Biochimica et Biophysica Acta 1818 (2012) 1135–1141

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Biochimica et Biophysica Acta

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A calcium-permeable non-selective cation channel in the thick ascending limb apicalmembrane of the mouse kidney

Romain Guinamard a,⁎, Marc Paulais b,c,d, Stéphane Lourdel b,c,d, Jacques Teulon b,c,d

a Université de Caen, Groupe Signalisation, Electrophysiologie et imagerie des lésions d'ischémie reperfusion myocardique, Franceb UPMC Univ Paris 06, UMR_S 872, Laboratoire de génomique, physiologie et physiopathologie rénales, F-75005, Paris, Francec INSERM, UMR_S 872, Laboratoire de génomique, physiologie et physiopathologie rénales, F-75005, Paris, Franced CNRS, ERL 7226, Laboratoire de génomique, physiologie et physiopathologie rénales, F-75005, Paris, France

Abbreviations: CTAL, cortical thick ascending limb; Enamic acid; IMCD, inner medullary collecting duct; NSCchannel; NSCCa channel, calcium-activated non-selectiveability; TRP, transient receptor potential⁎ Corresponding author at: Groupe Signalisation, Elec

lésions d'ischémie reperfusion myocardique, UniversitéD, Salle SD 060, Esplanade de la Paix, 14032 CAEN, Frafax: +33 2 31 56 54 53.

E-mail address: romain.guinamard@unicaen.fr (R. G

0005-2736/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.bbamem.2011.12.024

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 November 2011Received in revised form 22 December 2011Accepted 23 December 2011Available online 31 December 2011

Keywords:Cation channelCalciumKidneyEpitheliumTRPM7

Non-selective cation channels have been described in the basolateral membrane of the renal tubule, but lit-tle is known about functional channels on the apical side. Apical membranes of microdissected fragments ofmouse cortical thick ascending limbs were searched for ion channels using the cell-free configuration of thepatch-clamp technique. A cation channel with a linear current–voltage relationship (19 pS) that was per-meable both to monovalent cations [PNH4(1.7)>PNa (1.0)=PK (1.0)] and to Ca2+ (PCa/PNa≈0.3) wasdetected. Unlike the basolateral TRPM4 Ca2+-impermeable non-selective cation channel, this non-selective cation channel was insensitive to internal Ca2+, pH and ATP. The channel was already activeafter patch excision, and its activity increased after reduced pressure was applied via the pipette. Externalgadolinium (10−5 M) decreased the channel-open probability by 70% in outside-out patches, whereas ex-ternal amiloride (10−4 M) had no effect. Internal flufenamic acid (10−4 M) inhibited the channel ininside-out patches. Its properties suggest that the current might be supported by the TRPM7 protein thatis expressed in the loop of Henle. The conduction properties of the channel suggest that it could be involvedin Ca2+ signaling.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Cation channels are a heterogeneous molecular family includingligand-gated channels [1], cAMP- and cGMP-dependent channels[2], mechanosensitive channels and TRPs [3], many of which are per-meable at least to some extent to calcium. Many cation channels havebeen identified on native tissues using the patch-clamp technique. Inthe renal tubule, patch-clamp studies have revealed the functionalexpression of the Ca2+-activated non-selective cation channelTRPM4 [4–6], of a cGMP-inhibited channel[7], of a pressure activatedcation channel, and of a volume sensitive channel [8–10]. There is alsonon-electrophysiological evidence for the presence of P2X receptor-channels [11].

r, reversal potential; FA, flufe-channel, non-selective cationcation channel; Po, open prob-

trophysiologie et imagerie desde CAEN, Campus 1, Sciencesnce. Tel.: +33 2 31 56 51 39;

uinamard).

rights reserved.

One specific domain of research concerns the channels that medi-ate the entry of calcium into the cell, and are involved in hormonal/paracrine transduction cascades [12,13]. Store-operated channelsand agonist-activated channels with high selectivity for calciumhave been reported in several tissues, and although their preciseidentity is still a matter of debate, this has made it possible to someextent to disentangle the modes of hormonal activation in these tis-sues. Electrophysiological data on Ca2+ permeable channels are espe-cially sparse for the renal tubule, where research has so far focused onchannels involved in the transepithelial absorption of calcium andmagnesium: TRPV5, TRPV6 and TRPM6 [14].

Here we report the properties of a non-selective cation (NSC)channel in the cortical thick ascending limb (CTAL), which has hither-to unprecedented properties in the renal tubule: this channel is locat-ed at the apical membrane, and it has a conductance of 20 pS that issimilar to that of a previously described Ca2+-dependent, non-selective, cation channel located at the basolateral membrane of themouse CTAL, but which is not permeable to Ca+ [4–6], in contrastto this new channel. Its electrophysiological properties are also clear-ly different from those of TRPV5, TRPV6 and TRPM6, which are highlyselective for calcium and magnesium. The physiological role of thechannel requires further investigation, but its location near the keyion transport systems (Na+–K+–Cl− and K+ channels) suggests apossible role in calcium signaling and ion transport regulation.

