Pfl/igers Arch (1992) 422:112-119
Journal ysiology 9 Springer-Verlag 1992
A calcium-permeable channel in the apical membrane of primary cultures of the rabbit distal bright convoluted tubule V. Poncet, J. Merot, and P. Poujeol D~partement de Biologie Cellulaire et Mol6culaire, Service de Biologie Cellulaire, Centre d'Etudes de Saclay, F-91191 Gif sur Yvette, France Received March 2, 1992/Received after revision July 6, 1992/Accepted July 13, 1992
Abstract. Calcium is actively reabsorbed in the distal nephron segments and recent studies have demonstrated the presence of Ca 2+ channels in these epithelial cells, which could be involved in transepithelial transport. To test this possibility, single-channel currents were recorded by the patch-clamp technique in the apical membrane of primary cultures of the rabbit distal bright convoluted tubule cells (DCTb). In the cell-attached mode with 100 mmol/1 BaC12 in the pipette and 145 mmol/1 NaC1 in the bath, inward negative currents, consistent with Ba2+ currents, were recorded. In these conditions, the single-channel conductance was 15 pS. In excised insideout patches, the single-channel conductance was 13 pS and the current reversal potential of + 60 mV was close to the Nernst equilibrium potential for Ba2+ (> +58 mV). Similar experiments conducted with Ca2+ as the main charge carrier showed that this ion was less permeant through the channel than Ba2+ ( P B a / P c a ,'..~1.4). We also showed that the Ca2+-channel blocker, lanthanum (1 gmol/1 La3+), added on the cytosolic side of the membrane, reversibly blocked the channel activity. On the other hand, verapamil (0.1 mmol/1) and nifedipine (10 gmol/1), perfused on the cytosolic side of the membrane, abolished the channel activity but this effect was not reversible. Another type of channel was also identified in the apical membrane of cultured DCTb cells. Ionsubstitution experiments showed that this 21-pS conductance channel did not discriminate between Na + and K + and did not conduct Ba2+. 4'-Methyl-2-diphenylaminecarboxylic acid (10 ~mol/1), added on the cytosolic side of the membrane, reversibly blocked this cationic channel whereas La3+ (10 ~mol/1) had no effect on its activity. We conclude that (a) La3+-sensitive Ca 2+ channels are present in the apical membrane of DCTb cells and may represent the apical uptake pathway in the transepithelial calcium transport. (b) These Ca 2+ channels are distinct from the non-selective cationic channels that do not show Baz+ permeability. Correspondence to: E Poujeol
Introduction In the mammalian nephron 65% and approximately 20% of the filtered calcium is reabsorbed by the proximal tubule and the loop of Henle respectively, whereas 1% - 3 % is normally excreted in the final urine [I 3, 18]. Consequently, 10% - 15 % of the filtered calcium must be reabsorbed in the terminal segments. This supposition was confirmed by several investigators who also showed that the calcium transport in these segments is modulated by the parathyroid hormone and/or by calcitonin depending on the species studied [9, 16, 27, 28]. In the rabbit, the distal tubule consists of at least three well-defined segments: the early distal bright convoluted tubule (DCTb), the connecting tubule and the cortical collecting duct. In elegant studies, Shimizu and colleagues successfully perfused each of these segments in vitro and demonstrated an active calcium transport in them all [28]. This confirmed the results of Shareghi and Stoner [27], who described a hormonal regulation differing from that advanced by Shimizu et al. [28]. Nevertheless, the involvement of distal segments in calcium reabsorption is now well documented, although the cellular mechanisms involved in the calcium transport of renal epithelial cells remain to be clarified. In the two-step process generally accepted, calcium enters the cell through the apical membrane and is extruded across the basolateral membrane. The latter mechanism probably involves a basolateral Na+/Ca 2+ exchanger [4, 17, 27, 29, 301 and calcium ATPase [6]. The apical uptake pathway, on the other hand, remains largely unknown. However, recent data suggest the presence of voltage-independent Ca 2+ channels in renal cells, which may be involved in the uptake mechanism [1, 29]. In the present study we investigated this possibility using the patch-clamp technique on the apical membrane of primary cultures of rabbit DCTb cells. In a previous paper, we showed that these cultured cells express many of the properties of the original epithelium [22] and therefore represent a useful model to study the transport properties of this segment.
113
Materials and methods Primary cell cultures. The primary cell cultures were prepared as described previously [22]. Briefly, the kidneys were removed under sterile conditions from 4- to 5-week-oldmale New Zealand rabbits and perfused with Hank's solution (Eurobio-France) containing 600-700kU/1 collagenase (Worthington biochemical corporation-Freehold, N.J.). They were cut into small pyramids and incubated for 45 -60 rain in dissection medium containing 150 kU/1 collagenase. The bright part of the distal tubule was carefully microdissected and seeded in collagen-coated 35 mm petri dishes filled with primary culture medium. The latter consisted of equal quantities of Dulbecco's modified Eagle medium and HAMF 12 medium (Gibco) and contained: 15mmol/1 NaHCO 3, 20 mmol/1 HEPES (pH = 7.5), 2mmol/1 glutamine, 50 nmol/1 dexamethasone, 30nmol/1 sodium selenite, 10nmol/1 triiodothyronine, 5 mg/1 insulin, 10 ~tg/1 epidermal growth factor and 5 mg/1 transferrin. Cultures were maintained at 37 ~ in a 5~ CO2/95% air water-saturated atmosphere. The medium was changed 4 days after seeding and then every 2 days. Patch-clamp experiments. Single-channel currents were recorded according to the method described by Hamill et al. [141. Pipettes were pulled in two steps from borosilicate glass (outer diameter = 1.5 mm; inner diameter 1.1 mm; Clay Adams, USA) using a vertical puller (David Kopf, Tujunga, Calif. USA). Electrode tips were coated with Sylgard 184 (Dow Coming, Belgium) to reduce electrical noise. The pipette resistances were 5-10 Mr2. They were connected via an Ag/AgC1 wire to the headstage of a RK 300 patch-clamp amplifier (Biologic, France). Seals were achieved spontaneously or by applying a slight mouth suction to the patch pipette. Channel currents were filtered with a low-pass five-pole Tchebicheff filter at 0.3 - 1 kHz, stored on digital audio tapes using a DTR 1200 recorder (Biologic, France) and visualized on a digital oscilloscope (Nicolet Instruments, Madison Wis., USA). Throughout this report, the potential (Vm) indicates the potential on the cytoplasmic face of the membrane relative to the pipette. In cell-attached experiments, the intracellular potential that contributes to the actual membrane potential was assumed to be -60 mV. On the recordings given in the figures, upward deflections from the closed state represent the flow of positive charges moving from the cytoplasmic to the external side of the patch membrane (outward currents). All experiments were performed at room temperature. Data analysis. Channel current amplitudes were measured by replaying the tape onto the digital oscilloscope. The current/voltage relationships of the channel were established from the average amplitude of ten welldefined transitions between closed and open current levels at each potential. The Goldman Hodgkin and Katz equation or a straight line was fitted to the data by the least-square method. The best fit obtained is shown as a solid line on the 1/V plots. For divalent cation currents the data were fitted according to Rorsman et ai. [26]. For channel kinetics analysis, the data were transferred on a Hewlett Packard computer at a sampling frequency of 1- 3 kHz. To estimate the channel-open probability, current-amplitude histograms were created and analysed with Biopatch software (Biologic, France). The ratio of the areas of the apparently Gaussian current-amplitude histograms corresponding to the closed and open states of the channel was taken as an estimate of the channel-open probability and denoted Po. The values are presented as means_+SE. Solutions and drugs. During seal formation, the cells were bathed with a Na+-rich solution containing (in mmol/1): 145NaC1, 1 CaC1z, 1 MgC12, 5 glucose, 10 HEPES (pH = 7.4 adjusted with NaOH). In some experiments gluconate replaced chloride (in mmol/1): 145 sodium gluconate, 3 calcium gluconate, 5 glucose, 10 HEPES (pH = 7.4 adjusted with NaOH). To obtain Na+-free and K+-rich solutions, the 145 mmol/1 NaC1 of the Na+-rich solution was replaced by 145mmol/1 N-methyl-Dglucaminechloride and 145 mmol/1 KC1 respectively. To measure unitary Ca2+ currents the pipette was filled with (in mmol/1): 100CaC12, 10 HEPES (pH = 7.4 adjusted with TRIS) or
100 BaC12. Mannitol was added to the solution to keep the osmolarity close to 300 mosmol/1. Lanthanum chloride (Merck) and verapamil (Sigma) were directly dissolved in external saline. To dissolve nifedipine (Sigma) in aqueous solution it was first dissolved in ethanol (107o final (ethanol)). 4'-Methyl-2-diphenylaminecarboxylic acid (4'-Me-DPC) was the gift of Dr. Hans-J. Lang from Hoechst (FRG). A stock solution of 4'-Me-DPC (10 mmol/l) was prepared in dimethylsulphoxide. Just before the experiments an aliquot was dissolved in the Na + -rich solution at a final concentration of 10 gmol/1.
