between WT and the mutant channels. These single-channel kinetic data and the dose-response relationships shown above strongly suggest that, once fully phosphorylated, the interaction between ATP and the NBDs in F508-CFTR is similar to that of wild-type channels.
 |
View larger version [in this window] [in a new window] |
|
|
A, the dose-response relationship between [ATP] and macroscopic WT-CFTR ( | ||
 |
View larger version [in this window] [in a new window] |
|
|
F508-CFTR
A, representative single-channel traces of WT- and | ||
Comparison of whole-cell current density between WT- and F508-CFTR
Our previous studies (Hwang et al. 1997) have shown that in cell-attached patches and in the presence of a saturating concentration of forskolin (10 µM), the steady-state single-channel Po for WT-CFTR is >10 times that for F508-CFTR. However, genistein, in the presence of forskolin, could stimulate both WT-CFTR and F508-CFTR activity to a similar Po value of 0·7. Similar magnitudes of potentiation of cAMP-dependent CFTR channel current by genistein can be observed in whole-cell patch-clamp experiments. Figure 7 shows representative whole-cell recordings from NIH3T3-CFTR and NIH3T3-F508 cells. With 10 mM ATP in the pipette, forskolin (10 µM) induced a significant outward current at a 0 mV holding potential ([Cl]i = 24 mM, [Cl]o = 156 mM) from an NIH3T3-CFTR cell, and genistein (20 µM) further increased the current by about 2·3-fold (Fig. 7A, left panel). Figure 7A (centre panel) shows samples of resulting current traces over the entire voltage range of -100 to 100 mV at a, b and c, marked in the left panel of Fig. 7A. In the absence of forskolin (Fig. 7A, centre panel, a), the current amplitudes over the ±100 mV range were very small indicating minimal basal channel activity and a high whole-cell resistance. In the presence of forskolin, as well as in the presence of forskolin and genistein (Fig. 7A centre panel, b and c), currents were dramatically increased and nearly time independent at positive membrane potentials. An outward rectification of the whole-cell CFTR chloride conductance, which is also shown in the current-voltage (I-V) relationship (Fig. 7A, right panel), is seen mostly because of the imposed Cl- gradient across the membrane (see Methods).
 |
View larger version [in this window] [in a new window] |
|
|
F508-CFTR channel current activated by forskolin (Fsk) and genistein (Gen)
A, activation of whole-cell WT-CFTR current with 10 µM forskolin and 20 µM genistein. Pipette [Cl-] and bath [Cl-] were 24 and 156 mM, respectively, and the holding potential was 0 mV. A 12 mV junction potential, calculated with the Patcher's Power Tools software (HEKA, Germany), was corrected for the I-V relationships. Current traces (taken as marked in the left panel) over 100 mV at 20 mV increments are shown in the centre panel. The I-V relationships of forskolin-dependent current (b - a) and genistein-potentiated current (c - a) are shown. Series resistance = 4·1 m | ||
In NIH3T3-F508 cells grown at 37°C, forskolin (10 µM) elicited a very small current while genistein potentiated the current dramatically and the whole-cell F508-CFTR currents showed similar properties of outward rectification and time independence (Fig. 7B) to WT-CFTR (Fig. 7A). Compared to WT-CFTR, however, the absolute current amplitude from F508-CFTR cells is much smaller (notice the different current scales in Fig. 7A and B). Figure 8 summarizes the results from these whole-cell experiments. Current density was used to normalize differences in the cell size. When cells were incubated at 37°C, genistein increased forskolin-induced WT-CFTR current density by 2-fold (21·3 to 46·9 pA pF-1). For F508-CFTR, genistein gave a 40-fold increase in forskolin-induced current density (0·1 to 4·3 pA pF-1). In the presence of forskolin and genistein, the whole-cell current density of NIH3T3-CFTR cells is 10-fold higher than that of NIH3T3-F508 cells. However, when NIH3T3-F508 cells were incubated at 27°C for 2-3 days before the experiment, the whole-cell current density was greatly increased. In the presence of forskolin plus genistein, the current density of NIH3T3-F508 cells is 70 % of that from NIH3T3-CFTR cells (although they are not different significantly, P = 0·22, Student's t test). Thus, as reported previously (Hwang et al. 1997), lowering the culture temperature, although increasing the channel density in the cell membrane, does not rectify the defective response of F508-CFTR channels to the cAMP stimulation. Since the whole-cell current density reflects the number of functional channels in the plasma membrane, these results allow us to compare quantitatively channel density between WT and F508 channels (see Discussion).
