Na+-alanine uptake activates a Clminus conductance in frog renal proximal tubule cells via nonconventional PKC

I. D. Millar and L. Robson

Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom


    ABSTRACT
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INTRODUCTION
METHODS
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Hyposmotically induced swelling of frog renal proximal tubule cells activates a DIDS-sensitive, outwardly rectifying Cl- conductance via a conventional protein kinase C (PKC). This study examines whether Na+-alanine cotransport similarly activates a DIDS-sensitive Cl- conductance in frog renal proximal tubule cells. On stimulation of Na+-alanine cotransport, the DIDS-sensitive current (IDIDS-Ala) increased markedly over time. IDIDS-Ala exhibited outward rectification, a Na+/Cl- selectivity ratio of 0.19 ± 0.03, and an anion selectivity sequence Br- = Cl- > I- > gluconate-. Activation of IDIDS-Ala was dependent on ATP hydrolysis and PKC-mediated phosphorylation and was inhibited by hyperosmotic conditions. Activation could be not ascribed to a conventional PKC isoform, as IDIDS-Ala was not affected by removing Ca2+ or by phorbol ester treatment, suggesting a role for a nonconventional PKC isoform, either novel or atypical. We conclude that Na+-alanine cotransport activates a DIDS-sensitive Cl- conductance via a nonconventional PKC isoform. This contrasts with the hyposmotically activated Cl- conductance, which requires conventional PKC activation.

cell volume; chloride channels; cotransport; 4,4' diisothiocyanostilbene-2,2' disulfonic acid; protein kinase C


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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AS EPITHELIAL CELLS ABSORB and secrete large quantities of solutes and water, the composition of their intracellular environment and volume is constantly under challenge. Accordingly, epithelial cells possess mechanisms to regulate excessive changes in their volume (14). In most cells, swelling activates solute exit pathways that drive the loss of excess cell water, allowing cell volume to return to preswollen levels. This is termed a regulatory volume decrease (RVD). Indeed, cell swelling induced by hyposmotic media in renal proximal tubule cells of different species activates both conductive and cotransport pathways, allowing the efflux of K+, Cl- and/or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (1, 17, 21, 39).

Exposure of single frog renal proximal tubule cells to a hypotonic shock leads to cell swelling followed by RVD (26). The RVD is inhibited by barium (an inhibitor of K+ channels), DIDS (an anion transport inhibitor), and gadolinium (Gd3+, an inhibitor of stretch-activated channels) (26). On the basis of these findings, it was suggested that hyposmotically stimulated RVD was mediated by K+, Cl-, and nonselective stretch-activated channels (SACs). Indeed, two whole cell conductances that are activated by hyposmolality and inhibited by Gd3+ have been attributed to SACs in these cells (27). An investigation into the identity of the swelling-activated K+ conductance is ongoing (30, 32). In addition, a Cl- conductance that is sensitive to DIDS and whose activity increases under hyposmotic conditions (GDIDS-Hypo) has also been characterized (28, 29). GDIDS-Hypo exhibits outward rectification with an anion selectivity sequence I- > Br- > Cl- (29). Furthermore, activation is dependent on ATP hydrolysis and phosphorylation, mediated by a protein kinase C (PKC)- and Ca2+-dependent mechanism (28, 29).

However, cells in vivo are rarely subjected to large changes in medium osmolality, except in the cases of specialized epithelia (e.g., renal medullary cells) or systemically during certain pathological conditions (6). In the case of renal proximal tubule cells, the more pertinent challenges to cell volume are changes in the rate of transepithelial flux of substrate (36). In general, transepithelial substrate flux is typified by concentrative, secondary active uptake from the tubule lumen followed by facilitated efflux at the basolateral face (34). The majority of concentrative substrate uptake from the lumen is energized by the Na+ gradient (3, 13, 15, 25, 33, 35). Any disparity in uptake at the apical membrane and efflux at the basolateral membrane will lead to changes in intracellular composition, and, consequently, cell volume. Therefore, cells must possess mechanisms to counteract excessive changes in their volume due to altered substrate uptake, a situation analogous to hyposmotically induced cell swelling and subsequent volume regulation (14).

In several cell types, Na+-amino acid cotransport has been found to induce cell swelling and subsequent volume regulation (8, 16, 18, 24). However, some apparent differences exist between the signaling of volume regulation stimulated by Na+-amino acid uptake and hyposmolality. In guinea pig jejunum and frog renal proximal tubule cells, RVD induced by hyposmotic shock is dependent on extracellular Ca2+, whereas RVD induced by Na+-alanine uptake is dependent on intracellular Ca2+ (19, 24, 26). Also, RVD in guinea pig jejunal enterocytes induced by Na+-alanine uptake is PKC dependent, whereas hyposmotically activated RVD is PKC insensitive (20).

