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Originally published In Press as doi:10.1074/jbc.M301096200 on March 11, 2003

J. Biol. Chem., Vol. 278, Issue 21, 19230-19236, May 23, 2003
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A Novel Voltage-dependent Chloride Current Activated by Extracellular Acidic pH in Cultured Rat Sertoli Cells*

Céline Auzanneau {ddagger}, Vincent Thoreau §, Alain Kitzis § and Frédéric Becq {ddagger} 

From the {ddagger} Laboratoire des Biomembranes et Signalisation Cellulaire CNRS UMR 6558, Université de Poitiers, 40 Avenue du Recteur Pineau, § Laboratoire de Génétique Cellulaire et Moléculaire, UPRES EA 2622, CHU de Poitiers, 86022 Poitiers, France

Received for publication, January 31, 2003
   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sertoli cells from mammalian testis are key cells involved in development and maintenance of stem cell spermatogonia as well as secretion of a chloride- and potassium-rich fluid into the lumen of seminiferous tubules. Using whole-cell patch clamp experiments, a novel chloride current was identified. It is activated only in the presence of an extracellular acidic pH, with an estimated half-maximal activation at pH 5.5. The current is strongly outwardly rectifying, activated with a fast time-dependent onset of activation but a slow time-dependent kinetic at depolarization pulses. The pH-activated chloride current was not detected at physiological or basic pH and is not sensitive to intracellular or extracellular Ca2+ variation. Diphenylamine-2-carboxylic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid blocked the induced currents, and its anionic selectivity sequence was Cl— > Br— > I—> gluconate. We have performed a reverse transcription-PCR analysis to search for voltage-dependent chloride rClC channels in cultured rat Sertoli cells. Among the nine members of the family only rClC-2, rClC-3, rClC-6, and rClC-7 have been identified. The inwardly rectifying rClC-2 chloride current was activated by hyperpolarization but not by pH variation. A different depolarization-activated outwardly rectifying chloride current was activated only by hypotonic challenge and may correspond either to rClC-3 or rClC-6. Immunolocalization experiments demonstrate that rClC-7 resides in the intracellular compartment of Sertoli cells. This study provides the first functional identification of a native acid-activated chloride current. Based on our molecular analysis of rClC proteins, this new chloride current does not correspond to rClC-2, rClC-3, rClC-6, or rClC-7 channels. The potential physiological role of this native current in an epithelial cell from the reproductive system is discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sertoli cells from mammalian testis are involved in the development and maintenance of spermatogenesis, support, and nourishment of germ cells (1). The conversion of stem cell spermatogonia into differentiated spermatozoa in the testis is also critically dependent on the Sertoli cells (2). Among the factors involved in stem cell differentiation is the composition of the surrounding medium of sperm cells. Sperm cells remain immobile with a very low level of metabolism before their release into the external environment, where the spermatozoa become mobile and metabolically active.

One function of Sertoli cells in this process is the synthesis and release of several proteins as well as the secretion of a potassium- and chloride-rich fluid into the lumen of seminiferous tubules (3). Despite the fact that the composition of the fluid secreted by Sertoli cells remains undetermined, its pH plays an important role (4). Particularly, an acidic microenvironment is important for the survival of germ cells and for the proliferation of spermatogonia (2). The secretion of a large amount of lactate by Sertoli cells appears to be involved in the maintenance of this acidic pH (5, 6). The basic fibroblast growth factor stimulates the production of lactate by Sertoli cells through the regulation of glucose transport, lactate dehydrogenase activity, and GLUT1 and lactate dehydrogenase A mRNA levels (6). The production of lactate by Sertoli cells may be used as an energetic substrate for germ cells and as a promoter of their survival and proliferation through the production of the Stem Cell Factor SCF (2).

Sertoli cells express a variety of ionic channels, among them voltage-dependent Ca2+ channels (7, 8), calcium-dependent Cl— channels (9), and cystic fibrosis transmembrane conductance regulator chloride channels (10). The superfamily of voltage-dependent chloride channel named ClC1 is composed of nine members in mammals (for review, see Ref. 11), but none of them have been previously described in Sertoli cells. ClC-1, ClC-2, ClC-K1, and ClC-K2 are closely related, and ClC-3, ClC-4, and ClC-5 form a distinct branch of the family. Finally, ClC-6 and ClC-7 comprised an other separate branch. The tissue distribution for these different proteins suggests either a broad expression (e.g. for ClC-2) or a very restrictive expression. For example, ClC-1 is only present in muscle tissue, and ClC-K1 and -K2 are restricted to the kidney (for review, see Ref. 11).