 |
View larger version [in this window] [in a new window] |
|
|
F508-CFTR whole-cell current density
| ||
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Incremental activation of the CFTR channel by PKA-dependent phosphorylation
Applying different concentrations of PKA to excised inside-out membrane patches and monitoring CFTR channel current before reaching the steady state allowed us to quantify the PKA phosphorylation-dependent activation of CFTR. Just like using ATP-dependent gating parameters to extract biochemical information about the ATP-hydrolysis reaction (e.g. Zeltwanger et al. 1999; Weinreich et al. 1999), this real-time recording of the PKA-dependent CFTR activation process is another example of using electrophysiological methods to tackle (albeit indirectly) biochemical processes. The time course of PKA-dependent CFTR activation could be best fitted with a sigmoidal function, suggesting that multiple kinetic steps are involved in reaching the maximal function of the CFTR channel. Our previous studies (Wang et al. 1998) have demonstrated that CFTR can assume partially phosphorylated states upon pulse applications of PKA. The partially phosphorylated CFTR channel has a lower Po with characteristic prolonged closed times even at millimolar [ATP] (cf. Hwang et al. 1993, 1994). Mathews et al. (1998) also reported a prolonged closed time associated with CFTR mutants where some of the PKA consensus serine residues are converted to alanine. Conversely, when the CFTR channel is strongly phosphorylated, the Po is increased with a concomitant shortening of the closed time (Wang et al. 1998). Since multiple serine residues in the R domain seem to be involved in PKA-dependent phosphorylation activation of the CFTR channel (Rich et al. 1993; Chang et al. 1994; Seibert et al. 1997; Winter & Welsh, 1997; Mathews et al. 1998), it is expected that, in excised inside-out patches, the channel should sojourn through many intermediate states before it reaches a maximal level of phosphorylation. Thus, the time course can be sigmoidal provided that the Po increases with an increased level of phosphorylation (cf. Wilkinson et al. 1997). The curvilinear relationship between [PKA] and the apparent activation rate (Fig. 3) further supports a co-operative relationship between multiple phosphorylation sites and the CFTR channel activity.
A slower PKA-dependent phosphorylation activation with the F508 mutation
Our data demonstrated that F508-CFTR has a slower apparent PKA-dependent activation rate than WT-CFTR in excised inside-out membrane patches. The simplest interpretation of this observation is that PKA phosphorylates F508-CFTR channels at a slower rate, although we cannot rule out the possibility of a slower conformational change that occurs after phosphorylation. In other words, compared to WT-CFTR, F508-CFTR proteins are not such good substrates for PKA. (Note that the rate of an enzymatic reaction is determined by the concentrations of the enzyme and the substrate as well as the intrinsic properties of the substrate.) This conclusion then predicts that, in an intact cell where kinases are constantly counteracted by cellular phosphatases, the critical phosphorylation site(s) for CFTR function will be phosphorylated to a lesser extent in F508-CFTR than in WT-CFTR. Thus, the steady-state Po of F508-CFTR in cell-attached patches is much lower than that of WT-CFTR despite a normal ATP-dependent gating with the F508 mutation (see below). This conclusion also predicts that when cellular PKA activity is lowered experimentally (e.g. using lower [forskolin]), WT-CFTR should have a lower Po with similar kinetic behaviour to F508-CFTR channels in cell-attached patches. Indeed, we have shown previously that in cell-attached patches, the behaviour of F508-CFTR in the presence of a saturating concentration of forskolin mimics that of WT-CFTR in the presence of a submaximal concentration of forskolin (Hwang et al. 1997; Al-Nakkash & Hwang, 1999). In the current report, we show that, in excised inside-out patches, the PKA-dependent activation rate of F508-CFTR is similar to that of WT-CFTR at a much lower [PKA] (Fig. 3). Our conclusion that F508-CFTR is defective in PKA-dependent phosphorylation is interesting since F508 is located in NBD1 while PKA-dependent phosphorylation is believed to happen mostly in the R domain (Cheng et al. 1991; Chang et al. 1994; Seibert et al. 1997). Thus, this conclusion implies a functional perturbation in the R domain secondary to a structural change in NBD1 (i.e. an interdomain interaction). Our results also appear to resolve a long-standing puzzle in which the Po of purified F508-CFTR is not significantly different from that of WT-CFTR in lipid bilayers (Li et al. 1993) while the mutant channel behaves abnormally in cell-attached patches (Dalemans et al. 1991; Haws et al. 1996).