Results T h e f o r m a t i o n o f a g i g a o h m seal b et w een the p a t c h pipette and t h e apical cell m e m b r a n e was achieved in 65~ o f the trials (n = 531), 27~ o f w h i ch c o n t a i n e d active ionic channels. Ca 2+ channels In the first series o f experiments, we investigated C a 2+ channels using C a 2+ as the m a i n charge carrier. We occasionally observed single-channel currents in the cell-att a c h e d c o n f i g u r a t i o n , b u t m o s t o f the t i m e the recordings with a g o o d signal-to-noise ratio did n o t last m o r e t h a n a few tenths o f a second and the s p o n t a n e o u s f o r m a t i o n o f a vesicle at t h e tip o f the p i p et t e prevented f ur t he r analysis o f the channel. W h e n the p at ch m e m b r a n e was excised in the N a + - r i c h s o l u t i o n a s p o n t a n e o u s c h a n n e l activity was observed in 6~ o f the trials. Figure 1 A shows the single-channel currents recorded at different m e m b r a n e h o l d i n g potentials. A t 0 mV c h a n n e l o p e n i n g s a p p e a r e d as d o w n w a r d deflections. In o u r conditions, this represented either positive charges (Ca 2+) flowing o u t o f the pipette or the o p p o s i t e m o v e m e n t o f negative charges (C1-). As the m e m b r a n e p o t e n t i a l was c h a n g e d f r o m 0 mV towards negative values ( - 20 mV) the current a m p l i t u d e s increased, whereas they decreased at a positive h o l d i n g p o t e n t i a l ( + 2 0 mV). T h e c u r r e n t / v o l t a g e rel a t i o n s h i p o f the c h a n n e l in this e x p e r i m e n t is s h o w n in Fig. 1 B. T h e best fit to the G o l d m a n H o d g k i n Katz e qua t i o n was o b t a i n e d with a p e r m e a b i l i t y c o e f f i c i e n t P c a = 9.45 X 10-15 cm3/s. T h e c o n d u c t a n c e o f the channel, calculated as the slope o f the line b et w een - 60 mV a n d - 4 0 m V , was 7.8___0.2pS (n = 3). T h e extrapolated cu r r en t reversal p o t e n t i a l ( + 6 0 mV) was consistent with the N e r n s t e q u i l i b r i u m p o t e n t i a l for C a 2§ (Vca = + 58 mV). In the next series o f experiments we investigated C a 2+ channels, using Ba 2+ as the m a i n c o n d u c t i n g ion. T h e pipette was filled with BaCI a a n d the b a t h c o n t a i n e d the N a + - r i c h solution. In this c o n d i t i o n we were able to detect single-channel activity in t h e cell-attached c o n f i g u ration. Figure 2 A shows t h e single-channel currents rec o r d e d at different m e m b r a n e h o l d i n g potentials. A t - 80 mV, ch an n el o p e n i n g s a p p e a r e d as d o w n w a r d deflections an d were consistent with Ba 2+ currents. As the m e m b r a n e p o t e n t i a l was c h a n g e d f r o m - 8 0 m V to - 1 2 0 m V the current a m p l i t u d e s increased. T h e corr e s p o n d i n g c u r r e n t / v o l t a g e r e l a t i o n s h i p is s h o w n in Fig. 2B. T h e c o n d u c t a n c e calculated b et w een - 1 0 0 m V an d - 1 2 0 m V was 1 5 + 1 p S (n = 9).
114
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( C - ) on the left (sampling rate 1 kHz, 0.3 kHz filter). B Single-channel current/voltage relationship (I/V) with the same solutions as in A. The line is a least-square fit according to the Goldman Hodgkin Katz equation
Fig. 1. A Single Ca 2+ channel currents recorded from an inside-out patch. The pipette contained 100mmol/l CaC1z and the bath the Na + -rich solution. The membrane holding potentials (Vm) are indicated on the right of each recording and the closed state of the channel
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B right of each recording and the closed state of the channel is indicated on the left ( C - ) (sampling rate 1 kHz, 0.3 kHz filter). B I/Vrelation-
Fig. 2. A Single Ca 2+ channel currents recorded from a cell-attached patch with 100 retool/1 BaCI2 in the pipette and 145 mmol/1 NaC1 in the bath. The membrane holding potentials (Vm) are indicated on the
ship of the channel with the same solutions as in A
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Fig. 3.A Single Ca2+ channel currents recorded from an inside-out patch with I00 mmo]/l BaCI2 in the pipette and the Na +-rich solution in the bath. The membrane holding potentials (Vm) are indicated on the right of each recording and the close state of the channel (C-) on
the left (sampling rate 1 kHz, 0.3 kHz filter). B I/V relationship of the channel with NaC1 ( 9 ) and sodium gluconate ( 9 ) in the bath. The lines are the least-square fit of the data to the Goldman Hodgkin Katz equation (PBa = 1.22 10 -14 cm3/s and PBa = 1.18 10 -14 cm3/s respectively)
115
Non-selective cationic channel
The recordings obtained when the patch was excised in the Na§ solution are shown in Fig. 3A. Downward channel openings were observed at 0 mV holding potential and the current amplitude increased as the membrane was hyperpolarized from 0 mV to - 80 mV. At variance with the previous experiments, the current amplitudes recorded were almost twice those measured with CaC12. The I~ V relationship (Fig. 3 B filled circles) followed the Goldman Hodgkin Katz equation for a current carried by Ba 2§ . The permeability coefficient, PBa, was 1.33+0.06• 10 -14 cm3/s and the slope conductance between - 4 0 mV and - 6 0 mV was 13_+0.9 pS (n = 8). The current reversal potential measured in these conditions is consistent with the expected current reversal potential for Ba 2+ (VBa> +58 mV). To demonstrate that the observed currents were not carried by anions, we conducted ion-substitution experiments. When the bath chloride was substituted by gluconate the channel currents remained unchanged. The I~ V relationship (Fig. 3 B open circles) in this condition was similar to that obtained in the presence of C1-. This result indicates a high selectivity of the channel for divalent cations rather than anions. The effect of the Ca 2+ channel blocker lanthanum (La 3§ was tested on the channel activity. In five experiments, the application of La 3§ (1 gmol/1) to the cytoplasmic side of the channel abolished its activity (Fig. 4). This effect was partially reversed when the La 3+ was rinsed out. We also examined the effect of two other Ca 2§ channel blockers. Verapamil (0.1mmol/l) and nifedipine (10 ~tmol/1), when perfused on the cytosolic side of the membrane, abolished the channel activity (n = 3) but this effect was irreversible when the inhibitors were rinsed out (Fig. 4).