The RVD that is observed after Na+-alanine cotransport stimulation in single proximal tubule cells isolated from frog kidney has previously been shown to be inhibited by DIDS, suggesting that cotransport-induced cell swelling activates an anion efflux pathway (24). A recent study has identified an anion-selective whole cell conductance (GVD) in single proximal tubule cells that is activated by stimulation of Na+-alanine cotransport, although its DIDS sensitivity was not investigated (31). The activity of GVD is both voltage and time dependent. It activates at potentials more positive than +60 mV and shows a characteristic slow time course of activation on stepping cell potential. As GVD is three times more selective for anions than cations, it could provide the anion efflux pathway during Na+-alanine-induced volume regulation. However, GVD is only observed at very positive, nonphysiological potentials. Therefore, another, as yet unidentified, anion conductance could be activated at more physiological potentials by stimulation of Na+-alanine cotransport. Certainly, hypotonic shock activates a DIDS-sensitive anion conductance at physiological potentials that has very different properties from those of GVD (28, 29). Therefore, the purposes of the following study were to investigate the effects of Na+-alanine cotransport on DIDS-sensitive currents in frog renal proximal tubule cells and the DIDS sensitivity of GVD.


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METHODS
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Single proximal tubule cells were isolated from frog kidney by enzyme digestion, modified from the method of Hunter (9). Briefly, Rana temporaria were killed by decapitation, the brain and spinal column were destroyed, and the kidneys were removed. The excised kidneys were then perfused for 30 min with a divalent cation-free amphibian Ringer solution, containing (in mM) 101 NaCl, 3 KCl, and 10 NaHEPES (pH 7.4), to disrupt cell-to-cell tight junction integrity. The kidneys were then perfused retrogradely with 10 ml in total of the above solution plus collagenase (130 U/ml) and pronase (0.8 U/ml) to digest connective tissue. The kidneys were minced and further incubated in the enzyme mixture for 10 min at 20°C and gassed with 100% O2. A mixed cell population was liberated by trituration, resuspended in experimental bath solution, and kept on ice until required. Single proximal tubule cells were identified by their polarized morphology (26).

Suspensions of single cells were placed in a Perspex superfusion bath at room temperature on the stage of an inverted microscope (Olympus IX70). Whole cell currents were investigated by using standard patch-clamp techniques (5). Patch pipettes were pulled from soda lime glass and had resistances of ~5 MOmega when filled with pipette solution. Whole cell patches were obtained via the basolateral membrane, with access resistance being <25 MOmega . Success of achieving the whole cell configuration was dependent on the effectiveness of daily cell isolation and bath and pipette solutions, but when conditions were optimum success rates of up to 90% were possible. In all cases, patched cells were free floating. Voltage protocols were driven from an IBM-compatible computer with a Digidata 1200 interface using pCLAMP software Clampex6 (Axon Instruments). Cell recordings were made via a Heka EPC-7 patch-clamp amplifier, subjected to low-pass filtering at 5 kHz and saved directly to the computer hard drive. Data were expressed as either current normalized to the cell capacitance or conductance per unit cell area. Cell area was calculated from capacitance, using the assumption that membrane capacitance corresponded to 1 µF/cm2. Cell capacitance was estimated from cancellation of slow capacitance transients, with mean capacitance being 55.8 ± 1.6 pF (n = 142). To aid capacitance transient cancellation, all patch pipettes were coated with Sylgard (Dow Corning).

Solutions

Unless stated in the text, bath and pipette solutions were asymmetrical with regard to Na+ and Cl-. Thus the control bathing solution was a high-Ca2+ amphibian Ringer containing (in mM) 92 NaCl, 2 CaCl2, 1 MgCl2, 16 mannitol, and 10 HEPES, titrated to pH 7.4 with NaOH. L-Alanine was included in the bathing solution at a concentration of 5 mM when required, with equimolar substitution of mannitol. Except where stated in the text, the pipette contained a low- NaCl, nominally Ca2+-free amphibian Ringer which contained (in mM) 20 NaCl, 80 Cs gluconate, 0.5 EGTA, 2 MgATP, and 10 HEPES, titrated to pH 7.4 with NaOH. Cs+ was included to inhibit K+ currents in the cell (Millar and Robson, unpublished observations). These solutions generated an inwardly directed Na+ gradient and thus promoted Na+-alanine cotransport (31). The pipette solution was 4-8 mosmol/kgH2O hyposmotic to the bathing solution to inhibit spontaneous activation of the osmotically sensitive Cl- conductance previously characterized in these cells (28, 29). Osmolality of all solutions was determined by using a Roebling osmometer, and osmolalities were adjusted to the desired value by altering Cs gluconate or mannitol concentration, respectively, as appropriate. All chemicals were purchased from Sigma, except alkaline phosphatase (AP), PKC-ps, Gö 6983, and Gö 6850 (Calbiochem), and were of analytical grade.

Experimental Protocol: Effect of Alanine and Mechanism of Activation

When the whole cell configuration was obtained, residual currents were allowed to run down to steady state over ~2 min. Cells were clamped at a holding potential of -70 mV and episodically stepped to +39 mV for 200 ms every 5 s during recording. This voltage approximated to the reversal potential for alanine transport in 11 cells tested. At this potential, current was predominantly Cl- selective. In all cases, the magnitude of the Cl- current was determined by measuring the current sensitive to 100 µM Na2DIDS, which was added to the bathing solution. DIDS sensitivity was tested just before alanine superfusion (control) and at steady-state activation. The increase in DIDS-sensitive current observed on addition of alanine was termed the alanine-activated, DIDS-sensitive current (IDIDS-Ala). Na+-alanine cotransport currents were determined from the instantaneous currents generated immediately on addition of alanine at -70 mV (holding potential) given that the cotransport current was found to be directly proportional to voltage over the range -10 to -100 mV (results not shown). These currents were not sensitive to DIDS or Gd3+. All current values were normalized to the capacitance of the cell and were expressed as pA/pF.