ATP-dependent gating behaviour of F508-CFTR
In addition to a defective PKA-dependent phosphorylation, is the ATP-dependent gating also defective with the F508 mutation? ATP-dependent gating for phosphorylated F508-CFTR has been studied (Li et al. 1993; Schultz et al. 1999), but results from these two reports are quite different. While Li et al. (1993) showed no difference in channel kinetics at 1 mM ATP between WT- and F508-CFTR, Schultz et al. (1999) demonstrated a 2-fold difference in the maximal Po and a 9-fold difference in K½ of the ATP dose-response relationship. Our results are more in line with those of Li et al. (1993). We found no differences in Po, single-channel kinetics or the ATP dose-response relationship between WT- and F508-CFTR channels. Although it is unclear what accounts for these discrepancies, different levels of membrane-associated protein phosphatases in systems used by different groups may be partly responsible.
Li et al. (1993) characterized purified CFTR incorporated in lipid bilayers, where there is likely to be little, if any, phosphatase contamination. The current studies were performed in excised membrane patches from NIH3T3 cells where minimal membrane-associated phosphatase activity was noted (Zeltwanger et al. 1999). On the other hand, Schultz et al. (1999) studied F508-CFTR in excised inside-out membrane patches from L-cells or HEK 293 cells. Schultz et al. (1999) clearly demonstrated a fast dephosphorylation of F508-CFTR upon removal of PKA in excised patches, suggesting a strong phosphatase activity associated with F508-CFTR channels in the membrane patch. Because of this robust dephosphorylation of CFTR, Schultz et al. (1999) could not consistently obtain truly stationary conditions for their kinetic analysis of F508-CFTR. Thus, they considered their estimates of kinetic parameters as 'first approximations' (Schultz et al. 1999). Indeed, if the membrane patches contain significant phosphatase activity, the Po as well as the ATP dose-response relationships will be affected in such a way that the kinetic parameters should reflect an average behaviour of different phospho-isoforms of CFTR (a situation mimicking CFTR in intact cells). This might explain their reported long closed time associated with F508-CFTR.
Although we cannot rule out the possibility that F508-CFTR may have an abnormally higher dephosphorylation rate than the WT channel as suggested by Schultz et al. (1999), our data indicate that F508-CFTR channels still could be strongly phosphorylated by PKA in excised patches due to the fact that the mutant channel activity reached a steady state with a similar Po to that of WT-CFTR. Furthermore, the conclusion that the ATP-dependent gating of F508-CFTR is not affected by the mutation is consistent with a recent homologous model of NBD1 of CFTR, in which the F508 residue was placed in an 
helix that was >10 Å away from the ATP-binding pocket. This model also suggests that deletion of F508 causes little change in the ATP-binding pocket and speculates that this region where F508 is located may be involved in inter-domain interactions (Armstrong et al. 1998; also see Hung et al. 1998).
Relative roles of the trafficking defect and the functional defect with the F508 mutation
It has been well established that deletion of F508 causes a trafficking defect and a functional defect, but the relative roles of these two abnormalities in cystic fibrosis pathogenesis is unknown. In theory, the defective trafficking will result in a reduced number of functional F508-CFTR channels in the plasma membrane, whereas the abnormal response to the cAMP stimulation causes a lower Po of the existing channels. Measurements of whole-cell CFTR current density could yield quantitative information about the number of functional channels in the plasma membrane since the macroscopic whole-cell current amplitude is determined by the number of channels, the single-channel Po and the single-channel amplitude. It has been shown that WT- and F508-CFTR chloride channels have identical single-channel conductances (Dalemans et al. 1991) and very similar Po values in the presence of forskolin and genistein in intact cells (Hwang et al. 1997). Therefore, the whole-cell current density in the presence of forskolin and genistein provides a parameter that can be used to compare plasma membrane channel densities between cells expressing WT- or F508-CFTR channels. With known values for Po and single-channel current amplitude, one can even estimate precisely the channel density in the cell membrane.