In the course of our study, we observed another type of channel that was only active in excised patches. A typical recording is shown in Fig. 5 A. In this experiment, the pipette contained BaC1z and the bath the Na+-rich solution. In contrast to the observations on the Ca 2+ channels, in these experiments the openings were upward at 0 mV membrane holding potential and the current amplitudes increased when the membrane was depolarized from 0 to +80mV. A slope conductance of 21_+1pS (n = 5) was calculated from the I~ Vrelationship (Fig. 5 B) between + 60 mV and + 80 mV. In this condition, the I~ V relationship followed the Goldman Hodgkin Katz equation for a Na + current. The extrapolated current reversal potential was close to the expected reversal potential for Na + ( V N a > - 6 0 m V ) suggesting that the observed current was carried by Na § . The permeability of the channel to other cations was investigated. From the substitution experiments shown in Fig. 6A and the corresponding I / V plot (Fig. 6B) it is clear that K § but not N-methyl-D-glucamine or Ba 2§ was conducted by the channel. The slope conductance calculated between +60mV and +80mV was 20.3 + 0.3 pS (n = 5). Thus it appears that the channel was equally permeable to Na + and K + but had a very low permeability to divalent cations. Over the range of potentials utilized in these experiments, the channel activity appeared only poorly dependent on the membrane potential. Typically the channel-open probabilities (Po) at +20 mV and +80 mV were 0.67_+0.04 and 0.70+0.02 (n = 3) respectively. This cationic channel was next studied in the presence of conducting ions on both sides of the membrane. Figure 7A shows the recordings obtained with the pipette
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Fig. 4A, B. Single Ca 2+ channel current recorded from an inside-out patch at 0 m V membrane holding potential with 100 mmol/l BaCI 2 in the pipette and 145 mmol/l NaCI in the bath. A La 3+ (I ~tmol/1) is added to the cytosolic side of the channel (middle trace). B In the sec-
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ond and fourth traces, verapamil 0.1 mmol/l and nifedipine 10 ~mol/l are added to the bath respectively. C-, on the leftof each recording, indicates the closed state of the channel (sampling rate I kHz, 0.3 kI-Izfilter)
116
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500ms Fig. 5. A Single-channel current recordings of the non-selective cation channel in an excised inside-out patch. The pipette contained 100 retool/1 BaC12 and the bath the Na+-rich solution. The membrane holding potentials (Vm) are indicated on the right of the recordings and
( C - ) indicates the closed state of the channel (sampling rate 2.5 kHz, 1 k H z filter). B I / V relationship of the channel with the same solutions as in A. The line is the least-square fit of the data to the Goldman Hodgkin Katz equation for a monovalent cation channel
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Fig. 6. A Single non-selective cation channel recordings from an insideout patch at + 60 mV membrane holding potential. The pipette contained 1 0 0 m m o l / l BaC12 and the bath 145 mmol/1 KC1, N-methyl-D-glucamine chloride, or BaC12. C - on the left of the recordings in-
dicates the closed state o f the channel (sampling rate 2.5 kHz, 1 kHz filter). B I / V curves in the presence of K+-rich solution ( e ) , methylglucamine ( 9 or 100 mmol/1 BaC12 ( ~ )
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200ms Fig. 7. A Single-channel current recordings of the non-selective cation channel from an inside-out patch. The pipette contained 145 m m o l / l NaC1 and the bath 145 m m o l / l KC1. The different membrane potentials (Vm) are indicated on the right of each recording. C - indicates the closed state of the channel (sampling rate 2.5 kHz, 1 kHz filter). B I / V
relationships of the channel, e , Experiments in which pipette and bath contained the same solutions as in A; 0 , experiments in which the bath contained a choline chloride solution. The data are fitted by the G o l d m a n Hodgkin Katz equation for a monovalent cationic channel
117
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Fig. 8 A, B. Single-channel cationic currents recorded from a inside-out patch at + 60 mV m e m b r a n e holding potential. C - indicates the closed state of the channel (sampling rate 2.5 kHz, 1 k H z filter). A The pipette contained 100 mmol/1 BaC12 and the bath 145 mmol/1 NaC1. 4'-meth-
yl-2-diphenylaminecarboxylic acid (10 gmol/1) and La 3+ (I0 gmol/1) were added to the bath. B Same experiments as in A except that the pipette was filled with 140 mmol/1 NaC1
containing the Na+-rich solution and the bath the K+-rich one. Upward and downward single-channel currents were observed at negative and positive membrane holding potentials respectively. The channel conductance calculated from the I~ V relationship (Fig. 7 B, filled circle) was 22.9_+ 1.2 pS (n = 5) and the reversal potential corresponded to the equilibrium potential of cations (Vca t = 0 mV). The cationic selectivity of the channel was confirmed in experiments in which 140 mmol/1 potassium in the bath was replaced by the impermeant cation, choline. As shown in Fig. 7 B (filled square), the I / V relationship followed the Goldman Hodgkin Katz equation for a cation-selective channel. This manoeuvre resulted in a shift of the current-reversal potential toward the expected Nernst equilibrium potential of cations (Vcat = + 85 mV). The channel activity appeared to be strongly voltage-dependent at a negative membrane holding potential (Fig. 7A). Po was 0.02_+0.01 and 0.57_+0.06 (n = 5) at - 6 0 mV and +60 mV holding potentials respectively. These results suggest that it was the same channel under study in the two sets of experiments, a hypothesis confirmed by the sensitivity of the channel to 4"Me-DPC and La 3+. Figure 8 shows that the currents observed with either BaC12 or NaC1 in the pipette were reversibly blocked when 4"Me-DPC (10 gmol/1) was applied on the cytoplasmic side of the channel (n = 5) whereas La 3+ (10 gmol/1) applied on the cytoplasmic side (n = 5) had no effect on the channel activity.