Na+ dependence and stereospecificity of IDIDS-Ala activation. When the sodium dependence of the activation of IDIDS-Ala was examined, NaCl in bath and pipette was replaced with equimolar amounts of N-methyl-D-glucamine (NMDG) Cl, and all solutions were titrated to pH 7.4 with free NMDG. The stereospecificity of activation by alanine was examined by the addition of 5 mM D-alanine to the bath (substitution of mannitol).

Osmotic sensitivity of IDIDS-Ala activation. The osmotic sensitivity of IDIDS-Ala activation was tested by exposing cells to 5 mM L-alanine under hyperosmotic conditions throughout (addition of 40 mM mannitol to bathing solutions).

ATP dependence of IDIDS-Ala activation. The dependence of IDIDS-Ala activation on ATP was investigated. The pipette contained either 2 mM MgATP (control), no ATP (addition of 2 mM Cs gluconate), 2 mM 5'-adenylylimidodiphosphate (AMP-PNP, nonhydrolysible ATP analog), or 2 mM MgATP plus 25 U/ml AP (to catalyze dephosphorylation).

PKC dependence of IDIDS-Ala activation. The dependence of IDIDS-Ala on PKC was examined by inclusion of 10 µM PKC-ps (a broad-range PKC inhibitor), 0.6 µM Gö 6983 (an inhibitor of PKC isoforms alpha , beta , gamma , delta , and zeta , but not µ) (4), or 2 µM Gö 6850 (an inhibitor of PKC isoforms alpha ,beta , gamma , delta , epsilon , and zeta ) (7) in the pipette. In a second series of experiments, activation of PKC was examined by including 0.1 µM phorbol 12-myristate 13-acetate (PMA, activator of conventional and novel isoforms) in the bath (7). To allow full activation of PKC, cells were preincubated in experimental solutions for ~10 min.

Ca2+ dependence of IDIDS-Ala activation. To test the dependence of IDIDS-Ala on extracellular Ca2+, CaCl2 was omitted from the bathing solution. To test the dependence of IDIDS-Ala activation on intracellular Ca2+, 5 mM EGTA was included in the pipette solution to buffer intracellular Ca2+.

Experimental Protocol: Properties of IDIDS-Ala

Na+/Cl- selectivity ratio. The Na+/Cl- selectivity ratio of the DIDS-sensitive current was examined in the asymmetrical solutions described earlier, with extracellular and intracellular concentrations of Na+ being 98 and 29 mM, respectively, and those of Cl- being 98 and 20 mM, respectively. Clamp potential was stepped between +40 mV and -100 mV, in -10-mV steps in the absence and presence of L-alanine. The current sensitive to 100 µM DIDS was measured under both circumstances. The reversal potential (Vrev) of IDIDS-Ala was determined by using polynomial regression with either 80 mM Cs-gluconate or 80 mM NMDG-gluconate in the pipette. The Na+/Cl- selectivity ratio was calculated by using the Goldman equation. This calculation used the measured selectivity ratio for gluconate (see RESULTS) and, as the Vrev of the conductance did not alter between Cs+ and NMDG+ pipette solutions (see RESULTS), permeation of Cs+ through the conductance was assumed to be minimal and was omitted from the selectivity calculation.

Current-voltage relationship. The current-voltage relationship of IDIDS-Ala was investigated by using symmetrical Cl- solutions. The bath solution was a high-Na+, high-Ca2+ amphibian Ringer containing (in mM) 95 NaCl, 2 CaCl2, 1 MgCl2, 5 mannitol, and 10 HEPES, titrated to pH 7.4 with NaOH. The pipette contained a low-Na+, nominally Ca2+-free amphibian Ringer containing (in mM) 99 CsCl, 0.5 EGTA, 2 MgATP, and 10 HEPES, titrated to pH 7.4 with NaOH. On achievement of the whole cell configuration, residual currents were allowed to run down to steady state. Whole cell potential was held at -70 mV and stepped between +15 and -60 mV in -5-mV steps. DIDS sensitivity was tested before 5 mM L-alanine superfusion (control) and when currents had activated to a steady state in the presence of L-alanine. Outward slope conductance was determined between +15 and +5 mV, whereas inward slope conductance was determined between -5 and -60 mV. Conductance was expressed as a function of cellular area (µS/cm2).