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2. Methods

2.1. Tubule isolation

All animal experiments were performed according to the Europe-an Commission directive 2010/63/EU. Male mice (15–20 g) were eu-thanized by cervical dislocation, and renal tubules were obtained aspreviously described [6]. One kidney was perfused via the renal veinwith Leibowitz medium (Eurobio, Les Ulis, France) containing300 U/ml Worthington collagenase (CLS II). Small kidney slices ofvarying mass were incubated in the same solution at 37 °C for 45–75 min. Fragments of CTAL were dissected out from tissue immersedin ice-cold Leibowitz medium under a stereomicroscope. The frag-ments were placed on a plastic dish coated with poly-L-lysine in a re-cording chamber, and cut open along their long axis with amicropipette to expose the apical membrane.

2.2. Solutions and chemicals

The standard solution (140 mM NaCl solution) used in the bathand in the pipette contained (in mM): 140 NaCl, 4.8 KCl, 1.2 MgCl2,1 CaCl2, 10 glucose, and 10 HEPES. Low NaCl solutions had the samecomposition, except that the NaCl concentration was reduced to 42or 14 mM NaCl (200 or 256 mM sucrose was added to maintained os-molarity and, no KCl was included).

For tests of the selectivity between monovalent cations, 140 mMNaCl was replaced by the same concentration of KCl or NH4Cl. TheCa2+ permeability was determined using a solution containing (inmM) 100 CaCl2, 10 glucose, and 10 HEPES. All solutions were adjustedto pH 7.2. Internal Ca2+ concentrations below 10−5 Mwere producedwith CaCl2 and Ca-EGTA buffers [6]. Flufenamic acid, nucleotides,EGTA, gadolinium tetrachloride and ammonium chloride wereobtained from Sigma-Aldrich-Fluka (L'Isle d'Abeau Chesnes, France),other chloride salts were from Merck (Nogent-sur-Marne, France),and amiloride was from LC laboratories (MA, USA).

2.3. Measurements

Single-channel currents were recorded with List LM-EPC7 (ListElectronics, Darmstadt, Germany) or RK 400 (Bio-logic, Claix,France) patch-clamp amplifiers from patches of apical membranesusing the inside-out and outside-out variants of the patch-clamptechnique. Patch pipettes with a tip resistance of 7–12 MΩ (in140 mM NaCl solution) were made from microhematocrit borosili-cate glass tubes, and were coated with Sylgard (Dow Corning,Seneffe, Belgium). The signal was displayed on an oscilloscope (Tek-tronix, Beaserton, CA, USA) or a computer-generated scope (Axo-scope, Axon, CA) and stored on a DAT recorder (Sony PCM 701ES). The bath reference was an Ag/AgCl pellet connected to an agarbridge filled with a 0.5-M KCl solution. Liquid junction potentialswere determined by placing a patch pipette filled with 2.7 M KClsuccessively in standard and test solutions, and measuring the volt-age deflections in zero current clamp. The applied potentials(Vm=Vbath−Vpipette) were corrected accordingly. The pipette-filling solution differed from the initial bath solution (140 mMNaCl) for one experimental protocol in which a Ca2+-rich solutionwas used instead of a solution containing 140 mM NaCl solutionwas used. In this case, we took into account the fact that the liquidjunction potential disappeared after sealing, unmasking the compen-sating voltage used to offset the junction potential before sealing.Currents due to the migration of cations from the inner to theouter surface of the membrane were positive, and were registeredas upward deflections in single-channel current tracings. Experi-ments were conducted at room temperature (20–24 °C).

2.4. Data analysis

Signals for analysis were filtered at 300 Hz with an 8-pole Besselfilter (Frequency Devices, Haverhill, MA, USA), and digitized at1 kHz using a Digidata 1200A analog–digital interface and Axoscopesoftware (Axon Instruments, Foster City, CA, USA). The channel-open probability (Po) was calculated from the equation I=N Po i,where i is the amplitude of the unit current, and N the apparent num-ber of channels of the patch. Themean current (I) passing through theN channels present in the patch was estimated from current ampli-tude histograms. Relative permeabilities and associated reversal po-tentials for current flow (Er) were estimated by fitting experimentalmeasurements to the Goldman–Hodgkin–Katz equation:

i ¼ gV∑sðPs=PNaÞzs2 S½ �ie

zFVRT− S½ �o

� �= Na½ �o e

zFVRT−1

� �

with g=PNa[Na]oF2/RT, where V, PNa, F, R and T have their usualmeaning. Ps represents the permeability of the channel to the S ion;subscripts i and o refer to the inside and outside, respectively. The rel-ative permeabilities of the non-selective cation channel to monova-lent cations were deduced from Er values after linear fitting of thedata points since no current rectification was apparent when K+ orNH4

+ was substituted for Na+ in the bath. The activity coefficientsused to correct the ion concentrations were taken from Robinsonand Stokes [15], and had the following values: 0.75 (140 mM mono-valent chloride salts), 0.83 (40 mM NaCl), 0.89 (20 mM NaCl) and0.52 (100 mM divalent chloride salts). Experimental values aregiven as means±SEM, and n denotes the number of results. Thegroups were compared using Student's t-test or Mann–WhitneyRank sum test (SigmaStat; SpSS GmbH, Erkrath, Germany).

