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(The FASEB Journal. 2001;15:927-931.)
© 2001 FASEB

A src-like kinase activates outwardly rectifying chloride channels in CFTR-defective lymphocytes

ALBECHT LEPPLE-WIENHUES*,12, ULRICH WIELAND*,1, TILMANN LAUN, LUZIA HEIL, MARTIN STERN{dagger} and FLORIAN LANG

Department for
* Physiology and of
{dagger} Pediatric Medicine, University of Tübingen, Tübingen, Germany

2Correspondence: Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany. E-mail: alepplew{at}uni-tuebingen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Defective activation of chloride channels is a hallmark of cystic fibrosis (CF). Recently we have described activation of a volume-sensitive, outwardly rectifying chloride conductance (IOR) through the src-like tyrosine kinase p56lck. Here we show that p56lck activates IOR independently of CFTR. In lymphocytes from healthy donors, chloride channels could be opened by either intracellular cAMP, p56lck or osmotic swelling. In CF lymphocytes, p56lck and cell swelling but not cAMP could activate chloride channels. Regulation of IOR by p56lck thus represents an alternative pathway of stimulating membrane chloride conductance that is left intact in cystic fibrosis.—Lepple-Wienhues, A., Wieland, U., Laun, T., Heil, L., Stern, M., Lang, F. A src-like kinase activates outwardly rectifying chloride channels in CFTR-defective lymphocytes.


Key Words: cystic fibrosis • tyrosine kinase • human lymphocytes • cyclic adenosine monophosphate • cell volume


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CYSTIC FIBROSIS (CF) IS a frequent hereditary disorder resulting from mutations in the CFTR gene (1 2 3) . CFTR encodes for a multifunctional membrane protein that serves as a Cl- channel (4 5 6 7 8 9) , but also regulates separate chloride channels and ion transporters (7 , 10 11 12 13) . CF mutations lead to reduced transepithelial transport of Cl- and HCO3- and altered Na+ channel activity (14 15 16 17 18 19) . Specifically, anion secretion and reabsorption activated by cAMP are impaired in CF (20 , 21) . In CF respiratory epithelium, forskolin or cAMP fail to activate a Cl- current that can be restored by retransfection of intact CFTR (22) . The most frequent CF mutation {Delta}F508 disrupts trafficking of the CFTR protein to the outer membrane (23) .

An alternative chloride conductance is supposed to prevent the development of lung disease in CFTR knockout mice. In murine respiratory epithelium, a Ca2+-activated anion channel can substitute for the defective cAMP-dependent Cl- channel (24 , 25) whereas no alternative channel is expressed in murine intestine (26 , 27) . While lung and pancreas are only slightly affected, the animals suffer from severe pathology in the gut (28 , 29) . Therefore, alternative pathways of chloride transport represent an intriguing therapeutic concept for this lethal hereditary disease. In a recent study, chloride currents were restored by overexpressing CLC-2 channels in human airway epithelial cells (30) .

Outwardly rectifying chloride channels are found in airway epithelia (31 32 33) as well as in lymphocytes (34) , representing an alternative Cl- conductive pathway. These channels can be activated by cAMP in intact tissue (31 32 33) but not in CF knockout mice (35) . Similarly, in CF lymphocytes, activation of outwardly rectifying chloride current by cAMP is lacking although the channel is present (34) .

Biophysical and pharmacological differences have been observed between outwardly rectifying anion channels (36 37 38 39) . Some channels can be activated by excision and depolarization of the patch, but they do not mediate the swelling-activated chloride conductance in several cell lines (40) .