From the data presented in Fig. 8, several conclusions can be made. (1) In the presence of cAMP stimulation alone (i.e. 10 µM forskolin), the current density of NIH3T3-F508 cells is almost negligible (0·1 ± 0·1 pA pF-1 at 37°C and 0·6 ± 0·2 pA pF-1 at 27°C). However, with forskolin and genistein together, significant currents can be elicited (4·3 ± 2·1 pA pF-1 at 37°C and 33·6 ± 14·2 pA pF-1 at 27°C). Thus, using cAMP-dependent chloride current to quantify F508-CFTR in the cell membrane is likely to produce an underestimation of the channel density (e.g. Rich et al. 1993). (2) In the presence of forskolin and genistein, when there is little difference in the Po between WT- and F508-CFTR, the current density of NIH3T3-F508 cells increases from 9 % (4·3/46·9) to 72 % (33·6/46·9) of that from NIH3T3-CFTR cells as the culture temperature is lowered. This result provides a quantitative estimation of the role of the trafficking defect and the degree of its correction by the low culture temperature. (3) Assuming a single-channel amplitude of 0·2 pA and a Po of 0·7 in the presence of forskolin and genistein (Hwang et al. 1997), the current density values can be converted to channel density given a specific membrane capacitance of 1 µF cm-2 (3·35 µm-2 for WT-CFTR, 0·31 µm-2 for F508-CFTR at 37°C and 2·40 µm-2 for F508-CFTR at 27°C). Thus, the trafficking defect caused by the F508 mutation decreases the number of functional F508-CFTR channels to 10 % of that of WT-CFTR. However, in the presence of cAMP, the physiological stimulus, alone, the current density from NIH3T3-F508 cells is only 0·4 % that of NIH3T3-CFTR cells because of a combination of the trafficking defect and the functional defect. Therefore, at least in these two NIH3T3 cell lines where similar amounts of total CFTR proteins (mature plus immature) have been demonstrated (Denning et al. 1992), the functional defect of F508-CFTR plays a role of equal importance in the pathophysiology of cystic fibrosis to that of the trafficking defect.
One should note, however, that the relative role of the trafficking defect may vary in different expression systems (c.f. Rich et al. 1993; Lukacs et al. 1994; Haws et al. 1996) and in different tissues in patients with cystic fibrosis (Kälin et al. 1999). Using immunocytochemical methods, Kälin et al. (1999) demonstrated that the amount of CFTR proteins in the cell membrane is drastically reduced in sweat ducts isolated from F508 homozygotes, but the expression of the mutant protein is indistinguishable from the WT-CFTR in airway and intestinal epithelial cells. This result strongly suggests that the abnormal trafficking may not be the major determinant for F508 cystic fibrosis disease. Thus, a better understanding of the molecular mechanism for the functional defect of F508-CFTR will aid in the design of pharmacological agents for therapeutical intervention in cystic fibrosis.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Al-Nakkash, L. & Hwang, T.-C. (1999). Activation of wild-type and F508-CFTR by phosphodiesterase inhibitors through cAMP-dependent and -independent mechanisms. Pflügers Archiv 437, 553-561 |
[Medline] |
| Armstrong, S., Tabernero, R. L., Zhang, H., Hermodson, M. & Stauffacher, C. V. (1998). Powering the ABC transporter: The 2.5 A crystallographic structure of the ABC domain of RBSA. Pediatric Pulmonology Supplement 17, 91-92. | [Medline] |
| Berger, H. A., Anderson, M. P., Gregory, R. J., Thompson, S., Howard, P. W., Maurer, R. A., Smith, A. E. & Welsh, M. J. (1991). Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel. Journal of Clinical Investigation 88, 1422-1431 | [Medline] |