Anion selectivity. Anion selectivity was determined, in the presence of 5 mM L-alanine, from changes in the Vrev of IDIDS-Ala when 95 mM NaCl in the bathing solution was substituted with 95 mM NaBr, NaI, or Na-gluconate. Clamp potential was varied between +15 and -60 mV in -5-mV steps in the presence of Cl-, Br-, or I-, or varied between +70 and -20 mV in -10-mV steps in the presence of gluconate-, all from a holding potential of -70 mV. When the +70 to -20-mV voltage step protocol was employed, each voltage step was separated by 10 s, as opposed to 1 s, to allow inactivation of the voltage-activated conductance GVD, which is activated at by potentials over +60 mV (27). To rule out any contamination by GVD in the gluconate selectivity calculation, the effect of DIDS on GVD was examined at +70 mV in the presence of L-alanine. Current was recorded initially on stepping the potential to +70 mV and then within 20 ms of the end of the potential step, in the absence and presence of 100 µM DIDS. The difference between the current recorded initially on stepping the potential and the current recorded at the end of the potential step (after full activation of GVD) was taken as an index of GVD (see Fig. 4, inset). Selectivity was determined from shifts in Vrev by using the Goldman equation. Vrev was calculated from polynomial regression of IDIDS-Ala. For all selectivity calculations, the Vrev for the experimental anion was compared with the Vrev measured in the presence of Cl- immediately before bath solutions were changed. Vrev was corrected for junction potential changes associated with bath Cl- substitution. Junction potential changes were determined by using a flowing 3 M KCl reference electrode and were found to be 1.8, 10.1, and 10.7 mV for substitution of Cl- with Br-, I-, and gluconate-, respectively.

Dose response to DIDS. To test the nature of the inhibition of IDIDS-Ala induced by DIDS, the effect of a range of concentrations of DIDS, from 2 to 500 µM, was tested for their potency of inhibition. The bath and pipette solutions were the asymmetrical solutions described earlier. Whole cell currents were allowed to activate to steady state in response to 5 mM L-alanine. The voltage protocol utilized a holding potential of -70 mV and was stepped to +40, 0, and -40 mV. The data were fitted to the Hill equation (Eq. 1)
I<SUB>DIDS</SUB><IT>=</IT><FR><NU><IT>I</IT><SUB>min</SUB><IT>+</IT>(<IT>I</IT><SUB>max</SUB><IT>−I</IT><SUB>min</SUB>)</NU><DE><IT>1+</IT>([DIDS]<IT>/K</IT><SUB>d</SUB>)<SUP><IT>−n</IT></SUP></DE></FR> (1)
where IDIDS is the DIDS-sensitive current, Imax and Imin are the maximum and minimum DIDS-sensitive current, respectively, Kd is the concentration of DIDS capable of inhibiting 50% of IDIDS, and n is the Hill coefficient.

Gd3+ sensitivity of IDIDS-Ala. A previous study has demonstrated that the hyposmotically activated Cl- conductance is inhibited by Gd3+ (28). The sensitivity of IDIDS-Ala to Gd3+ was examined at a clamp potential of +39 mV after the current had activated to a steady state in the presence of 5 mM L-alanine. The magnitude of the DIDS-sensitive current was measured before and during exposure of the cells to 100 µM GdCl3.

Role of PKC in Na+-Alanine-Induced RVD

The length of intact cells was monitored by using a photodiode array as described by Mounfield and Robson (24). Briefly, the length of cells adhered to a coverslip was determined by measuring the distance between the changes in light intensity that occur at the start and end of the cell. The bathing solution was an amphibian Ringer containing (mM) 90 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 15 mannitol, and 10 HEPES, titrated to pH 7.4 with NaOH. RVD induced by 5 mM L-alanine (equimolar substitution of mannitol) was examined under the control circumstance, in the presence of 0.1 µM PMA or in the presence of 2 µM Gö 6850 (a cell-permeable inhibitor of PKC isoforms alpha , beta , gamma , delta , epsilon , and zeta ).

Simultaneous Cell Length and Patching

To determine whether stimulation of Na+-alanine cotransport altered cell length in the whole cell configuration, simultaneous measurement of cell length and whole cell current recordings were carried out in response to addition of 5 mM L-alanine to the extracellular solution. Whole cell recordings were obtained by patching the side of free-floating cells and current recorded at -70 mV to give an estimate of the Na+-alanine cotransport current. Cell length was recorded as above.

Statistical Significance

Statistical significance was determined by using Student's t-test or ANOVA, as appropriate, and was assumed at the 5% level. All values are expressed as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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Na+-Alanine Cotransport Current

Application of 5 mM L-alanine to frog renal proximal tubule cells induced large instantaneous increases in the whole cell currents when recorded at -70 mV (Fig. 1 and Table 1; Na+-alanine cotransport currents). The instantaneous currents generated by application of L-alanine were not inhibited by DIDS (data not shown). Exposure of cells to 5 mM D-alanine also generated a significant current, but this was reduced compared with L-alanine. When Na+ in the bath and pipette solutions was replaced with NMDG+, no significant L-alanine-induced currents were observed. These findings suggest that alanine is transported by proximal tubule cells via Na+-coupled cotransport. In all other experimental conditions, there was no effect of treatment on the magnitude of the Na+-alanine cotransport currents (Table 1).


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Fig. 1.   Effect of 5 mM L-alanine on whole cell current in a single cell. A: typical traces from a single cell showing whole cell current recorded when cell potential was stepped from -70 to +39 mV for 200 ms. Traces shown are (from left to right) under the control circumstance, initially on addition of L-alanine to the bath, when IDIDS-Ala was at steady state, and on washout of L-alanine. Relative time of recording the traces during the experiment is indicated in relation to the condition bar. The dashed line indicates zero current. B: corresponding whole cell current in response to addition of L-alanine to the bath recorded episodically at +39 mV throughout the time course. Thick solid dots indicate when 100 µM DIDS was present in the bathing solution.