therefore constitute a suitable material for analysing its transport properties [22, 23]. The microdissection technique used to isolate the tubule limited the possible contamination by cells from neighbouring segments. This is of importance since a different calcium transport mechanism probably occurs in the cortical thick ascending limb (CTAL) of the loop of Henle [5, 13]. In our study, we characterized a C a 2§ channel with a conductance of approximately 8 pS in excised but not in cell-attached patches when Ca 2§ was used as the main charge carrier. We thus decided to use Ba 2§ as the permeant ion because this had two advantages: first, it has been shown to be more permeant in Ca 2+ channels than Ca 2§ itself, thus increasing the signal-to-noise ratio in the current recordings [2, 15], and, secondly, Ba 2+ blocks K + channels. In fact, in cell-attached or excised inside-out configurations, with 100 mmol/1 BaCI 2, in the pipette, the channel conductance was 15 pS and 13 pS respectively and a permeability ratio (PBa/Plca~l.4) could be determined. In our ion-substitution experiments we did not detect any significant permeability to Na § or C I - , which thus indicates that the channel is highly selective for Ca 2+ . We also showed that cytoplasmic La 3+ (1 gmol/1) reversibly blocked the channel activity whereas verapamil (0.1 mmol/1) and nifedipine (10gmol/1) irreversibly blocked the channel. Taken together these results are reminiscent of those described for the b t y p e Ca 2+ channels in excitable tissues [2, 26], except that most of the latter Ca 2+ channels were shown to be voltage-dependent whereas, in our experiments, neither in cell-attached nor excised inside-out patches did we detect any modulation of the channel activity by the membrane potential. To odr knowledge, this is the first study describing Ca 2§ channels in epithelial kidney cells. However, in preliminary reports, Matsunaga et al. and Lau et al. [19, 21] characterized Ca 2+ channels in the apical membrane of cultured distal cells. Both groups of workers described dihydropyridine-sensitive Ca 2§ channels activated by the
Discussion In the present study we have shown the presence of Ca 2§ channels in the apical membrane of primary cultures of the rabbit distal bright convoluted tubule cells. We used cultured cells as a model because they offer a direct access to the apical cell membrane. Their use is justified since, as we have already shown, cultured DCTb cells retain most of the properties of the original epithelium and
118 cyclic AMP pathway. More data are required to ascertain that the channel they describe is the same as that under study. Even though a thorough characterization of epithelial Ca 2+ channels has never been made, their involvement in the transport of calcium by kidney cells has been suggested by many investigators. In single microdissected rat cortial collecting tubules, Taniguchi et al. [29] concluded that the calcium uptake they observed was best explained by the presence of voltage-independent Ca 2+ channels in the cell membranes. In cultured mouse cTAL and DCT cells Bacskai and Friedman [1] showed that treatment with parathyroid hormone led to the recruitment of dihydropyridine-sensitive but voltage-independent Ca 2+ channels. However, none of these studies established the localization of the channel nor its involvement in the transepithelial transport of calcium. More studies are definitely needed to clarify these points. We also investigated the calcium permeability of a non-selective cation channel. In a previous paper [23], we characterized this channel and showed that: (a) it does not discriminate between Na + and K + , (b) its activity is dependent on cytoplasmic [Ca 2§ and (c) the diuretic amiloride does not block the channel activity. These properties are also shared by Ca 2§ non-selective cation channels in various cell types [3, 10, 20, 24] suggesting that this channel could represent a calcium-uptake pathway in DCTb cells. We studied the channel with either 100 mmol/1 BaC12 or 145 retool/1 NaC1 in the pipette. The question arose as to whether it was the same channel that we observed in the two experimental conditions. There are several lines of evidence that this was the case. (a) The channel conductance and open probability were not significantly different in the two series of experiments (22.9 pS versus 21.0 pS and Po = 0.70 versus 0.57 at positive holding potentials). (b) The pharmacological profiles of the channels were the same: 4" Me-DPC but not La 3+ (10 ~mol/1) blocked the channel in its closed state when applied on its cytoplasmic face. Such derivatives of DPC have been shown to block nonselective cation channels though there are different reports about the side from which they act. Thus, while DPC blocked the non-selective cation channel from the cytoplasmic side in the exocrine pancreas [12] it did so from the external side in mandibular cells [7]. In our experiments we did not detect any significant permeability of the channel to Ba 2+ . This result agrees with similar reports concerning non-selective cation channels in mouse mandibular gland [7], mouse fibroblasts [11] and brown adipocytes [31], but disagrees with the results obtained on Necturus proximal tubule [10], liver cells [3], choroid plexus [8], Balb/3T3 cells [20] or endothelial cells [24]. Thus, on the basis of calcium permeability two types of non-selective cation channel may be assumed to exist. In an attempt to assign physiological roles to these different channels, it is worth noting that most of the Ca2+-permeable non-selective cation channels have been shown to be activated by membrane stretch [10, 32, 3] or ligand binding [20]. The former are probably involved in the cell-volume-regulatory mechanism whereas the latter may be linked to cell differentiation or
secretion processes [25]. As far as the distal convoluted tubule is concerned, the physiological role of the non-selective cation channels remains uncertain. In conclusion, we have described a Ca 2+ channel in the apical membrane of cultured DCTb cells that could represent the apical pathway for the transepithelial calcium transport occurring in this segment. This channel shares some of the properties of the L-type Ca 2+ channel expressed in excitable tissues except for its voltage independence. It may therefore represent a tissue-specific isoform of an L-type Ca 2+ channel in epithelial cells. It is interesting that recent studies, using the polymerase chain reaction with selected regions of nucleotide sequences from cardiac and skeletal muscle Ca 2+ channels, identified multiple Ca 2+ channel isoforms in the kidney [331.
References 1. Bacskai BJ, Friedman PA (1990) Activation of latent Ca 2+ channels in renal epithelial cells by parathyroid hormone. Nature 347:388-391 2. Bean BP (1989) Classes of calcium channels in vertebrate ceils. Annu Rev Physiol 51:367-384 3. Bear CE (1990) A nonselective cation channel in rat liver cells is activated by membrane stretch. Am J Physiol 258:C421-C428 4. Bindels RJM, Hartog A, Timmermans J, Van os CH (1991) Active Ca 2+ transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D 3 and PTH. Am J Physiol 261: F799- F807 5. Bourdeau JE, Burg MB (1979) Voltage dependence of calcium transport in the thick ascending limb of Henle's loop. Am J Physiol 236:F357-F364 6. Carafoli E (1991) Calcium pump of the plasma membrane. Physiol Rev 71:129-153 7. Cook DI, Poronnik P, Young JA (1990) Characterization of a 25-pS nonselective cation channel in cultured secretory epithelial cell line. J Membr Biol 114:37-52 8. Christensen O (1987) Mediation of cell volume regulation by Ca 2+ influx through stretch-activated channels. Nature 330:66-68 9. Costanzo LS (1984) Comparison of calcium and sodium transport in early and late rat distal tubules: effect of amiloride. Am J Physiol 246:F937- 945 10. Filipovic D, Sackin H (1991) A calcium-permeable stretch-activated cation channel in renal proximal tubule. Am J Physiol 260: Fllg-F129 11. Frace AM, Gargus JJ (1989) Activation of single-channel currents in mouse fibroblasts by platelet-derived growth factor. 86: 2511-2515 12. GOgelein H, Pfannmtiller B (1989) The nonselective cation channel in basolateral membrane of rat exocrine pancreas. Inhibition by 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) and activation by stilbene disulfonates. PfRigers Arch 413:287-298 13. Greger R (1985) Ion transport mechanisms in thick ascending limb of Henle's loop of mammalian nephron. Physiol Rev 65:760-797 14. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfliigers Arch 391:85-100 15. Hess P, Lansman JB, Tsien RW (1986) Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells. J Gen Physiol 88:293-319 16. Imai M (1981) Effects of parathyroid hormone and N 6, O2'-dibu tyryl cyclic AMP on Ca 2+ transport across the rabbit distal nephron segments perfused in vitro. Pfltigers Arch 390:145-151