In the present study, the complete inventory of ClC channels in cultured rat Sertoli cells was performed using RT-PCR analysis and whole-cell patch clamp recordings. We identified four voltage-dependent ClC chloride channels, ClC-2, ClC-3, ClC-6, ClC-7, and a novel acidic pH-activated chloride channel in which properties and localization do not match those for the cloned ClC channels.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Sertoli Cells—Experiments were performed on cultured Sertoli cells isolated in sterile conditions from 13–15-day-old Wistar rats. Animals, raised at constant temperature (20 °C) under a 12-h light/12-h dark cycle were killed by decapitation, and testis were removed aseptically. After decapsulation, the parenchyma of 6–10 testis was submitted at 34 °C to two enzymatic treatments by collagenase and pancreatin (7, 12). The interstitial tissue was separated from tubules by incubation with continuous shaking (90 cycles/min, 10 min) in 25 ml of medium A supplemented with 4.7 mg of collagenase (Worthington Biochemical Corp.; 390 units/mg). Medium A was a Ca2+- and Mg2+-free modified Earle's solution containing 116.3 mM Na+, 5.4 mM K+, 121.7 mM Cl—, 0.9 mM H2PO4, and 5.5 mM glucose supplemented with 53.5 mM mannitol and 20 mM Hepes and with streptomycin sulfate (100 mM) and penicillin G (100 IU/ml) (Sigma). The pH was adjusted to 7.4, and the osmolarity was adjusted to 300 mosmol by mannitol. The medium was sterilized by filtration through a 0.22-µm-pore filter (Millipore, Molsheim, France). Tubules were then allowed to sediment for 5 min, and the supernatant was removed. Then they were resuspended in 10 ml of medium A and shaken by hand for 10 s. The washing procedure was repeated four or five times. The tissue was minced with scissors, washed 5 times with 10 ml of medium A, then allowed to sediment for 5 min. The second dissociation was carried out to detach peritubular cells, comprising myoid cells and fibroblasts embedded in a collagen network, from the tubular wall. This was performed under continuous shaking (90 cycles/min, 5 min) in 25 ml of medium A containing 12.5 mg of pancreatin (grade VI; Sigma). The pancreatin solution was then discarded, and peritubular cells were separated from fragments of seminiferous tubules by gentle shaking in the presence of 10 ml of medium A (5 times). Fragments of seminiferous tubules were discarded after centrifugation. Sedimented fragments were resuspended in 10 ml of medium A and passed about 10 times through a syringe needle (17-gauge) at a slow rate. The cell suspension was adjusted to a volume of 10 ml with the following culture medium: Dulbecco's modified Eagle's medium/NUT mix F-12 (Ham's medium) (Invitrogen) supplemented with insulin (0.01 mg/ml; Sigma), bovine serum albumin (1 mg/ml; Sigma), transferrin (0.005 mg/ml; Sigma), and streptomycin sulfate (10 mg/100 ml) and penicillin G (100 IU/ml). Final cell preparations were plated at low density in 35-mm plastic dishes (Nunclon, Nunc, Roskilde, Denmark) for patch clamp experiments. Cells were incubated at 34 °C for 2–6 days in a humidified CO2 incubator (5% CO2, 95% ambient air). The medium was renewed at 2-day intervals.

Analysis of rClC mRNA Expression—Tissue and cell samples were assayed for rClC mRNA expression via RT-PCR. Total RNA was extracted using RNABIe® (Eurobio), according to the protocol provided by the manufacturer. 10 µg of RNA was reverse-transcribed in a reaction containing 400 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen), 2.4 µg of random hexanucleotide primers, 1 mM dNTPs, and 4 units of RNAguard (Amersham Biosciences) in 25 µl of final volume. After incubation at 37 °C for 1 h, the volume was adjusted to 50 µl with H2O, and the reaction was heated at 100 °C for 2 min. cDNAs were used as template in a polymerase chain reaction with one pair of PCR primers specific for rClC genes. Primers for rClC-1/2/3/5/K1/K2 have been described previously (13, 14, 15); primers for rClC-4/7 were designed according to cDNA sequences (accession number NM_022198 [GenBank] , nucleotide 1091–1600, and Z67744 [GenBank] , nucleotide 1523–2029, respectively); primers for rClC-6 were chosen on a rat genomic sequence (accession number AC113917 [GenBank] .2) by homology with human ClC-6 cDNA sequence. These primer pairs produced no amplification on rat genomic DNA. For RT-PCR, 0.5 µl of cDNA was mixed with 1.25 units of Taq DNA polymerase (Promega), 250 µM dNTPs, and 200 nM each primer in 50 µl of final volume. The temperature cycling conditions were initial melting at 95 °C for 5 min, annealing at specific temperature (55 °C for rClC-1/2/3/5 primers, 60 °C for rClC-4/6/7/K1/K2 primers) for 5 min, followed by 30 cycles of 72 °C for 30 s, 95 °C for 30 s, annealing for 30 s, and a final extension at 72 °C for 5 min. PCR products were visualized by 1.5% agarose gel electrophoresis. RT-PCR bands were sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). Reactions were run on an ABI 310 automatic sequencer (Applied Biosystems). The primers used to identify rClC mRNA in Sertoli cells are presented Table I.