                              
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Table 1.   Dependence of Na+-alanine cotransport current and alanine-activated DIDS-sensitive current on amino acid stereoisomer, Na+, osmolality, ATP, and PKC

Mechanism of Activation of IDIDS-Ala

Effect of alanine on IDIDS-Ala. In control conditions, the DIDS-sensitive current recorded at +39 mV was small (Fig. 1). On application of 5 mM L-alanine, there was a progressive increase in the DIDS-sensitive current to a plateau, which subsequently decreased when L-alanine was removed from the bath solution (Fig. 1). In paired cells, the DIDS-sensitive current was 0.04 ± 0.02 pA/pF in the control bath, increasing to 2.64 ± 0.49 pA/pF in the presence of 5 M L-alanine, 36 ± 3 min after L-alanine superfusion was started (n = 10). Subsequent removal of L-alanine from the bathing solution caused the DIDS-sensitive current to decrease slowly again to a steady state of 0.86 ± 0.22 pA/pF, which was significantly lower than the peak DIDS-sensitive current in the presence of L-alanine, but remained significantly higher than prestimulation values. The DIDS-sensitive current activated by alanine, IDIDS-Ala, was taken as the difference between the DIDS-sensitive current recorded under the control conditions and at steady state in the presence of alanine (Table 1).

Na+ dependence and stereospecificity of IDIDS-Ala activation. Application of 5 mM L-alanine to proximal tubule cells activated IDIDS-Ala. This activation was dependent on the presence of extracellular Na+ and was stereospecific. When NMDG+ was substituted for Na+, IDIDS-Ala was still capable of activation. However, this was significantly reduced compared with Na+-containing solutions (Table 1). Five millimolar D-alanine significantly activated IDIDS-Ala, although at a level reduced from that induced by L-alanine (Table 1). Furthermore, the magnitude of IDIDS-Ala activation varied proportionately with either L- or D-alanine cotransport currents (Fig. 2). When IDIDS-Ala was calculated as a function of the Na+-alanine cotransport current (IDIDS-Ala divided by the Na+-alanine cotransport current), there was no significant difference between the DIDS-sensitive current activated by either L- or D-alanine. (IDIDS-Ala = 1.23 ± 0.17 and 1.10 ± 0.24 pA/pF per pA/pF Na+-alanine cotransport current for L- and D-alanine, respectively.)


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Fig. 2.   Relationship between Na+-alanine cotransport current recorded at -70 mV and DIDS-sensitive current recorded at +39 mV, in the presence of 5 mM L-alanine or D-alanine.

Osmotic sensitivity of IDIDS-Ala activation. IDIDS-Ala activation was completely blocked under hyperosmotic conditions compared with isosmotic conditions (Table 1).

ATP dependence of IDIDS-Ala activation. Activation of IDIDS-Ala was dependent on the presence of ATP (Table 1). Omission of MgATP from the pipette solution abolished activation of IDIDS-Ala. Activation of IDIDS-Ala was similarly inhibited by replacement of pipette MgATP with 2 mM AMP-PNP. The addition of 25 U/ml AP to the pipette solution, to stimulate dephosphorylation, in the presence of 2 mM MgATP gave rise to significant activation of IDIDS-Ala in response to 5 mM L-alanine, although activation was reduced compared with 2 mM MgATP alone.

PKC dependence of IDIDS-Ala activation. Activation of IDIDS-Ala was dependent on the stimulation of PKC (Table 1). The broad range PKC inhibitor PKC-ps (10 µM) significantly blunted IDIDS-Ala activation. The PKC isoform-specific inhibitors Gö 6983 (0.6 µM) and Gö 6850 (2 µM) also inhibited activation. However, inclusion of 0.1 µM PMA in bath and pipette solutions had no effect on activation.

Ca2+ dependence of IDIDS-Ala activation. Removal of extracellular Ca2+ had no effect on IDIDS-Ala (Table 1). This was also true for increased buffering of intracellular Ca2+ by increasing pipette EGTA from 0.5 to 5 mM (Table 1).

Properties of IDIDS-Ala

Na+/Cl- selectivity ratio. In asymmetrical solutions with Cs-gluconate in the pipette, the Vrev of IDIDS-Ala was -18.8 ± 1.4 mV (n = 6). This was not significantly different from the Vrev recorded when Cs-gluconate was replaced with NMDG-gluconate (-20.9 ± 2.6 mV, n = 4). Combining these data gave a mean Vrev of -19.6 ± 1.2 mV and a Na+/Cl- selectivity ratio of 0.19 ± 0.03 (n = 10).

Current-voltage relationship. In symmetrical solutions, IDIDS-Ala demonstrated outward rectification (Fig. 3). GDIDS-Ala: out and GDIDS-Ala: in were 95.99 ± 10.64 and 30.33 ± 3.63 µS/cm2, respectively (n = 24). The reversal potential of IDIDS-Ala in symmetrical Cl- was found to be 4.31 ± 0.42 mV. This was not significantly different from 4.17 mV, which corresponds to the calculated reversal potential, taking the partial Na+ permeability into account.