We have recently identified a novel activation mechanism for an outwardly rectifying lymphocyte chloride channel opened by osmotic swelling (41) and during CD95-induced apoptosis in Jurkat T cells (42) . The channel is activated by the Src-like kinase tyrosine kinase p56lck. Genetic knockout or pharmacological inhibition of p56lck both prevent activation of the channel during cell swelling and apoptosis, thus abolishing cell volume regulation and inhibiting CD95-induced apoptosis (41 , 42) . Moreover, cytosolic application of purified p56lck activates and addition of anti-phosphotyrosine antibodies inhibits the channel in excised and whole cell patches (42) . The channel lacks inactivation, is blocked by 500 µM DIDS, is selective for Cl-, and shows outward rectification. Unitary conductances of 25–28 pS obtained from single channel transitions in whole cell recordings activated by either swelling (42) or p56lck corresponded well to 31 pS in excised membrane patches when adding purified p56lck to the cytosolic surface (41 , 42) . Thus, cell swelling and p56lck seem to activate the same chloride channel in lymphocytes judged by biophysical and pharmacological profiles. However, molecular cloning of the channel protein in lymphocytes will be required to finally prove this concept.

Tyrosine kinase inhibitors block osmotic activation of Cl- channels in cardiac, epithelial and endothelial cells (43 44 45) . The present study has been performed to test whether p56lck is capable to stimulate Cl- channels in lymphocytes lacking functional CFTR. If so, activation of Cl- channels through p56lck could represent an alternative anion conductance pathway in CFTR-defective tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Human peripheral blood lymphocytes were harvested from healthy donors or from patients with cystic fibrosis ({Delta}F508) with informed consent. Peripheral T lymphoblasts were prepared using a Ficoll gradient and purified with nylon wool columns (>90% CD3+). The cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 mM HEPES (pH 7.4), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin (all purchased from Life Technologies, Eggenstein, Germany), and 50 µM ß-mercaptoethanol for up to 3 days. All experiments were performed at 37°C.

Solutions
Cells were superfused with modified Ringer’s containing (mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4 (310 mOsmol/kg). To separate Cl- currents, the internal pipette solution contained (mM) 160 Cs glutamate, 0.1 CaCl2, 2 Mg Cl2, 1.1 mM EGTA, 4 Na2ATP, and 10 HEPES, (330 mOsmol/kg, pH 7.2). For symmetrical chloride concentrations, CsCl replaced Cs glutamate. For hypotonic intracellular conditions, this solution was diluted as indicated. For hypertonic conditions, sucrose was added to obtain the indicated osmolality. Purified p56lck was purchased from UBI (Happauge, N.Y.). All other chemicals were obtained from Sigma (Deisenhofen, Germany). Osmolality was measured using a freezing point osmometer (Knauer, Berlin, Germany).

Patch-clamp recordings
Whole cell currents were recorded at 31°C using an EPC-9 patch-clamp amplifier and Pulse software (Heka, Lambrecht, Germany). Experiments were performed on an Axiovert 135 microscope and cells were observed using a video system in order to note volume changes. Pipettes were pulled to a resistance of 2–5 M{Omega} from borosilicate glass. For high-resolution recordings, pipettes were coated with Sylgard (Dow Corning, Midland, Mich.). Capacitative transients were canceled using the Cslow compensation of the amplifier. Series resistance was not compensated. Cells were held at 0 mV and voltage ramps or pulses of the indicated size were applied every 5 or 20 s. The current signal was filtered at 1 kHz and digitized at 5 kHz sampling rate. By convention, anionic inward fluxes are shown as positive (outward) currents.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cyclic AMP activates chloride channels in normal, but not in CF lymphocytes
Intracellular cAMP applied through the pipette activates an outwardly rectifying anion conductance in wild-type lymphocytes (Fig. 1A ). Cell swelling was prevented by using a hypotonic pipette solution. The shift of reversal potential toward -40 mV and the small inward current indicate poor permeability for intracellular glutamate. Even in symmetrical Cl- solutions, the current shows outward rectification, does not visibly inactivate during 200 ms pulses, and is blocked by 500 µM DIDS (Fig. 1B ). The current shares these properties with the swelling-activated current but differs from CFTR currents. The current induced by cAMP seems smaller (6.8±1.2 pA at 35 mV, n=10) than outwardly rectifying chloride conductance (IOR) induced by swelling (100 s after break in 10.1±3.9 pA; 250 s: 32.8±18.2 pA, n=3). However, this comparison is limited since in dialyzed lymphocytes the persistent osmotic gradient prevents development of a steady state (see also Fig. 2C and ref 46 ).