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  		TABLE I
Forward (sense) and reverse primers for rat ClC

 

Patch Clamp Experiments—Whole cell recording was performed on Sertoli cells at room temperature. Currents were recorded with a RK300 patch clamp amplifier (Biologic, Grenoble, France). I-V relationships were build by clamping the membrane potential to —40 mV and by pulses from —100 mV to +100 mV or alternatively from +60 mV to —140 mV (20 mV increments). Pipettes with resistance of 3–5 mega-ohms were pulled from borosilicate glass capillary tubing (GCL150-TF10, Clark Electromedical Inc., Reading, UK) using a two-step vertical puller (Narishige, Japan). They were connected to the head stage of the patch clamp amplifier through an Ag-AgCl pellet. Seal resistances ranging from 3 to 15 gigaohms were obtained. Pipette capacitance was electronically compensated in cell-attached mode. Membrane capacitance and series resistances were measured in the whole-cell mode by fitting capacitance currents, obtained in response to a hyperpolarization of 6 mV, with a first-order exponential and by integrating the surface of the capacitance current. They were not compensated. Results were analyzed with the pCLAMP5.5 package software (pCLAMP, Axon Instruments). The external bath solution contained 161 mM NaCl, 5 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, and 10 mM HEPES-NaOH, pH 7.4, 370 mosmol). Two intrapipette solutions were used with different EGTA-calcium buffers to hold the intracellular free calcium concentration at 1 nM (solution A) and 1 µM (solution B). They consisted of the following; for solution A, 1 mM NaCl, 57 mM CsCl, 88 mM cesium glutamate, 3 mM MgCl2, 0.2 mM EGTA, 10 mM HEPES/CsOH buffer (pH 7.2, 300 mosmol); for solution B, 1 mM NaCl, 39 mM CsCl, 81 mM cesium glutamate, 9.1 mM CaCl2, 3 mM MgCl2, 10 mM EGTA, 10 mM HEPES (pH 7.2, 300 mosmol). The osmolarity was corrected with mannitol. For hypo-osmotic challenge, solution A in which the osmolarity was modified to 200 mosmol was used. To determine the anionic selectivity, NaCl from the external bath solution was replaced in equimolar amounts by NaBr, NaI, or sodium gluconate. For experiments using chloride transport inhibitors, diphenylamine-2-carboxylic acid (DPC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) were dissolved in dimethyl sulfoxide Me2SO (final Me2SO concentration, 0.1%). The currents were not altered by Me2SO alone. DPC and DIDS were from Sigma).

ClC-7 Antibodies—Two different antibodies were used to analyze the expression and localization of rClC-7 protein. A commercial rabbit anti-rat ClC-7 generated from a 23-amino acid peptide sequence near the C terminus of rClC-7 was purchased from Alpha Diagnostic International (San Antonio, TX). The second, generously given by T. J. Jentsch (ZMNH, Hamburg, Germany), was raised against a different N-terminal sequence (16).

Immunoprecipitation and Western Blot—Cell lysates (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 20 µM leupeptin, 0.8 µM aprotinin, 10 µM pepstatin, 1.25 mM phenylmethyl-sulfonyl fluoride) were incubated overnight at 4 °C with 6 µl of rClC-7 antibodies or an equal amount of non-immune rabbit IgG (Sigma-Aldrich). Immune complexes were incubated with 3 µg of protein A-Sepharose (Amersham Biosciences) for 1 h at 4 °C. Bead-bound complexes were denatured in Laemmli buffer and separated by 10% polyacrylamide SDS-PAGE. Proteins were transferred from gel to nitrocellulose membrane (Sartorius) and Western-blotted to detect rClC-7. Horseradish peroxide-conjugated donkey anti-rabbit IgG (Amersham Biosciences) was used as secondary antibody and was revealed with ECL Western blotting Detection Reagent (Amersham Biosciences).