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Fig. 3.   Current-voltage relationship of IDIDS-Ala recorded with symmetrical Cl-. A: typical traces obtained from the same cell in the absence (top) or presence (bottom) of 5 mM L-alanine and in the absence (left) and presence (right) of 100 µM DIDS. Clamp potential was stepped between +15 and -60 mV. B: current-voltage relationship plot of the control and alanine-activated DIDS-sensitive current.

Anion selectivity. The anion selectivity sequence of IDIDS-Ala was Br- = Cl- > I- >> gluconate (Table 2).

                              
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Table 2.   Anion selectivity of IDIDS-Ala

Contribution of GVD. The voltage- and time-dependent conductance GVD was not sensitive to DIDS (Fig. 4). There was no significant difference between the activation of GVD in the absence and presence of DIDS. The magnitude of current flow through GVD at +70 mV was 11.1 ± 1.9 pA/pF in the absence of DIDS and 11.9 ± 1.5 pA/pF in its presence (n = 5).


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Fig. 4.   Effect of DIDS on anion-selective whole cell conductance (GVD). Typical traces taken from the same cell showing GVD recorded at +70 mV in the absence and presence of 100 µM DIDS. The inset shows how GVD was calculated.

Dose-response to DIDS. Inhibition of IDIDS-Ala by DIDS was dose dependent (Fig. 5). There were no significant differences between the Hill coefficients determined at +40, 0, and -40 mV: 1.6 ± 0.4, 1.0 ± 0.2, and 1.8 ± 0.7, respectively (n = 7). These values were also not significantly different from one. However, inhibition was voltage dependent. The Kd values determined at +40, 0, and -40 mV were 4.8 ± 1.2, 13.0 ± 3.1, and 26.9 ± 3.1 µM, respectively (n = 7). These values were significantly different from each other (F2,18 = 21.06).


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Fig. 5.   Dose response to DIDS. The mean DIDS-sensitive current (in pA/pF) at concentrations of DIDS ([DIDS]) between 2 and 500 µM. The lines are best fits of Eq. 1 recorded at +40, 0, and -40 mV (r2= 0.99 in each case).

Sensitivity of IDIDS-Ala to Gd3+. IDIDS-Ala was inhibited by Gd3+. Maximally activated DIDS-sensitive current before addition of Gd3+ to the bath was 3.76 ± 1.28 pA/pF (n = 5). This was reduced to 1.54 ± 0.64 pA/pF in the presence of Gd3+ (n = 5).

Role of PKC in Na+-Alanine-Induced RVD

Addition of L-alanine to the cells initially increased cell length to a peak (Fig. 6). This was followed by decrease in length back toward prealanine levels (Fig. 6). Neither the degree of cell swelling (a), nor the magnitude of RVD during L-alanine superfusion (b), nor its rate (T1/2) was affected by PMA (Table 3). However, RVD was inhibited by the presence of the PKC inhibitor Gö 6850 (Table 3).


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Fig. 6.   The PKC dependence of L-alanine-stimulated regulatory volume decrease (RVD). Typical traces of cell length demonstrating the effects of alanine alone (top), + 0.1 µM phorbol-12-myristate-13-acetate (PMA; middle), or + 100 µM Gö 6850 (bottom). The traces depict increases in cell length on application of alanine to a peak (a), which was followed by a decrease to steady state (b). T1/2 represents the time taken from attainment of peak cell length to half-regulated steady state. See Table 2 for summary data.


                              
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Table 3.   The role of PKC in L-alanine-stimulated volume regulation

Simultaneous Cell Length and Patching

Addition of L-alanine to the extracellular solution increased both the whole cell current recorded at -70 mV and cell length (Fig. 7). In the absence of L-alanine, whole cell current was -186.9 ± 134 pA and cell length was 21.2 ± 1.3 µm (n = 5). On the addition of L-alanine, activation of the Na+-alanine cotransporter significantly increased the current by -65.1 ± 19.7 pA and cell length by 0.70 ± 0.27 µm (n = 5). The increase in current and cell length was reversible. On removal of L-alanine from the bath, current and length fell by 95.0 ± 18.7 pA and 0.65 ± 0.14 µm, respectively (n = 5).


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Fig. 7.   Effect of L-alanine on cell length and whole cell current in a patched cell. Top: effect of 5 mM L-alanine on the length (in µm) of a cell held in the whole cell configuration. Bottom: simultaneous effect of L-alanine on the whole cell current (in pA) recorded at -70 mV in the same cell. The condition bar (top) indicates the presence of L-alanine in bathing solution.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Application of alanine to frog renal proximal tubule cells induced instantaneous currents. The induced current was found to be Na+ dependent and stereoselective for L-alanine over D-alanine. These findings suggest that the instantaneous current generated by alanine is due to Na+-alanine cotransport, in common with mammalian proximal tubule (3, 13). Indeed, the instantaneous current generated by application of alanine (Fig. 1) cannot be attributed to a K+ conductance as K+ was absent from all solutions. Similarly, the current cannot be attributed to a nonselective conductance, GVI, identified in these cells as it has been shown to be unaffected by alanine application (31). Furthermore, DIDS or Gd3+ did not inhibit the alanine current.