| Chang, X.-B., Hou, Y.-X., Jensen, T. J. & Riordan, J. R. (1994). Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion. Journal of Biological Chemistry 269, 18572-18575 | [Abstract] |
| Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., O'Riordan, C. R. & Smith, A. L. (1990). Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 65, 827-834. | |
| Cheng, S. H., Rich, D. P., Marshall, J., Gregory, R. J., Welsh, M. J. & Smith, A. E. (1991). Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell 66, 1027-1036 | [Medline] |
| Dalemans, W., Barbry, P., Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G., Pavirani, A., Lecocq, J.-P. & Lazdunski, M. (1991). Altered chloride ion channel kinetics associated with the F508 cystic fibrosis mutation. Nature 354, 526-528 |
[Medline] |
| Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J., Smith, A. E. & Welsh, M. J. (1992). Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358, 761-764 | [Medline] |
| Drumm, M. L., Wilkinson, D. J., Smit, L. S., Worrell, R. T., Strong, T. V., Frizzell, R. A., Dawson, D. C. & Collins, F. S. (1991). Chloride conductance expressed by F508 and other mutant CFTRs in Xenopus oocytes. Science 254, 1797-1799 |
[Medline] |
| Flockhart, D. A. & Corbin, J. D. (1984). Preparation of the catalytic subunit of cAMP-dependent protein kinase. In Brain Receptor Methodologies: Amino Acids, Psychoactive Drugs. Part A: General Methods and Concepts. Amines and Acetylcholine, ed. Marangos, P. J., Campbell, I. C. & Cohen, R. M., pp. 209-215. Academic Press, Orlando, FL, USA. | |
| Gadsby, D. C., Nagel, G. & Hwang, T.-C. (1995). The CFTR chloride channel of mammalian heart. Annual Review of Physiology 57, 387-416 | [Medline] |
| Gadsby, D. C. & Nairn, A. C. (1999). Control of CFTR gating by phosphorylation and nucleotide hydrolysis. Physiological Reviews 79, S77-107 | [Medline] |
| Haws, C. M., Nepomuceno, I. B., Krouse, M. E., Wakelee, H., Law, T., Xia, Y., Nguyen, H. & Wine, J. J. (1996). F508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines. American Journal of Physiology 270, C1544-1555 |
[Medline] |
| He, Z., Raman, S., Guo, Y. & Reenstra, W. W. (1998). Cystic fibrosis transmembrane conductance regulator activation by cAMP-independent mechanisms. American Journal of Physiology 275, C958-966 | [Medline] |
| Hung, L.-W., Wang, I. X., Nikaido, K., Liu, P.-Q., Ames, G. F. & Kim, S.-H. (1998). Crystal structure of the ATP-binding subunit of an ABC transporter. Nature 396, 703-707 | [Medline] |
| Hwang, T.-C., Horie, M. & Gadsby, D. C. (1993). Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl conductance in mammalian heart. Journal of General Physiology 101, 629-650 | [Abstract] |
| Hwang, T.-C., Nagel, G., Nairn, A. C. & Gadsby, D. C. (1994). Regulation of the gating of cystic fibrosis transmembrane conductance regulator Cl channels by phosphorylation and ATP hydrolysis. Proceedings of the National Academy of Sciences of the USA 91, 4698-4702 | [Abstract] |
| Hwang, T.-C., Wang, F., Yang, I. C.-H. & Reenstra, W. W. (1997). Genistein potentiates wild-type and F508-CFTR channel activity. American Journal of Physiology 273, C988-998 |
[Medline] |
| Kälin, N., Claaß, A., Sommer, M., Puchelle, E. & Tümmler, B. (1999). F508 CFTR protein expression in tissues from patients with cystic fibrosis. Journal of Clinical Investigation 103, 1379-1389 |
[Abstract/Full Text] |
| Kelley, T. J., Al-Nakkash, L., Cotton, C. U. & Drumm, M. L. (1996). Activation of endogenous F508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition. Journal of Clinical Investigation 98, 513-520 |
[Abstract/Full Text] |
| Li, C., Ramjeesingh, M., Reyes, E., Jensen, T., Chang, X., Rommens, J. M. & Bear, C. E. (1993). The cystic fibrosis mutation (F508) does not influence the chloride channel activity of CFTR. Nature Genetics 3, 311-316 |