Immunofluorescence—Sertoli cells were washed 3 times with phosphate-buffered saline (PBS), fixed with 3% paraformaldehyde in PBS for 20 min, and then soaked in 0.1% Triton X-100 in PBS for 10 min at room temperature. Cells were washed 3 times with PBS, soaked in blocking solution (PBS containing 10% bovine serum albumin) for 1 h, and then incubated with primary antibody (rabbit anti-rClC-7, 1:100) for 1 h in a moist chamber. After 3 washes, cells were incubated with the fluorescein isothiocyanate-conjugated goat anti-rabbit (Jackson ImmunoResearch, Interchim, 1:400) secondary antibody for 1 h at room temperature (stained in green). In all experiments, preparations were mounted in the same antifade solution Vectashield mounting medium (Vector Laboratories, Burlingame, VT). To identify cell nuclei nucleic acids were stained in blue with TO-PRO-3 iodide (Molecular Probes, Eugene, Oregon) for 15 min at room temperature (1:200 in PBS). In the control the primary antibody was omitted. Fluorescence was detected using confocal laser-scanning microscopy on a Bio-Rad MRC 1024.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of an Acid pH on Chloride Currents in Sertoli Cells— Because an acidic extracellular pH (pHe) plays a role in the stem cell differentiation associated to the physiology of Sertoli cells (2) and because some of the ClC chloride channels are regulated by pH (11), we first examined the effect of pHe on the activity of chloride channels in rat Sertoli cells in culture using the patch clamp technique in the whole-cell configuration. Currents are elicited by stepping from a holding potential of —40mV to a series of test potentials from —100 mV to +100 mV in 20-mV increments. With a bath containing a physiological solution at pH 7.4, Sertoli cells showed a very small basal current having a density measured at +60 mV of 1.76 ± 0.167 pA/picofarads (n = 25) as shown Fig. 1. When the pH of the bath solution was acidify from 7.4 to pH 5, a current rapidly activated with a density measured at +60 mV of 67.45 ± 9.95 pA/picofarads (n = 25, Fig. 1, B–D). The acidic pH-induced currents were activated instantaneously by depolarizing pulses with a further slow activation as shown Fig. 1B. The predicted reversal potential of this current was near the equilibrium potential for Cl— (ECl— = —25 mV), suggesting that the current was carried mainly by Cl—. Steady-state current curves of acidic pH-induced currents revealed a strong outward rectification (n = 25, Fig. 1, B–D). An example time course of reversible activation and inactivation of the current is shown Fig. 2. The activation occurred within 20 s with a bath solution at pH 5 (first three traces in Fig. 2A). Once activated, the maximal current amplitude remained stable, as shown Fig. 2A (last five traces). Increasing the bath pH from 5 back to 7.4 instantaneously inactivated the induced current as illustrated Fig. 2B. In 16 experiments of 16 total, the current could be re-activated after a second application of an acidic pH solution with the same amplitude than the first one (compare the current levels after the change in pH in Fig. 2B). The pH dependence of the induced current was studied in the presence of bath solutions in which we varied the pH value. The current was activated at pH 5 and 5.5 (Fig. 3, B–D) but not at pH 6 (Fig. 3, A–D). We can estimate a half-maximal stimulation around pH 5.5.



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  		FIG. 1.
External acidification activates an outwardly rectifying chloride current in Sertoli cells. Shown in whole cell patch clamp recordings performed on a rat Sertoli cell bathed with a NaCl-rich solution at pH 7.4 (A) and pH 5 (B). The corresponding current density/voltage relationship is given in C, as indicated. The control and the wash solution was at pH 7.4. Note the strong outward rectification of the current, the rapid onset of activation, and the further slow activation at depolarization pulses. D, histograms of the current density at +60 mV and —60 mV for 25 experiments in which the pH was varied from pH 7.4 to 5 as indicated. In Figs. 2, 3, 4, 5, 6, 7 the holding potential was —40 mV, and the currents were elicited from —100 mV to +100 mV in 20-mV increments. Data are presented as mean ± S.E. pF, picofarad.

 


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  		FIG. 2.
Reversible activation of the pH-induced chloride current. A, eight consecutive whole cell patch clamp traces obtained by a pulse from —40 mV to +60 mV at 10-s intervals showing the time course of activation of the chloride current from a Sertoli cell bathed with a NaCl-rich solution at pH 5. Note that after the third trace (20'') the current is maximum and remains stable thereafter. B, shown is the plot as a function of time of the current at +60 mV using an extracellular bath solution at either pH 7.4 (noted control) or pH 5 as indicated by the bar on the top of the graph. Note that changing the solution resulted in a rapid activation or inactivation of the current and that the current amplitude remained stable after the second pH 5 solution.