119 17. Lajeunesse D, Brunette MG (1991) The hypocalciuric effect of thiazides: subcellular localization of the action. Pfl~igers Arch 417:454- 462 18. Lassiter WE, Gottschalk, Mylle M (1963) Micropuncture study of renal tubular reabsorption of calcium in normal rodent. Am J Physiol 204:771-775 19. Lau K, Quamme G, Tan S (1991) Patch-clamp evidence for a Ca channel in apical membrane of cortical thick ascending limb (cTAL) and distal tubule (DT) cells (abstract). J Am Soc Nephrol 2:775-46P 20. Matsunaga H, Nishimoto I, Kojima I, Yamashita N, Kurokawa K, Ogata E (1988) Activation of a calcium-permeable cation channel by insulin-like growth factor II in BALB/c 3T3 cells. Am J Physiol 255:C442- C446 21. Matsunaga H, Friedman PA, Kiser N, Stanton BA (1991) Calcium channels in apical membranes of distal convoluted tubule cells (abstract). Am J Soc Nephrol 2:627-24P 22. Merot J, Bidet M, Gachot B, Le Maout S, Koechlin N, Tauc M, Poujeol P (1989) Electrical properties of rabbit early distal convoluted tubule in primary culture. Am J Physiol 257:F288-F299 23. Merot J, Tauc M, Bidet M, Poujeol P (1991) Ion channels in the distal convoluted tubule in primary culture. Nephrology 2:1698-1712 24. Nilius R (1990) Permeation properties of a non-selective cation channel in human vascular endothelial cells. Pflt~gers Arch 416: 609- 611
25. Partridge LD, Swandulla D (1988) Calcium-activated cation channels. Trends Neurosci 11:69-72 26. Rorsman P, Ashcroft FM, Trube G (1988) Single Ca channel currents in mouse pancreatic B-cells. Pfliigers Arch 412:597-603 27. Sharegi GR, Stoner LC (1978) Calcium transport across segments of the rabbit distal nephron in vitro. Am J Physiol 235:F367-F375 28. Shimizu T, Yoshitomi K, Nakamura M, Imai M (1990) Effects of PTH, calcitonin, and cAMP on calcium transport in rabbit distal nephron segments. Am J Physiol 259:F408-F414 29. Taniguchi S, Marchetti J, Morel F (1989) Cytosolic free calcium in single microdissected rat collecting tubules. Pfltigers Arch 414: 125-133 30. van Os CH (1987) Transcellular calcium transport in intestinal and renal epithelial cells. Biochim biophys Acta 906:195-222 31. Weber A, Siemen D (1989) Permeability of the non-selective channel in brown adipocytes to small cations. Pfliigers Arch 414: 564- 570 32. Yang XC, Sachs F (1989) Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243:1068-1071 33. Yu A, Saltzberg SN, Hebert SC, Lytton J (1991) Multiple voltagedependent Ca 2+ channels (VDCC) are expressed in rodent kidney (abstract). J Am Soc Nephrol 756:24P
Journal ysiology 9 Springer-Verlag 1992
A calcium-permeable channel in the apical membrane of primary cultures of the rabbit distal bright convoluted tubule V. Poncet, J. Merot, and P. Poujeol D~partement de Biologie Cellulaire et Mol6culaire, Service de Biologie Cellulaire, Centre d'Etudes de Saclay, F-91191 Gif sur Yvette, France Received March 2, 1992/Received after revision July 6, 1992/Accepted July 13, 1992
Abstract. Calcium is actively reabsorbed in the distal nephron segments and recent studies have demonstrated the presence of Ca 2+ channels in these epithelial cells, which could be involved in transepithelial transport. To test this possibility, single-channel currents were recorded by the patch-clamp technique in the apical membrane of primary cultures of the rabbit distal bright convoluted tubule cells (DCTb). In the cell-attached mode with 100 mmol/1 BaC12 in the pipette and 145 mmol/1 NaC1 in the bath, inward negative currents, consistent with Ba2+ currents, were recorded. In these conditions, the single-channel conductance was 15 pS. In excised insideout patches, the single-channel conductance was 13 pS and the current reversal potential of + 60 mV was close to the Nernst equilibrium potential for Ba2+ (> +58 mV). Similar experiments conducted with Ca2+ as the main charge carrier showed that this ion was less permeant through the channel than Ba2+ ( P B a / P c a ,'..~1.4). We also showed that the Ca2+-channel blocker, lanthanum (1 gmol/1 La3+), added on the cytosolic side of the membrane, reversibly blocked the channel activity. On the other hand, verapamil (0.1 mmol/1) and nifedipine (10 gmol/1), perfused on the cytosolic side of the membrane, abolished the channel activity but this effect was not reversible. Another type of channel was also identified in the apical membrane of cultured DCTb cells. Ionsubstitution experiments showed that this 21-pS conductance channel did not discriminate between Na + and K + and did not conduct Ba2+. 4'-Methyl-2-diphenylaminecarboxylic acid (10 ~mol/1), added on the cytosolic side of the membrane, reversibly blocked this cationic channel whereas La3+ (10 ~mol/1) had no effect on its activity. We conclude that (a) La3+-sensitive Ca 2+ channels are present in the apical membrane of DCTb cells and may represent the apical uptake pathway in the transepithelial calcium transport. (b) These Ca 2+ channels are distinct from the non-selective cationic channels that do not show Baz+ permeability. Correspondence to: E Poujeol
Introduction In the mammalian nephron 65% and approximately 20% of the filtered calcium is reabsorbed by the proximal tubule and the loop of Henle respectively, whereas 1% - 3 % is normally excreted in the final urine [I 3, 18]. Consequently, 10% - 15 % of the filtered calcium must be reabsorbed in the terminal segments. This supposition was confirmed by several investigators who also showed that the calcium transport in these segments is modulated by the parathyroid hormone and/or by calcitonin depending on the species studied [9, 16, 27, 28]. In the rabbit, the distal tubule consists of at least three well-defined segments: the early distal bright convoluted tubule (DCTb), the connecting tubule and the cortical collecting duct. In elegant studies, Shimizu and colleagues successfully perfused each of these segments in vitro and demonstrated an active calcium transport in them all [28]. This confirmed the results of Shareghi and Stoner [27], who described a hormonal regulation differing from that advanced by Shimizu et al. [28]. Nevertheless, the involvement of distal segments in calcium reabsorption is now well documented, although the cellular mechanisms involved in the calcium transport of renal epithelial cells remain to be clarified. In the two-step process generally accepted, calcium enters the cell through the apical membrane and is extruded across the basolateral membrane. The latter mechanism probably involves a basolateral Na+/Ca 2+ exchanger [4, 17, 27, 29, 301 and calcium ATPase [6]. The apical uptake pathway, on the other hand, remains largely unknown. However, recent data suggest the presence of voltage-independent Ca 2+ channels in renal cells, which may be involved in the uptake mechanism [1, 29]. In the present study we investigated this possibility using the patch-clamp technique on the apical membrane of primary cultures of rabbit DCTb cells. In a previous paper, we showed that these cultured cells express many of the properties of the original epithelium [22] and therefore represent a useful model to study the transport properties of this segment.
113
Materials and methods Primary cell cultures. The primary cell cultures were prepared as described previously [22]. Briefly, the kidneys were removed under sterile conditions from 4- to 5-week-oldmale New Zealand rabbits and perfused with Hank's solution (Eurobio-France) containing 600-700kU/1 collagenase (Worthington biochemical corporation-Freehold, N.J.). They were cut into small pyramids and incubated for 45 -60 rain in dissection medium containing 150 kU/1 collagenase. The bright part of the distal tubule was carefully microdissected and seeded in collagen-coated 35 mm petri dishes filled with primary culture medium. The latter consisted of equal quantities of Dulbecco's modified Eagle medium and HAMF 12 medium (Gibco) and contained: 15mmol/1 NaHCO 3, 20 mmol/1 HEPES (pH = 7.5), 2mmol/1 glutamine, 50 nmol/1 dexamethasone, 30nmol/1 sodium selenite, 10nmol/1 triiodothyronine, 5 mg/1 insulin, 10 ~tg/1 epidermal growth factor and 5 mg/1 transferrin. Cultures were maintained at 37 ~ in a 5~ CO2/95% air water-saturated atmosphere. The medium was changed 4 days after seeding and then every 2 days. Patch-clamp experiments. Single-channel currents were recorded according to the method described by Hamill et al. [141. Pipettes were pulled in two steps from borosilicate glass (outer diameter = 1.5 mm; inner diameter 1.1 mm; Clay Adams, USA) using a vertical puller (David Kopf, Tujunga, Calif. USA). Electrode tips were coated with Sylgard 184 (Dow Coming, Belgium) to reduce electrical noise. The pipette resistances were 5-10 Mr2. They were connected via an Ag/AgC1 wire to the headstage of a RK 300 