[Medline] |
| Lukacs, G. L., Mohamed, A., Kartner, N., Chang, X.-B., Riordan, J. R. & Grinstein, S. (1994). Conformational maturation of CFTR but not its mutant counterpart (delta F508) occurs in the endoplasmic reticulum and requires ATP. EMBO Journal 13, 6076-6086 | [Abstract] |
| Mathews, C. J., Tabcharani, J. A., Chang, X.-B., Jensen, T. J., Riordan, J. R. & Hanrahan, J. W. (1998). Dibasic protein kinase A sites regulate bursting rate and nucleotide sensitivity of the cystic fibrosis transmembrane conductance regulator chloride channel. The Journal of Physiology 508, 365-377 | [Abstract/Full Text] |
| Rich, D. P., Berger, H. A., Cheng, S. H., Travis, S. M., Saxena, M., Smith, A. E. & Welsh, M. J. (1993). Regulation of the cystic fibrosis transmembrane conductance regulator Cl channel by negative charge in the R domain. Journal of Biological Chemistry 268, 20259-20267 | [Abstract] |
| Riordan, J. R., Rommens, J. M., Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Iannuzzi, M. C., Collins, F. S. & Tsui, L.-C. (1989). Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066-1073 | [Medline] |
| Sato, S., Ward, C. L., Krouse, M. E., Wine, J. J. & Kopito, R. R. (1996). Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. Journal of Biological Chemistry 271, 635-638 | [Abstract/Full Text] |
| Schultz, B. D., Frizzell, R. A. & Bridges, R. J. (1999). Rescue of dysfunctional F508-CFTR chloride channel activity by IBMX. Journal of Membrane Biology 170, 51-66 |
[Medline] |
| Seibert, F. S., Jia, Y., Mathews, C. J., Hanrahan, J. W., Riordan, J. R., Loo, R. W. & Clarke, D. M. (1997). Disease-associated mutations in cytoplasmic loops 1 and 2 of cystic fibrosis transmembrane conductance regulator impede processing or opening of the channel. Biochemistry 36, 11966-11974 | [Medline] |
| Sheppard, D. N. & Welsh, M. J. (1999). Structure and function of the CFTR chloride channel. Physiological Reviews 79, S23-43 | [Medline] |
| Wang, F., Zeltwanger, S., Yang, I. C.-H., Nairn, A. C. & Hwang, T.-C. (1998). Actions of genistein on cystic fibrosis transmembrane conductance regulator channel gating: evidence for two binding sites with opposite effects. Journal of General Physiology 111, 477-490 | [Abstract/Full Text] |
| Ward, C. L., Omura, S. & Kopito, R. R. (1995). Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83, 121-127 | [Medline] |
| Weinreich, F., Riordan, J. R. & Nagel, G. (1999). Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites. Journal of General Physiology 114, 55-70 | [Abstract/Full Text] |
| Wilkinson, D. J., Strong, T. V., Mansoura, M. K., Wood, D. L., Smith, S. S., Collins, F. S. & Dawson, D. C. (1997). CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain. American Journal of Physiology 273, L127-133 | [Medline] |
| Winter, M. C. & Welsh, M. J. (1997). Stimulation of CFTR activity by its phosphorylated R domain. Nature 389, 294-296 | [Medline] |
| Yamamoto, T., Yamamoto, S., Osborne, J. C., Manganiello, V. C., Vaughan, M. & Hikada, H. (1983). Complex effects of inhibitors on cyclic GMP-stimulated cyclic nucleotide phosphodiesterase. Journal of Biological Chemistry 258, 14173-14177 | [Abstract] |
| Zeltwanger, S., Wang, F., Wang, G. T., Gillis, K. & Hwang, T.-C. (1999). Gating of CFTR chloride channels by ATP hydrolysis: quantitative analysis of a cyclic gating scheme. Journal of General Physiology 113, 541-554 | [Abstract/Full Text] |
This work was supported by the National Institute of Health, the Cystic Fibrosis Foundation, and the American Heart Association, Missouri Affiliate.
Corresponding author
T.-C. Hwang: DCRC Research Park, University of Missouri-Columbia, Columbia, MO 65211, USA.