 


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  		FIG. 3.
Effect of various pH on the chloride currents. Whole cell patch clamp recording performed on rat Sertoli cell bathed with a NaCl-rich solution at pH 6 (A) and pH 5.5 (B). The corresponding current density/voltage relationship for several pH values is given in C as indicated. D, histograms of the current density at +60 mV at pH 7.4 (n = 10), 6 (n = 8), 5.5 (n = 7), and 5 (n = 10). Data are presented as the means ± S.E.

 



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  		FIG. 4.
Effect of Ca2+ on the pH-induced chloride current. A, histograms of current densities for three different experimental conditions: 0 mM Ca2+ intrapipette and 2 mM Ca2+ in the bath (n = 6); 1 µM Ca2+ intrapipette and 0 mM Ca2+ extracellular (n = 6); 0 Ca2+ intrapipette and in the bath (n = 4). NS, no statistical significance. B, current density/voltage relationships for the conditions presented in A. Data are presented as the means ± S.E. pF, picofarad.

 


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  		FIG. 5.
Effect of DPC on the pH-induced chloride current. Whole cell patch clamp recordings from a Sertoli cell before (A), during (B), and after (C) perfusion of 500 µM DPC. D, corresponding current/voltage relationship for the currents recorded in A, B, and C. Note the reversible inhibition of the pH-activated chloride current. In D the inward current is magnify to show the inhibitory effect of DPC.

 


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  		FIG. 6.
Anionic selectivity of the pH-induced chloride current. A, whole cell patch clamp recordings from a Sertoli cell bathed in a NaBr-rich solution at pH 5. B, current/voltage relationships for the current activated in the presence of various anion as indicated.

 


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  		FIG. 7.
RT-PCR analysis of ClC mRNA expression in rat Sertoli cells. Primers were designed as described under "Experimental Procedures," and the list for rClC-1–7 and rClC-K1 and -K2 primers is presented in Table I. Note that in Sertoli cells four rClC were detected, rClC-2, -3, -6, and -7. Control expression for rClC-1, -4, -5, -K1, and -K2 was performed using rat muscles and kidney. PCR product length: rClC-1, 351 bp; rClC-2, 179 bp; rClC-3, 242 bp; rClC-4, 510 bp; rClC-5, 556 bp; rClC-6, 424 bp; rClC-7, 507 bp; rClC-K1, 275 bp; rClC-K2, 275 bp. The molecular size marker (noted M, first lane on top) was {phi}X174/HaeIII.

 

Properties of the Acid pH-activated Chloride Current in Sertoli Cells—The calcium sensitivity of the pH-induced chloride current was tested by manipulating the calcium content of either the extracellular bath (0 and 2 mM Ca2+, n = 6) or the intra-pipette (1 nM and 1 µM Ca2+, n = 6) solutions or both (n = 4 for 0 mM Ca2+ intrapipette and extracellular). The current density measured at +60mV was not different whatever the calcium content of our experimental solutions (Fig. 4A). Fig. 4B shows the lack of effect of these different protocols on the current-voltage relationship of the acid-induced chloride current, further supporting an insensitivity to calcium of the current. The presence of a bath solution with forskolin (10 µM) failed to stimulate any chloride current in this preparation (n = 5, not shown). Finally, we have tested the effect of the chloride channel inhibitors DPC and DIDS. Extracellular DPC (n = 5, 500 µM) reversibly decreased the acid-induced chloride currents (Fig. 5). Because of the strong rectification of the current, the blocking effect on the inward currents is magnified and presented Fig. 5E. Similar inhibitory effect was found using 500 µM DIDS (n = 4, not shown). Relative anion selectivity to Cl—, Br—, I—, and gluconate was compared in 15 experiments, and the results are presented Fig. 6. With solutions containing equimolar amount of each anions, the reversal potential for iodide anions was more positive than bromide and chloride (Fig. 6B) but more negative than gluconate (not shown), suggesting the following anion selectivity sequence: Cl— > Br— > I— > gluconate. From these experiments we concluded that a pHe more acidic than 6 activates a Ca2+-insensitive DPC- and DIDS-inhibitable outwardly rectifying chloride current.