patch-clamp amplifier (Biologic, France). Seals were achieved spontaneously or by applying a slight mouth suction to the patch pipette. Channel currents were filtered with a low-pass five-pole Tchebicheff filter at 0.3 - 1 kHz, stored on digital audio tapes using a DTR 1200 recorder (Biologic, France) and visualized on a digital oscilloscope (Nicolet Instruments, Madison Wis., USA). Throughout this report, the potential (Vm) indicates the potential on the cytoplasmic face of the membrane relative to the pipette. In cell-attached experiments, the intracellular potential that contributes to the actual membrane potential was assumed to be -60 mV. On the recordings given in the figures, upward deflections from the closed state represent the flow of positive charges moving from the cytoplasmic to the external side of the patch membrane (outward currents). All experiments were performed at room temperature. Data analysis. Channel current amplitudes were measured by replaying the tape onto the digital oscilloscope. The current/voltage relationships of the channel were established from the average amplitude of ten welldefined transitions between closed and open current levels at each potential. The Goldman Hodgkin and Katz equation or a straight line was fitted to the data by the least-square method. The best fit obtained is shown as a solid line on the 1/V plots. For divalent cation currents the data were fitted according to Rorsman et ai. [26]. For channel kinetics analysis, the data were transferred on a Hewlett Packard computer at a sampling frequency of 1- 3 kHz. To estimate the channel-open probability, current-amplitude histograms were created and analysed with Biopatch software (Biologic, France). The ratio of the areas of the apparently Gaussian current-amplitude histograms corresponding to the closed and open states of the channel was taken as an estimate of the channel-open probability and denoted Po. The values are presented as means_+SE. Solutions and drugs. During seal formation, the cells were bathed with a Na+-rich solution containing (in mmol/1): 145NaC1, 1 CaC1z, 1 MgC12, 5 glucose, 10 HEPES (pH = 7.4 adjusted with NaOH). In some experiments gluconate replaced chloride (in mmol/1): 145 sodium gluconate, 3 calcium gluconate, 5 glucose, 10 HEPES (pH = 7.4 adjusted with NaOH). To obtain Na+-free and K+-rich solutions, the 145 mmol/1 NaC1 of the Na+-rich solution was replaced by 145mmol/1 N-methyl-Dglucaminechloride and 145 mmol/1 KC1 respectively. To measure unitary Ca2+ currents the pipette was filled with (in mmol/1): 100CaC12, 10 HEPES (pH = 7.4 adjusted with TRIS) or
100 BaC12. Mannitol was added to the solution to keep the osmolarity close to 300 mosmol/1. Lanthanum chloride (Merck) and verapamil (Sigma) were directly dissolved in external saline. To dissolve nifedipine (Sigma) in aqueous solution it was first dissolved in ethanol (107o final (ethanol)). 4'-Methyl-2-diphenylaminecarboxylic acid (4'-Me-DPC) was the gift of Dr. Hans-J. Lang from Hoechst (FRG). A stock solution of 4'-Me-DPC (10 mmol/l) was prepared in dimethylsulphoxide. Just before the experiments an aliquot was dissolved in the Na + -rich solution at a final concentration of 10 gmol/1.
Results T h e f o r m a t i o n o f a g i g a o h m seal b et w een the p a t c h pipette and t h e apical cell m e m b r a n e was achieved in 65~ o f the trials (n = 531), 27~ o f w h i ch c o n t a i n e d active ionic channels. Ca 2+ channels In the first series o f experiments, we investigated C a 2+ channels using C a 2+ as the m a i n charge carrier. We occasionally observed single-channel currents in the cell-att a c h e d c o n f i g u r a t i o n , b u t m o s t o f the t i m e the recordings with a g o o d signal-to-noise ratio did n o t last m o r e t h a n a few tenths o f a second and the s p o n t a n e o u s f o r m a t i o n o f a vesicle at t h e tip o f the p i p et t e prevented f ur t he r analysis o f the channel. W h e n the p at ch m e m b r a n e was excised in the N a + - r i c h s o l u t i o n a s p o n t a n e o u s c h a n n e l activity was observed in 6~ o f the trials. Figure 1 A shows the single-channel currents recorded at different m e m b r a n e h o l d i n g potentials. A t 0 mV c h a n n e l o p e n i n g s a p p e a r e d as d o w n w a r d deflections. In o u r conditions, this represented either positive charges (Ca 2+) flowing o u t o f the pipette or the o p p o s i t e m o v e m e n t o f negative charges (C1-). As the m e m b r a n e p o t e n t i a l was c h a n g e d f r o m 0 mV towards negative values ( - 20 mV) the current a m p l i t u d e s increased, whereas they decreased at a positive h o l d i n g p o t e n t i a l ( + 2 0 mV). T h e c u r r e n t / v o l t a g e rel a t i o n s h i p o f the c h a n n e l in this e x p e r i m e n t is s h o w n in Fig. 1 B. T h e best fit to the G o l d m a n H o d g k i n Katz e qua t i o n was o b t a i n e d with a p e r m e a b i l i t y c o e f f i c i e n t P c a = 9.45 X 10-15 cm3/s. T h e c o n d u c t a n c e o f the channel, calculated as the slope o f the line b et w een - 60 mV a n d - 4 0 m V , was 7.8___0.2pS (n = 3). T h e extrapolated cu r r en t reversal p o t e n t i a l ( + 6 0 mV) was consistent with the N e r n s t e q u i l i b r i u m p o t e n t i a l for C a 2§ (Vca = + 58 mV). In the next series o f experiments we investigated C a 2+ channels, using Ba 2+ as the m a i n c o n d u c t i n g ion. T h e pipette was filled with BaCI a a n d the b a t h c o n t a i n e d the N a + - r i c h solution. In this c o n d i t i o n we were able to detect single-channel activity in t h e cell-attached c o n f i g u ration. Figure 2 A shows t h e single-channel currents rec o r d e d at different m e m b r a n e h o l d i n g potentials. A t - 80 mV, ch an n el o p e n i n g s a p p e a r e d as d o w n w a r d deflections an d were consistent with Ba 2+ currents. As the m e m b r a n e p o t e n t i a l was c h a n g e d f r o m - 8 0 m V to - 1 2 0 m V the current a m p l i t u d e s increased. T h e corr e s p o n d i n g c u r r e n t / v o l t a g e r e l a t i o n s h i p is s h o w n in Fig. 2B. T h e c o n d u c t a n c e calculated b et w een - 1 0 0 m V an d - 1 2 0 m V was 1 5 + 1 p S (n = 9).
114
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Fig. 1. A Single Ca 2+ channel currents recorded from an inside-out patch. The pipette contained 100mmol/l CaC1z and the bath the Na + -rich solution. The membrane holding potentials (Vm) are indicated on the right of each recording and the closed state of the channel
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ship of the channel with the same solutions as in A
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the left (sampling rate 1 kHz, 0.3 kHz filter). B I/V relationship of the channel with NaC1 ( 9 ) and sodium gluconate ( 9 ) in the bath. The lines are the least-square fit of the data to the Goldman Hodgkin Katz equation (PBa = 1.22 10 -14 cm3/s and PBa = 1.18 10 -14 cm3/s respectively)
115
Non-selective cationic channel
The recordings obtained when the patch was excised in the Na§ solution are shown in Fig. 3A. Downward channel openings were observed at 0 mV holding potential and the current amplitude increased as the membrane was hyperpolarized from 0 mV to - 80 mV. At variance with the previous experiments, the current amplitudes recorded were almost twice those measured with CaC12. The I~ V relationship (Fig. 3 B filled circles) followed the Goldman Hodgkin Katz equation for a current carried by Ba 2§ . The permeability coefficient, PBa, was 1.33+0.06• 10 -14 cm3/s and the slope conductance between - 4 0 mV and - 6 0 mV was 13_+0.9 pS (n = 8). The current reversal potential measured in these conditions is consistent with the expected current reversal potential for Ba 2+ (VBa> +58 mV). To demonstrate that the observed currents were not carried by anions, we conducted ion-substitution experiments. When the bath chloride was substituted by gluconate the channel currents remained unchanged. The I~ V relationship (Fig. 3 B open circles) in this condition was similar to that obtained in the presence of C1-. This result indicates a high selectivity of the channel for divalent cations rather than anions. The effect of the Ca 2+ channel blocker lanthanum (La 3§ was tested on the channel activity. In five experiments, the application of La 3§ (1 gmol/1) to the cytoplasmic side of the channel abolished its activity (Fig. 4). This effect was partially reversed when the La 3+ was rinsed out. We also examined the effect of two other Ca 2§ channel blockers. Verapamil (0.1mmol/l) and nifedipine (10 ~tmol/1), when perfused on the cytosolic side of the membrane, abolished the channel activity (n = 3) but this effect was irreversible when the inhibitors were rinsed out (Fig. 4).