Stimulation of the Na+-alanine cotransporter subsequently caused reversible activation of a predominantly Cl--selective conductance, IDIDS-Ala. There are several pieces of evidence that support this conclusion: 1) IDIDS-Ala was more selective for anions, with a Na+/Cl- selectivity ratio of 0.19, suggesting that the conductance was five times more selective for Cl- than Na+; 2) the conductance was sensitive to the anion transport blocker DIDS; 3) removal of extracellular Na+ or addition of the poorly transported isomer of alanine (D-alanine) reduced the activation of IDIDS-Ala; and 4) the magnitude of IDIDS-Ala activation was proportional to the Na+-alanine cotransport current, which was found to be the case irrespective of whether cells were exposed to L- or D-alanine. Given that D-alanine is poorly metabolized by cells, these findings would suggest that activation of IDIDS-Ala by alanine was directly due to Na+-alanine cotransport, rather than secondary metabolism of alanine leading to activation. This appears to contrast with the finding that D-alanine caused no significant swelling of frog renal proximal tubule cells (24). However, given that D-alanine induces only 10% of the increase in IDIDS-Ala compared with L-alanine, a comparable increase in cell length induced by D-alanine would be ~0.05 µm. This value is on the limit of detection of the optical technique used in the previous study to assess cell volume changes (26). Taken together, these data show that stimulation of Na+-alanine cotransport activated a predominantly Cl--selective conductance in the cells.

Properties of the DIDS-sensitive conductance. The DIDS-sensitive current activated during application of 5 mM L-alanine exhibited outward rectification in symmetrical Cl- solutions and was sensitive to DIDS in a concentration-dependent fashion. These characteristics are also shared by several volume-sensitive Cl- conductances in different cell types that are activated in hyposmotic conditions (37), including frog renal proximal tubule cells (29).

The DIDS-sensitive Cl- conductance was also found to be directly inhibited by Gd3+. This result explains the conflicting findings in a previous study that alanine-stimulated RVD in frog renal proximal tubule cells was inhibited by Gd3+, suggesting a role for stretch-activated channels in mediating RVD, but was unaffected by removal of extracellular Ca2+, which argued against the participation of stretch-activated channels (24).

The anion selectivity exhibited by IDIDS-Ala markedly differs from GDIDS-Hypo. IDIDS-Ala exhibited an anion selectivity sequence that closely corresponds to Eisenman's sequence III (Br- = Cl- > I- > gluconate-; 40), as opposed to GDIDS-Hypo, which exhibits Eisenman's sequence I (I- > Br- > Cl- > gluconate-; 29). Interestingly, a cloned chloride channel that appears to be volume sensitive, ClC-2, also exhibits an anion selectivity sequence Cl- > I-, although it does not show outward rectification or sensitivity to DIDS (11, 38). The only other study to examine a Cl- conductance in response to Na+-alanine cotransport found that the anion selectivity of an activated conductance in a rat hepatoma cell line (16) did not differ from that of the conductance activated in response to hyposmotic conditions (22). However, it is not clear from the study by Lidofsky and Roman (16) whether junction potential changes associated with altering bathing chloride concentrations were taken into account and, therefore, definitive evaluation of this data is not possible.

Although the observed cation-to-anion selectivity ratio is high compared with many volume-sensitive anion conductances (37), it is similar to the cation-to-anion selectivity of 0.2 exhibited by a swelling-activated anion conductance in skate hepatocytes (10). The possibility exists that the low anion selectivity of IDIDS-Ala was due to multiple conductances of varying anion selectivity being activated by alanine uptake. However, we feel that this is unlikely because the activated conductances would all have to show DIDS sensitivity plus exhibit similar pharmacologies with respect to their activation.

Mechanism of activation of the DIDS-sensitive conductance. Activation of IDIDS-Ala was inhibited by a hypertonic bath solution, suggesting that activation was dependent on cell swelling. Indeed, simultaneous cell length and patching experiments demonstrated that stimulation of Na+-alanine cotransport during whole cell patching was still capable of increasing cell length. However, it should be noted that the magnitude of swelling observed in patched cells (Fig. 7) was approximately four times that observed in intact cells. This can be explained by the fact that K+ was replaced in the pipette solution by Cs+, thereby meaning that the patched cells would be incapable of undergoing RVD. Indeed, the magnitude of cell swelling observed in patched cells is comparable to that in intact cells that fail to exhibit RVD (24).

Activation of IDIDS-Ala was dependent on ATP hydrolysis. This is in common with several other volume-sensitive Cl- channels (2, 23, 29), although not all (37). Furthermore, activation of IDIDS-Ala was inhibited by AP, in common with GDIDS-Hypo (29). A similar phenomenon was found in M-1 mouse cortical collecting duct cells, where activation of a swelling-activated chloride channel was inhibited by omission of intracellular Mg2+ (23). These findings suggest that activation of these pathways is mediated via ATP hydrolysis and subsequent phosphorylation.