Expression of rClC Channels in Sertoli Cells—To begin to search for the molecular identity of the native pH-activated chloride current in cultured Sertoli cells we performed an inventory of rClC channels at mRNA level. An RT-PCR analysis was done using the primers listed in Table I. We searched for mRNA for rClC-1, rClC-2, rClC-3, rClC-4, rClC-5, rClC-6, and rClC-7 as well as for the two rClC-K1 and rClC-K2 genes. As shown in Fig. 7, mRNA expression was demonstrated for four of them: rClC-2, rClC-3, rClC-6, and rClC-7. To ascertain that the lack of amplification for rClC-1, rClC-4, rClC-5, and rClC-K1 and -K2 in Sertoli cells was not due to PCR conditions, we confirmed the presence of the corresponding transcript by using rat tissues known to express these proteins, as shown in Fig. 7, for rClC-1 in muscle (17), and rClC-4, rClC-5, and rClC-K1 and -K2 in kidney (11, 14).

ClC-2 and Swelling-induced Chloride Currents in Rat Sertoli Cells—Because rClC-2 is expressed in Sertoli cells (see Fig. 7), we felt it important to characterize the corresponding chloride current that must be inwardly rectifying and activated by hyperpolarization (18, 19, 20). We modified our pulse potential protocol to search for such hyperpolarization-induced current by stepping from a holding potential of —40mV to a series of test potentials from +60 to —140 mV in 20-mV increments. Using this protocol we first recorded whole cell currents under control conditions (i.e. with a physiological extracellular bath solution at pH 7.4). Under such experimental conditions no current was stimulated by hyperpolarization pulses (Fig. 8A). Decreasing the bath pH to 5 stimulated outwardly rectifying chloride currents but not inwardly rectifying chloride currents as shown Fig. 8B. An hypo-osmotic challenge was also applied to Sertoli cells preparation. A strong outwardly rectifying chloride currents was rapidly activated in three experiments, showing in this case a voltage-dependent inactivation typical of swelling-activated chloride currents (for a review of swelling-activated chloride currents, see Ref. 21). A typical example of such current is shown Fig. 8C. In some experiments a chloride current was also activated with an inwardly rectifying current/voltage relationship, a slow time- and voltage-dependent activation at hyperpolarization pulses. This current is illustrated Fig. 8D and is clearly identified as ClC-2 currents (11).



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  		FIG. 8.
ClC-2- and swelling-activated chloride currents in Sertoli cells. Whole cell patch clamp recordings were performed using a pulse protocol with a holding potential of —40 mV and step potentials from +60 mV to —140 mV in 20 mV increments except for C in which the steps were —100 mV to +100 mV from a holding potential of —40 mV. For each family of current, the corresponding current density/voltage curve is presented on the right. In A and D Sertoli cells were bathed with the NaCl-rich extracellular solution at pH 7.4. B, the same conditions but at pH 5. In C a hypo-osmotic challenge activated an outwardly rectifying chloride channel with different kinetics. Note the inward rectification of the current recorded in D and the different kinetics for B and C. pF, picofarad.

 

ClC-7 Chloride Channels in Rat Sertoli Cells—rClC-7 has recently been cloned (22) and is expressed at mRNA level in Sertoli cells (see Fig. 7). A recent report by Diewald et al. (23) report that acidic pH activated a chloride current when the corresponding rClC-7 mRNA was injected into Xenopus laevis oocytes. We, therefore, wished to determine whether our current could be due to rClC-7 in Sertoli cells. We first performed immunoprecipitation and Western blot analysis to detect rClC-7 proteins. As shown Fig. 9A, we were able to detect the corresponding ~80-kDa rClC-7 protein (16, 23) in rat Sertoli cells. It is important to note that only the antibody provided by Thomas J. Jentsch gave good results. The commercial rabbit anti-rat ClC-7 antibody did not work in our hands. Because Kornak et al. (16) report the intracellular location of ClC-7 protein in osteoclasts of mice and man, we performed an immunolocalization study of rClC-7 in rat Sertoli cells. The results presented Fig. 9B, b confirms the intracellular location of the protein as compared with control (Fig. 9B, a). Incubation of cells in an acidic pH solution before immunofluorescence study gave a similar intracellular location of rClC-7 (not shown). We were not able to detect any membrane localization of rClC-7 but only vesicular structures throughout the cytoplasm (Fig. 9B, b). Again only the antibody developed by T. J. Jentsch was good. No staining could be detected with the commercial antibody. The intracellular location of rClC-7 in rat Sertoli cells is, therefore, not compatible with the electrophysiological signature of the native pH-activated chloride current identified in the present study, simply because the corresponding channel must be membranous.