In the course of our study, we observed another type of channel that was only active in excised patches. A typical recording is shown in Fig. 5 A. In this experiment, the pipette contained BaC1z and the bath the Na+-rich solution. In contrast to the observations on the Ca 2+ channels, in these experiments the openings were upward at 0 mV membrane holding potential and the current amplitudes increased when the membrane was depolarized from 0 to +80mV. A slope conductance of 21_+1pS (n = 5) was calculated from the I~ Vrelationship (Fig. 5 B) between + 60 mV and + 80 mV. In this condition, the I~ V relationship followed the Goldman Hodgkin Katz equation for a Na + current. The extrapolated current reversal potential was close to the expected reversal potential for Na + ( V N a > - 6 0 m V ) suggesting that the observed current was carried by Na § . The permeability of the channel to other cations was investigated. From the substitution experiments shown in Fig. 6A and the corresponding I / V plot (Fig. 6B) it is clear that K § but not N-methyl-D-glucamine or Ba 2§ was conducted by the channel. The slope conductance calculated between +60mV and +80mV was 20.3 + 0.3 pS (n = 5). Thus it appears that the channel was equally permeable to Na + and K + but had a very low permeability to divalent cations. Over the range of potentials utilized in these experiments, the channel activity appeared only poorly dependent on the membrane potential. Typically the channel-open probabilities (Po) at +20 mV and +80 mV were 0.67_+0.04 and 0.70+0.02 (n = 3) respectively. This cationic channel was next studied in the presence of conducting ions on both sides of the membrane. Figure 7A shows the recordings obtained with the pipette
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116
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( C - ) indicates the closed state of the channel (sampling rate 2.5 kHz, 1 k H z filter). B I / V relationship of the channel with the same solutions as in A. The line is the least-square fit of the data to the Goldman Hodgkin Katz equation for a monovalent cation channel
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dicates the closed state o f the channel (sampling rate 2.5 kHz, 1 kHz filter). B I / V curves in the presence of K+-rich solution ( e ) , methylglucamine ( 9 or 100 mmol/1 BaC12 ( ~ )
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relationships of the channel, e , Experiments in which pipette and bath contained the same solutions as in A; 0 , experiments in which the bath contained a choline chloride solution. The data are fitted by the G o l d m a n Hodgkin Katz equation for a monovalent cationic channel
117
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Fig. 8 A, B. Single-channel cationic currents recorded from a inside-out patch at + 60 mV m e m b r a n e holding potential. C - indicates the closed state of the channel (sampling rate 2.5 kHz, 1 k H z filter). A The pipette contained 100 mmol/1 BaC12 and the bath 145 mmol/1 NaC1. 4'-meth-
yl-2-diphenylaminecarboxylic acid (10 gmol/1) and La 3+ (I0 gmol/1) were added to the bath. B Same experiments as in A except that the pipette was filled with 140 mmol/1 NaC1
containing the Na+-rich solution and the bath the K+-rich one. Upward and downward single-channel currents were observed at negative and positive membrane holding potentials respectively. The channel conductance calculated from the I~ V relationship (Fig. 7 B, filled circle) was 22.9_+ 1.2 pS (n = 5) and the reversal potential corresponded to the equilibrium potential of cations (Vca t = 0 mV). The cationic selectivity of the channel was confirmed in experiments in which 140 mmol/1 potassium in the bath was replaced by the impermeant cation, choline. As shown in Fig. 7 B (filled square), the I / V relationship followed the Goldman Hodgkin Katz equation for a cation-selective channel. This manoeuvre resulted in a shift of the current-reversal potential toward the expected Nernst equilibrium potential of cations (Vcat = + 85 mV). The channel activity appeared to be strongly voltage-dependent at a negative membrane holding potential (Fig. 7A). Po was 0.02_+0.01 and 0.57_+0.06 (n = 5) at - 6 0 mV and +60 mV holding potentials respectively. These results suggest that it was the same channel under study in the two sets of experiments, a hypothesis confirmed by the sensitivity of the channel to 4"Me-DPC and La 3+. Figure 8 shows that the currents observed with either BaC12 or NaC1 in the pipette were reversibly blocked when 4"Me-DPC (10 gmol/1) was applied on the cytoplasmic side of the channel (n = 5) whereas La 3+ (10 gmol/1) applied on the cytoplasmic side (n = 5) had no effect on the channel activity.
therefore constitute a suitable material for analysing its transport properties [22, 23]. The microdissection technique used to isolate the tubule limited the possible contamination by cells from neighbouring segments. This is of importance since a different calcium transport mechanism probably occurs in the cortical thick ascending limb (CTAL) of the loop of Henle [5, 13]. In our study, we characterized a C a 2§ channel with a conductance of approximately 8 pS in excised but not in cell-attached patches when Ca 2§ was used as the main charge carrier. We thus decided to use Ba 2§ as the permeant ion because this had two advantages: first, it has been shown to be more permeant in Ca 2+ channels than Ca 2§ itself, thus increasing the signal-to-noise ratio in the current recordings [2, 15], and, secondly, Ba 2+ blocks K + channels. In fact, in cell-attached or excised inside-out configurations, with 100 mmol/1 BaCI 2, in the pipette, the channel conductance was 15 pS and 13 pS respectively and a permeability ratio (PBa/Plca~l.4) could be determined. In our ion-substitution experiments we did not detect any significant permeability to Na § or C I - , which thus indicates that the channel is highly selective for Ca 2+ . We also showed that cytoplasmic La 3+ (1 gmol/1) reversibly blocked the channel activity whereas verapamil (0.1 mmol/1) and nifedipine (10gmol/1) irreversibly blocked the channel. Taken together these results are reminiscent of those described for the b t y p e Ca 2+ channels in excitable tissues [2, 26], except that most of the latter Ca 2+ channels were shown to be voltage-dependent whereas, in our experiments, neither in cell-attached nor excised inside-out patches did we detect any modulation of the channel activity by the membrane potential. To odr knowledge, this is the first study describing Ca 2§ channels in epithelial kidney cells. However, in preliminary reports, Matsunaga et al. and Lau et al. [19, 21] characterized Ca 2+ channels in the apical membrane of cultured distal cells. Both groups of workers described dihydropyridine-sensitive Ca 2§ channels activated by the
Discussion In the present study we have shown the presence of Ca 2§ channels in the apical membrane of primary cultures of the rabbit distal bright convoluted tubule cells. We used cultured cells as a model because they offer a direct access to the apical cell membrane. Their use is justified since, as we have already shown, cultured DCTb cells retain most of the properties of the original epithelium and