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  		Figure 1. cAMP activates an outwardly rectifying chloride channel in wild-type but not in CF T lymphocytes. A) In normal lymphocytes, intracellular cAMP (200 µM) activates an outwardly rectifying current. No leak correction was performed. Instantaneous IV plots were obtained using 200 ms voltage ramps (glutamate inside, n=10). Numbers indicate seconds after breaking the patch. B) Chloride current activated by cAMP in another wild-type lymphocyte using symmetrical Cl- concentrations. The current does not inactivate (upper) and is blocked by 500 µM DIDS (lower). Voltage steps from -60 to 80 mV in 20 mV increments were applied (0 mV holding potential). Leak currents were subtracted. C) Lack of cAMP-induced currents in CF T cells. The representative traces shown here were recorded 1 and 100 s after break-in (n=7).



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  		Figure 2. Osmotic activation of IOR in CF T cells. A) Osmotic swelling activates IOR in T cells obtained from CF patients (no leak correction, glutamate inside). Numbers indicate seconds after breaking the patch. B) Activation of the swelling induced chloride current ({blacksquare}) in CF T cells can be prevented by treatment with the tyrosine kinase inhibitor lavendustin (10 µM, {circ}) (n=7 and 9, respectively). Leak-subtracted outward currents at 35 mV ± SE are plotted vs. time after establishing the whole-cell configuration. C) Lavendustin does not block the swelling induced current when applied extracellularly after activation. The slope of activation during lavendustin superfusion transiently decreased in 2 of 4 cells. Leak-subtracted currents at 35 mV are plotted against time after the start of activation.

In agreement with previous studies (47 , 48) , cAMP fails to activate chloride channels in CF lymphocytes (Fig. 1C ).

Osmotic swelling activates IOR in CF T cells through a tyrosine kinase
In T lymphocytes from CF patients, IOR is activated by swelling (Fig. 2A ). The inhibitor of src-like tyrosine kinases lavendustin (10 µM) was applied through the patch pipette. Lavendustin blocks osmotic current activation in CF cells (Fig. 2B ). Once the current is osmotically activated, extracellular lavendustin does not block the channel (Fig. 2C ).

The swelling-induced current can be inhibited by 500 µM DIDS and does not inactivate when using 200 ms voltage steps (not shown). Osmotically activated IOR in CF lymphocytes is indistinguishable from that observed in a CFTR-expressing lymphocyte cell line (41) as well as wild-type lymphocytes (not shown).

p56lck activates IOR in CF lymphocytes
We have previously shown that osmotic activation of IOR requires p56lck. If IOR activation by tyrosine phosphorylation was independent of CFTR, addition of p56lck to the cytosol should activate ORCC in wild-type as well as CF cells. Purified p56lck added to the intracellular solution activates IOR in CF T cells and wild-type T cells (Fig. 3A B C ). A hypotonic pipette solution (270 mOsmol/kg) was used to avoid swelling. Controls performed either without or with heat-inactivated p56lck (100°C, 5 min) did not show activation (Fig. 3C, D ). Again, the current does not inactivate and is blocked by 500 µM DIDS (not shown). We conclude that p56lck activates IOR in T cells regardless of the CF defect.