Activation of IDIDS-Ala was dependent on PKC, as evidenced by inhibition of activation by PKC-ps, Gö 6983, and Gö 6850. This is in common with the hyposmotically activated Cl- conductance in frog renal proximal tubule cells (28). PKC isoforms can be classified into three groups: conventional isoforms that are activated by phorbol esters and inhibited by Ca2+ removal; novel isoforms that are activated by phorbol esters but are unaffected by Ca2+; and atypical isoforms that are unaffected by phorbol esters and Ca2+. GDIDS-Hypo was additionally activated by phorbol ester and was Ca2+ dependent (28). These data suggested that GDIDS-Hypo was activated via a conventional PKC isoform. However, IDIDS-Ala was unaffected by phorbol ester treatment and was not Ca2+ dependent, suggesting it was activated by an atypical PKC. In addition, phorbol ester stimulation affected neither the magnitude nor the rate of Na+-alanine-induced RVD. This is in contrast to hypotonic shock-induced RVD, the rate of which was more than doubled by phorbol ester treatment (26). This lack of effect of phorbol ester suggests that neither conventional nor novel PKC isoforms are involved in Na+-alanine-induced RVD in the cells. However, Na+-alanine-induced RVD was inhibited by exposure to Gö 6850, which inhibits conventional, novel, and atypical PKC isoforms. Taken together, these data suggest that an atypical PKC isoform is involved in Na+-alanine-induced RVD. However, the situation could exist that both IDIDS-Ala and Na+-alanine-induced RVD were activated by an already maximally stimulated novel PKC and were hence not additionally stimulated by PMA. Indeed, both novel and atypical PKC isoforms have been identified in proximal tubule cells of the rat (12). Differences in the PKC dependence of volume regulation depending on how cells are swollen have also been reported in guinea pig jejunal enterocytes (20). RVD and associated Cl- efflux induced by L-alanine uptake was found to be dependent on PKC, whereas RVD and Cl- efflux induced by hyposmotic media was PKC insensitive.

Comparison with other anion-selective conductances in the cells. Previous studies have identified two other anion conductances in these cells. One of the conductances, GVD, is also activated by stimulation of Na+-alanine cotransport and is predominantly Cl- selective (3 times more selective for anions over cations) (27, 31). However, it is unlikely that IDIDS-Ala is attributable to GVD. There are several pieces of evidence to support this. In the first instance, GVD is only observed at potentials more positive than +60 mV and shows a characteristic slow, time-dependent activation on stepping cell potential (Fig. 4). IDIDS-Ala was active at all potentials tested and did not show any time-dependent activation on stepping cell potential. In addition, the activation of GVD by alanine does not require intracellular ATP (31). Activation of IDIDS-Ala has an absolute requirement for ATP. Finally, IDIDS-Ala is inhibited by DIDS, whereas GVD is DIDS insensitive.

GDIDS-Hypo is the DIDS-sensitive conductance activated by a hyposmotic shock (28, 29). It shares many properties with IDIDS-Ala. Both are DIDS sensitive, demonstrate outward rectification, and are Cl- selective. The magnitude and voltage dependence of Kd of DIDS inhibition and the Hill constants associated with that inhibition are similar between the two conductances (29). In addition, activation of both conductances requires the presence of ATP, ATP hydrolysis, phosphorylation, and activation of PKC. However, IDIDS-Ala is only four times more selective for anions over cations, whereas GDIDS-Hypo is 16 times more selective. The anion selectivity sequence of IDIDS-Ala is Br- = Cl- > I- > gluconate, as opposed to I- > Br- > Cl- > gluconate- for GDIDS-Hypo. Finally, activation of IDIDS-Ala seems to involve an atypical isoform of PKC, whereas GDIDS-Hypo involves a conventional PKC isoform. The differences in mechanism of activation between the conductances could simply reflect different regulatory pathways acting on the same conductance. However, the differences in cation-to-anion ratio and anion selectivity sequences suggest that they may represent different conductances within the cell.

Also, IDIDS-Ala cannot be attributed to GVI, another swelling-activated conductance in these cells (27). GVI is ohmic, cation selective, and only present in ~50% of cells, whereas IDIDS-Ala is outwardly rectifying, anion selective, and present in all cells examined. Furthermore, GVI has been found to be unaffected by exposure to alanine (31).

In conclusion, this study has provided evidence that stimulation of Na+-alanine cotransport in single proximal tubule cells activates an outwardly rectifying, predominantly Cl--selective conductance. The conductance exhibits many characteristics of volume-sensitive chloride conductances (outward rectification, DIDS sensitivity, inhibition of activation by hyperosmolality, and dependence on ATP). However, it exhibits a different anion selectivity sequence from the hyposmotically activated chloride conductance previously identified in the same cells and also exhibits differences in the mechanism of activation. The hyposmotically activated Cl- conductance requires activation of a conventional PKC isoform, whereas the Na+-alanine cotransport-activated conductance appears to require activation of an atypical PKC isoform. The DIDS and volume sensitivity of IDIDS-Ala, taken with the requirement for an atypical PKC isoform in conductance activation and Na+-alanine-induced RVD, suggest that IDIDS-Ala may play an important role in the regulation of cell volume that is observed in response to stimulation of Na+-alanine cotransport.


    ACKNOWLEDGEMENTS

We are grateful to the Wellcome Trust and the National Kidney Research Fund for financial support. We also thank Judith Hartley for excellent technical assistance.


    FOOTNOTES

Address for reprint requests and other correspondence: L. Robson, Dept. of Biomedical Science, Univ. of Sheffield, Western Bank, Sheffield S10 2TN, UK (Email: l.robson{at}sheffield.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 September 2000; accepted in final form 7 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 280(5):F758-F767
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