118 cyclic AMP pathway. More data are required to ascertain that the channel they describe is the same as that under study. Even though a thorough characterization of epithelial Ca 2+ channels has never been made, their involvement in the transport of calcium by kidney cells has been suggested by many investigators. In single microdissected rat cortial collecting tubules, Taniguchi et al. [29] concluded that the calcium uptake they observed was best explained by the presence of voltage-independent Ca 2+ channels in the cell membranes. In cultured mouse cTAL and DCT cells Bacskai and Friedman [1] showed that treatment with parathyroid hormone led to the recruitment of dihydropyridine-sensitive but voltage-independent Ca 2+ channels. However, none of these studies established the localization of the channel nor its involvement in the transepithelial transport of calcium. More studies are definitely needed to clarify these points. We also investigated the calcium permeability of a non-selective cation channel. In a previous paper [23], we characterized this channel and showed that: (a) it does not discriminate between Na + and K + , (b) its activity is dependent on cytoplasmic [Ca 2§ and (c) the diuretic amiloride does not block the channel activity. These properties are also shared by Ca 2§ non-selective cation channels in various cell types [3, 10, 20, 24] suggesting that this channel could represent a calcium-uptake pathway in DCTb cells. We studied the channel with either 100 mmol/1 BaC12 or 145 retool/1 NaC1 in the pipette. The question arose as to whether it was the same channel that we observed in the two experimental conditions. There are several lines of evidence that this was the case. (a) The channel conductance and open probability were not significantly different in the two series of experiments (22.9 pS versus 21.0 pS and Po = 0.70 versus 0.57 at positive holding potentials). (b) The pharmacological profiles of the channels were the same: 4" Me-DPC but not La 3+ (10 ~mol/1) blocked the channel in its closed state when applied on its cytoplasmic face. Such derivatives of DPC have been shown to block nonselective cation channels though there are different reports about the side from which they act. Thus, while DPC blocked the non-selective cation channel from the cytoplasmic side in the exocrine pancreas [12] it did so from the external side in mandibular cells [7]. In our experiments we did not detect any significant permeability of the channel to Ba 2+ . This result agrees with similar reports concerning non-selective cation channels in mouse mandibular gland [7], mouse fibroblasts [11] and brown adipocytes [31], but disagrees with the results obtained on Necturus proximal tubule [10], liver cells [3], choroid plexus [8], Balb/3T3 cells [20] or endothelial cells [24]. Thus, on the basis of calcium permeability two types of non-selective cation channel may be assumed to exist. In an attempt to assign physiological roles to these different channels, it is worth noting that most of the Ca2+-permeable non-selective cation channels have been shown to be activated by membrane stretch [10, 32, 3] or ligand binding [20]. The former are probably involved in the cell-volume-regulatory mechanism whereas the latter may be linked to cell differentiation or
secretion processes [25]. As far as the distal convoluted tubule is concerned, the physiological role of the non-selective cation channels remains uncertain. In conclusion, we have described a Ca 2+ channel in the apical membrane of cultured DCTb cells that could represent the apical pathway for the transepithelial calcium transport occurring in this segment. This channel shares some of the properties of the L-type Ca 2+ channel expressed in excitable tissues except for its voltage independence. It may therefore represent a tissue-specific isoform of an L-type Ca 2+ channel in epithelial cells. It is interesting that recent studies, using the polymerase chain reaction with selected regions of nucleotide sequences from cardiac and skeletal muscle Ca 2+ channels, identified multiple Ca 2+ channel isoforms in the kidney [331.
References 1. Bacskai BJ, Friedman PA (1990) Activation of latent Ca 2+ channels in renal epithelial cells by parathyroid hormone. Nature 347:388-391 2. Bean BP (1989) Classes of calcium channels in vertebrate ceils. Annu Rev Physiol 51:367-384 3. Bear CE (1990) A nonselective cation channel in rat liver cells is activated by membrane stretch. Am J Physiol 258:C421-C428 4. Bindels RJM, Hartog A, Timmermans J, Van os CH (1991) Active Ca 2+ transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D 3 and PTH. Am J Physiol 261: F799- F807 5. Bourdeau JE, Burg MB (1979) Voltage dependence of calcium transport in the thick ascending limb of Henle's loop. Am J Physiol 236:F357-F364 6. Carafoli E (1991) Calcium pump of the plasma membrane. Physiol Rev 71:129-153 7. Cook DI, Poronnik P, Young JA (1990) Characterization of a 25-pS nonselective cation channel in cultured secretory epithelial cell line. J Membr Biol 114:37-52 8. Christensen O (1987) Mediation of cell volume regulation by Ca 2+ influx through stretch-activated channels. Nature 330:66-68 9. Costanzo LS (1984) Comparison of calcium and sodium transport in early and late rat distal tubules: effect of amiloride. Am J Physiol 246:F937- 945 10. Filipovic D, Sackin H (1991) A calcium-permeable stretch-activated cation channel in renal proximal tubule. Am J Physiol 260: Fllg-F129 11. Frace AM, Gargus JJ (1989) Activation of single-channel currents in mouse fibroblasts by platelet-derived growth factor. 86: 2511-2515 12. GOgelein H, Pfannmtiller B (1989) The nonselective cation channel in basolateral membrane of rat exocrine pancreas. Inhibition by 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) and activation by stilbene disulfonates. PfRigers Arch 413:287-298 13. Greger R (1985) Ion transport mechanisms in thick ascending limb of Henle's loop of mammalian nephron. Physiol Rev 65:760-797 14. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfliigers Arch 391:85-100 15. Hess P, Lansman JB, Tsien RW (1986) Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells. J Gen Physiol 88:293-319 16. Imai M (1981) Effects of parathyroid hormone and N 6, O2'-dibu tyryl cyclic AMP on Ca 2+ transport across the rabbit distal nephron segments perfused in vitro. Pfltigers Arch 390:145-151
119 17. Lajeunesse D, Brunette MG (1991) The hypocalciuric effect of thiazides: subcellular localization of the action. Pfl~igers Arch 417:454- 462 18. Lassiter WE, Gottschalk, Mylle M (1963) Micropuncture study of renal tubular reabsorption of calcium in normal rodent. Am J Physiol 204:771-775 19. Lau K, Quamme G, Tan S (1991) Patch-clamp evidence for a Ca channel in apical membrane of cortical thick ascending limb (cTAL) and distal tubule (DT) cells (abstract). J Am Soc Nephrol 2:775-46P 20. Matsunaga H, Nishimoto I, Kojima I, Yamashita N, Kurokawa K, Ogata E (1988) Activation of a calcium-permeable cation channel by insulin-like growth factor II in BALB/c 3T3 cells. Am J Physiol 255:C442- C446 21. Matsunaga H, Friedman PA, Kiser N, Stanton BA (1991) Calcium channels in apical membranes of distal convoluted tubule cells (abstract). Am J Soc Nephrol 2:627-24P 22. Merot J, Bidet M, Gachot B, Le Maout S, Koechlin N, Tauc M, Poujeol P (1989) Electrical properties of rabbit early distal convoluted tubule in primary culture. Am J Physiol 257:F288-F299 23. Merot J, Tauc M, Bidet M, Poujeol P (1991) Ion channels in the distal convoluted tubule in primary culture. Nephrology 2:1698-1712 24. Nilius R (1990) Permeation properties of a non-selective cation channel in human vascular endothelial cells. Pflt~gers Arch 416: 609- 611
25. Partridge LD, Swandulla D (1988) Calcium-activated cation channels. Trends Neurosci 11:69-72 26. Rorsman P, Ashcroft FM, Trube G (1988) Single Ca channel currents in mouse pancreatic B-cells. Pfliigers Arch 412:597-603 27. Sharegi GR, Stoner LC (1978) Calcium transport across segments of the rabbit distal nephron in vitro. Am J Physiol 235:F367-F375 28. Shimizu T, Yoshitomi K, Nakamura M, Imai M (1990) Effects of PTH, calcitonin, and cAMP on calcium transport in rabbit distal nephron segments. Am J Physiol 259:F408-F414 29. Taniguchi S, Marchetti J, Morel F (1989) Cytosolic free calcium in single microdissected rat collecting tubules. Pfltigers Arch 414: 125-133 30. van Os CH (1987) Transcellular calcium transport in intestinal and renal epithelial cells. Biochim biophys Acta 906:195-222 31. Weber A, Siemen D (1989) Permeability of the non-selective channel in brown adipocytes to small cations. Pfliigers Arch 414: 564- 570 32. Yang XC, Sachs F (1989) Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243:1068-1071 33. Yu A, Saltzberg SN, Hebert SC, Lytton J (1991) Multiple voltagedependent Ca 2+ channels (VDCC) are expressed in rodent kidney (abstract). J Am Soc Nephrol 756:24P