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  		Figure 3. IOR is activated by p56lck in both normal and CF T cells. A) IOR in a CF T cell with 10 U/ml purified p56lck in the pipette (glutamate inside, no leak subtraction). Numbers indicate seconds after breaking the patch. The calculated time constant for diffusion of p56lck into the cytoplasm is 115 s assuming 5 M{Omega} access resistance and 6 pF membrane capacitance (51) . B) Activation of IOR by intracellular p56lck in a normal T cell (10 U/ml, glutamate inside, no leak subtraction). C) Maximal outward current at 35 mV in the absence or presence of 10 U/ml p56lck in the pipette (n=7) or with heat-inactivated p56lck (n=4). Leak currents were subtracted. D) Control showing lack of IOR with heat-inactivated p56lck in the pipette. Numbers indicate seconds after breaking the patch (no leak subtraction).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Defective chloride transport across respiratory and intestinal epithelia is believed to play a crucial role in the pathophysiology of the CF phenotype. We show here that the tyrosine kinase p56lck can open outwardly rectifying chloride channels in the absence of functional CFTR. Activation by tyrosine kinase of volume-sensitive chloride channels is intact in T lymphocytes derived from CF patients. In CF T cells, osmotic cell swelling activates the channel and the tyrosine kinase inhibitor lavendustin blocks osmotic activation as in normal cells. However, and in agreement with previous results, the PKA-mediated activation of chloride channels by cAMP is lacking in CF lymphocytes mimicking the defective Cl- transport in epithelial cells (24 , 47) . Although volume- and cAMP-activated currents can be distinguished by neither selectivity, DIDS sensitivity, nor lack of inactivation (24 , 47) , we cannot exclude the possibility they are mediated by different channels. Molecular identification and genetic manipulation of lymphocyte chloride channels will be required to map the respective currents to channel subtypes.

Activation of chloride channels by tyrosine kinase is shared by lymphocytes, epithelial, and endothelial as well as cardiac cells (41 , 43 44 45) . Therefore, activation of chloride channels by src family kinases may represent an alternative anion conductance pathway in CFTR-defective tissues. Further studies in CF respiratory and intestinal epithelial cells will be required to address a possible manipulation of transepithelial transport by src-kinase stimulation.

In addition to epithelial transport, activation of Cl- channels is generally involved in cell volume regulation (49) . We have previously shown that p56lck is crucial for osmotic activation of IOR and subsequent regulatory cell volume decrease (41) . Furthermore, knockout of p56lck (42) as well as several Cl- channel blockers interfered with CD95-induced apoptosis (42) .

Single channel recordings from CFTR have revealed regulation of this channel by tyrosine phosphorylation. The tyrosine kinase p60c-src increases the open probability of CFTR (50) . Although the IOR can be distinguished from CFTR by its intrinsic outward rectification in symmetrical chloride solutions and its sensitivity to DIDS, we could not determine earlier whether tyrosine phosphorylation of the CFTR protein was involved in the activation of IOR. In the present study, however, we clearly observed activation of chloride channels by p56lck, but not by cAMP, when functional CFTR in the membrane was lacking.

We conclude that CFTR is not involved in p56lck-mediated activation of IOR. Whereas cAMP fails to activate chloride channels in CF lymphocytes, the src-like kinase p56lck is capable of opening an outwardly rectifying chloride channel. This represents a novel pathway to increase the membrane anion conductance in the absence of functional CFTR.


   ACKNOWLEDGMENTS
 
This study was supported by grants from Deutsche Forschungsgemeinschaft (La 315/4–3, La 315/6–1, and Le 792/3–1), the Mildred Scheel Foundation (10–1452-Gu 1), IZKF, and Mukoviszidose e.V.


   FOOTNOTES
 
1 A.L.-W. and U.W. contributed equally to this study.

Received for publication May 9, 2000. Revision received October 23, 2000.
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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M. C. BURESI, A. G. BURET, M. D. HOLLENBERG, and W. K. MacNAUGHTON
Activation of proteinase-activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase- and cyclo-oxygenase-dependent pathway
FASEB J, October 1, 2002; 16(12): 1515 - 1525.
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