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  		FIG. 9.
Immunoprecipitation and immunolocalization of rClC-7 proteins in Sertoli cells. A, Western blot analysis of immunoprecipitated (IP) rClC-7 proteins. We used the rabbit anti-rat ClC-7 raised against the N terminus of rClC-7 provided by T. J. Jentsch (ZMNH, Hamburg, Germany). Note the ~80-kDa rClC-7 protein as reported in Kornak et al. (16) and Diewald et al. (23). B, immunofluorescence study of rClC-7 in Sertoli cells in the absence (a) or presence (b) of the same antibody as in A. Sertoli cells nucleus are stained in blue, and rClC-7 is stained in green as indicated under "Experimental Procedures." The bar scale is 10 µm.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we have investigated the expression and electrophysiological signature of voltage-dependent chloride channels in rat Sertoli cells in culture. Four ClC transcripts were identified, rClC-2, rClC-3, rClC-6, and rClC-7. Here we have identified a novel outwardly rectifying chloride current activated by strong acidic extracellular pH. We also detected the hyperpolarization-activated rClC-2 chloride current and swelling-induced chloride currents. Although ClC-7 was cloned in 1995 by Brandt and Jentsch (22), the physiological and electrophysiological signature of the native channel remained unknown. In Sertoli cells, because immunolocalization experiments support an intracellular location, we concluded that the native pH-activated chloride channel is not rClC-7. To our knowledge, we present here the first characterization of a native chloride current that is regulated by extracellular acidic pH.

rClC-7 Is Not the Chloride Channel Activated by Acidic pHe in Sertoli Cells—Seven years ago the rat and human ClC-7 were cloned together with their ClC-6 homologues (22). ClC-7 is a 89-kDa protein, and ClC-6 is a 97-kDa protein, both broadly expressed in brain, testis, muscle, and kidney. However, in attempts of functional expression both proteins failed to generate chloride channel activity using the X. laevis oocytes expression system (22, 24). Recently, however, Diewald et al. reported on the successful expression of rClC-7 in the same model (23). In oocytes injected with rClC-7-cRNA, acidification of the pHe between 6 and 4 revealed a strong outwardly rectifying chloride channel inhibited by DIDS and SITS (23). In rat Sertoli cells, we identified a native chloride current inhibited by DIDS and DPC and activated by acidic pHe between 6 and 5. At this stage of our study, an important question was to know whether rClC-7 could be responsible for this current. Importantly Kornak et al. (16) shown that ClC-7 resides in late endosomal and lysosomal compartments but not in the plasma membrane of osteoclasts. Our immunolocalization study of rClC-7 in rat Sertoli cells is in agreement with this since no plasma membrane staining could be detected. On the contrary, rClC-7 staining appeared vesicular throughout the cytoplasm. We conclude that rClC-7 is not the molecular entity supporting the native pHe-activated chloride current in rat Sertoli cells. We also excluded the possibility that ClC-6 might be involved since no specific plasma membrane currents could be detected upon its heterologous expression in Xenopus oocytes (22, 24). ClC-6 is probably predominantly localized intracellularly (11, 22, 24). For a similar reason we dismissed ClC-3 because it is mainly present in endosomes and synaptic vesicles (11).

pH Regulation of ClC Channels—Most of the ClC channels are pH-sensitive, being either activated, inhibited, or gated (11). ClC-1 from skeletal muscle is the only known ClC in which gating is affected by pH variation (11, 25). A low pHe diminished the deactivating inward currents without change in time constants, whereas the steady-state component is enhanced (25). However, a low intracellular pH slowed the deactivating current kinetics (25). Moreover, ClC-1 current was activated by hyperpolarization. The authors concluded that gating may be controlled by a Cl—-binding site accessible only from the exterior and possibly by modification of this site by external protonation (25). ClC-2 activity has been shown to be dependent on acidic pHe (26, 27). The regulation of ClC-2 remains, however, puzzling because other (apparently unrelated) mechanisms of activation exist, including cell swelling (see Ref. 11). Nevertheless, we have shown in this report that in rat Sertoli cells, a low pHe failed to activate ClC-2 (its regulation by cell swelling was not investigated here). Another argument for the absence of regulation by low pHe of ClC-2 in our preparation is the fact that the pH-induced chloride current was inhibited by DIDS, a drug that does not block ClC-2 (20). Conflicting results exist concerning the electrophysiological signature and pH regulation of ClC-4. Kawasaki et al. (28) report the identification of hClC-4sk, the human ClC-4 expressed from skeletal muscles. The human ClC-4sk is an acid-activated, DIDS-inhibited outwardly rectifying chloride channel in which halide selectivity is I— > Cl— > F— (28). More recently, hClC-4 was expressed in several mammalian cell lines (29). In each case, hClC-4 expression generated a novel outwardly rectifying chloride current with a different selectivity sequence of Cl— > Br— {approx} F— (29). Surprisingly, not only the halide selectivity was different but also the pH effects gave opposite results. In the second study the hClC-4 activity was inhibited by acidic pH (29) but not activated as in the first report (28). However, in both studies the current was apparently similar, i.e. it activated instantaneously by depolarizing pulses with a further slow activation (28, 29). Because rClC-4 is not present in our model, we rule out its participation in the observed pH-activated chloride current.