Email: dcrctch{at}showme.missouri.edu
This article has been cited by other articles:
|
|
 |
|
  J. Li, Y-Y. Xiang, L. Ye, L-C. Tsui, J. F. MacDonald, J. Hu, and W-Y. Lu Nonsteroidal anti-inflammatory drugs upregulate function of wild-type and mutant CFTR Eur. Respir. J., August 1, 2008; 32(2): 334 - 343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. S. Ostedgaard, C. S. Rogers, Q. Dong, C. O. Randak, D. W. Vermeer, T. Rokhlina, P. H. Karp, and M. J. Welsh Processing and function of CFTR-{Delta}F508 are species-dependent PNAS, September 25, 2007; 104(39): 15370 - 15375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. G. Bompadre, Y. Sohma, M. Li, and T.-C. Hwang G551D and G1349D, Two CF-associated Mutations in the Signature Sequences of CFTR, Exhibit Distinct Gating Defects J. Gen. Physiol., March 26, 2007; 129(4): 285 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Bebok, J. F. Collawn, J. Wakefield, W. Parker, Y. Li, K. Varga, E. J. Sorscher, and J. P. Clancy Failure of cAMP agonists to activate rescued {Delta}F508 CFTR in CFBE41o- airway epithelial monolayers J. Physiol., December 1, 2005; 569(2): 601 - 615. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Liu, A. Veilleux, L. Zhang, A. Young, E. Kwok, F. Laliberte, C. Chung, M. R. Tota, D. Dube, R. W. Friesen, et al. Dynamic Activation of Cystic Fibrosis Transmembrane Conductance Regulator by Type 3 and Type 4D Phosphodiesterase Inhibitors J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 846 - 854. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Wang, G. Li, J. P. Clancy, and K. L. Kirk Activating Cystic Fibrosis Transmembrane Conductance Regulator Channels with Pore Blocker Analogs J. Biol. Chem., June 24, 2005; 280(25): 23622 - 23630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. G. Bompadre, T. Ai, J. H. Cho, X. Wang, Y. Sohma, M. Li, and T.-C. Hwang CFTR Gating I: Characterization of the ATP-dependent Gating of a Phosphorylation-independent CFTR Channel ({Delta}R-CFTR) J. Gen. Physiol., March 28, 2005; 125(4): 361 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. L. Clarke, L. R. Gawenis, T.-C. Hwang, N. M. Walker, D. B. Gruis, and E. M. Price A domain mimic increases {Delta}F508 CFTR trafficking and restores cAMP-stimulated anion secretion in cystic fibrosis epithelia Am J Physiol Cell Physiol, July 1, 2004; 287(1): C192 - C199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ai, S. G. Bompadre, X. Wang, S. Hu, M. Li, and T.-C. Hwang Capsaicin Potentiates Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride-Channel Currents Mol. Pharmacol., June 1, 2004; 65(6): 1415 - 1426. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yang, A. A. Shelat, R. K. Guy, V. S. Gopinath, T. Ma, K. Du, G. L. Lukacs, A. Taddei, C. Folli, N. Pedemonte, et al. Nanomolar Affinity Small Molecule Correctors of Defective {Delta}F508-CFTR Chloride Channel Gating J. Biol. Chem., September 12, 2003; 278(37): 35079 - 35085. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A S Ramalho, S Beck, D Penque, T Gonska, H H Seydewitz, M Mall, and M D Amaral Transcript analysis of the cystic fibrosis splicing mutation 1525-1G>A shows use of multiple alternative splicing sites and suggests a putative role of exonic splicing enhancers J. Med. Genet., July 1, 2003; 40(7): e88 - 88. [Full Text] [PDF]
|
|
|
|
|
|
A. C. V. deCarvalho, L. J. Gansheroff, and J. L. Teem Mutations in the Nucleotide Binding Domain 1 Signature Motif Region Rescue Processing and Functional Defects of Cystic Fibrosis Transmembrane Conductance Regulator Delta F508 J. Biol. Chem., September 20, 2002; 277(39): 35896 - 35905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. V. J. Galietta, S. Jayaraman, and A. S. Verkman Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1734 - C1742. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. D. R. Setchell Soy Isoflavones--Benefits and Risks from Nature's Selective Estrogen Receptor Modulators (SERMs) J. Am. Coll. Nutr., October 1, 2001; 20(90005): 354S - 362. [Abstract] [Full Text]
|
|
|
|
|
|
L. Al-Nakkash, S. Hu, M. Li, and T.-C. Hwang A Common Mechanism for Cystic Fibrosis Transmembrane Conductance Regulator Protein Activation by Genistein and Benzimidazolone Analogs J. Pharmacol. Exp. Ther., April 13, 2001; 296(2): 464 - 472. [Abstract] [Full Text]
|
|
| ||||||||||||
) is presented for comparison. The number in parentheses next to each data symbol is the number of experiments (and also in the following figures).
) channel activity. For each patch (containing a different number of 
, 10 µM forskolin; 
, 10 µM forskolin + 20 µM genistein; WT, NIH3T3-CFTR cells;