Other ClC channels are inhibited rather than activated by low pHe. Apart from ClC-4, for which results are too confusing, this is particularly evident for the renal ClC-K1 and -K2 (30, 31, 32) and ClC-5 (33) that are inhibited by acid pH. Thus, among the ClC channels, a number of them are regulated by acid pHe. However, to our knowledge and according to the regulation reported here, none appears to be activated only by acid pHe as was observed in the present study.

Physiological Significance of pH-activated Chloride Currents in Spermatogenesis—Sertoli cells secrete a fluid in which the composition remains undetermined (2, 4). The role of pH was evidenced by the fact that an acidic (pH 6.3) microenvironment in Sertoli cells is associated in vitro with the survival of germ cells in the contact of Sertoli cells and proliferation of spermatogonia (2). Although the exact pH value of the fluid secreted by Sertoli cells is not known, the secretion of a large amount of lactate by Sertoli cells was proposed to be responsible for this acidic pH (5, 6). The basic fibroblast growth factor stimulates the production of lactate by Sertoli cells through the regulation of glucose transport, lactate dehydrogenase activity, and GLUT1 and lactate dehydrogenase A mRNA levels (6). The production of lactate by Sertoli cells may be used as an energetic substrate for germ cells and as a promoter of their survival and proliferation through the production of the stem cell factor SCF (2). The mechanism of transport of lactate is proton-dependent and occurs by proton-linked monocarboxylate transporter named MCT in various tissues (34, 35). MCT2 is highly express in rat testis (36).

An acidic pH in the testis is required for the maturation process of sperm cells because it maintains sperm quiescence and prevents the premature activation of acrosomal enzymes (2, 37). The native pH-activated chloride current in Sertoli cells may represent a chloride conductance involved in this acidification process. In osteoclasts, for example, ClC-7 may be responsible for the electrical shunt that is necessary for the pumping of the ruffled border H+-ATPase (16). Although additional experiments are required, the role of the pH-activated chloride current in Sertoli cells may be equivalent at the plasma membrane to that of ClC-7 in intracellular compartment by being an important player in proton-linked lactate production. An alternative role may be linked to inflammation of the testis in which Sertoli cells are important players (38), and many inflammatory conditions are accompanied by local tissue acidosis with severe acidification (pH < 6).

In conclusion, in the present report, through the inventory of voltage-dependent chloride channels in rat Sertoli cells, we identified four rClC members; they are rClC-2, activated by hyperpolarization but not by low pHe; rClC-3 and rClC-6, two proteins with unknown functions; and the intracellular rClC-7 chloride channel. A novel native chloride current was activated by acid pHe but not by cell swelling, intracellular cAMP, or Ca2+ variation. It is sensitive to DPC and DIDS and presents a strong outwardly rectification of the current with a Cl— > Br— > I—> gluconate selectivity. This chloride current may be involved in the proton-linked lactate production in Sertoli cells or, alternatively, in the severe acidification observed in various inflammatory conditions.


   FOOTNOTES
 
* This work was supported by a fellowship from Le Conseil Régional du Poitou-Charentes, CNRS (to C. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 33-549-45-37-29; Fax: 33-549-45-40-14; E-mail: frederic.becq{at}univ-poitiers.fr.

1 The abbreviations used are: ClC, voltage-dependent chloride channel; pHe, extracellular pH; DPC, diphenylamine-2-carboxylic acid; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; RT, reverse transcription; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank A. Cantereau for expertise with the confocal imaging and R. Robert and L. Bulteau-Pignoux for sharing immunolocalization methods. We thank Michel Joffre, Claire Mauduit, and Bernard Jégou for helpful discussions. We are indebted to T. J. Jentsch for the generous gift of the rat ClC-7 antibody and for critical reading of the manuscript and discussion and A. Marty for helpful suggestions concerning the calcium-dependent experiments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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