3. Results

A calcium-activated non-selective cationic channel has been char-acterized in basolateral membrane of mouse CTAL [6], but nothing isknown about the presence of non-selective cation channels in theapical membrane. To investigate this, we recorded single channel cur-rents in apical membranes from microdissected mouse CTAL.

Channel activity was recorded using the inside-out patch-clampconfiguration with 140 mM NaCl (1 mM CaCl2) on both sides of themembrane. Channel activity was detected in most excised patches(27 out of 36 patches, 75%), with Po=0.66±0.04 (n=24) and4.2±0.6 channels per patch (n=24) (Fig. 1).

3.1. Conductance and permeabilities

The current recordings in Fig. 1A show the activity of this channelat various voltages. No voltage dependence was apparent (Fig. 1A, B).The channel displayed a linear current–voltage relationship (Fig. 1C),with a unit conductance of 19±0.9 pS (n=10) and an Er (0.8±0.9 mV) not significantly different from 0 mV.

Ion selectivity was assessed by ion-substitution experiments.When a membrane patch was exposed to 140 mM NaCl in the pipette(extracellular side) and 42 or 14 mM NaCl in the bath (intracellularside), the current/voltage relationship was shifted toward the posi-tive voltage range (Fig. 1B, C). The reversal potentials were 22.1±1.1 mV (n=4) and 40.2±1.8 mV (n=7) for 42 and 14 NaCl, respec-tively, without any change in single channel conductance (seeTable 1). When mean reversal potentials were plotted as a functionof log ([cation]e/[cation]i), the points fell on a line with a slope of39.91±0.02 mV per decade (Fig. 1D), indicating that the channelhad substantial cation selectivity. The permeability ratio PNa/PCl calcu-lated from the reversal potentials using the Goldman–Hodkin–Katzequation was similar under both conditions (i.e., 0.08±0.02 and

Fig. 1. Conduction properties of the channel in the CTAL apical membrane. A, single-channel current recordings obtained at various voltages from an inside-out patch. Pi-pette and bath: 140 mM NaCl. Vm is the applied membrane potential. Dotted lines in-dicate the closed current level. B, channel current tracings in the same conditionsexcept that the bath contained 14 mM NaCl. C, current–voltage relationships obtainedwith 140 mM NaCl (open squares) or 14 mM NaCl (black circles) in the bath. Pipette:140 mM NaCl. The data points (means ± S.E.M., 6-10 patches) were fitted to a straightline for the 140 NaCl/140 NaCl condition (g=19.0 pS; Er=0.9 mV), and to theGoldman-Hodgkin-Katz equation for the 14 NaCl/140 NaCl condition (g=22.1 pS;PCl/PNa=0.09). Error bars are smaller than the symbols in some cases. D, Relationshipbetween mean reversal potential and log([cation]e/[cation]i). Linear regression on thedata indicates a slope of 39.9 mV per decade.

Fig. 2. Selectivity of the channel towards different monovalent cations. A, single-channel current recordings obtained at various voltages from inside-out patches with140 mM NaCl in the pipette. The bath NaCl was replaced by KCl (open circles, left) orNH4Cl (black triangles, right). Vm is the applied membrane potential. Dotted lines indi-cate the closed current level. B, Current–voltage relationships obtained in the sameconditions as in A. Points are means ± S.E.M. of 5-6 patches. Error bars are smallerthan the symbols in some cases. The lines are the linear regression of the data points(KCl: g=24.2 pS and Er=-1.3 mV; NH4Cl : g=23.7 pS and Er=-11.4 mV).

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0.09±0.01 (p>0.7) for 42 and 14 mM NaCl, respectively). This con-firmed the cation selectivity of the channel.

The selectivity among monovalent cations was assessed by repla-cing all the Na+ in the bath solution (140 mM) by the test cation(Fig. 2, Table 1). The unit conductances in the presence of K+ orNH4

+ were slightly but significantly higher than under the controlconditions (pb0.05). Substitution experiments gave a PK/PNa of1.2±0.2 (n=5, N.S. different from 1.0, p>0.1) and a PNH4/PNa of1.7±0.1 (n=6, significantly different from unity, pb0.001). Thisindicates that NH4

+ is more permeant than Na+ or K+ (Table 1),

Table 1Ion selectivity. G, Er and PX/PNa correspond to the unit conductance, reversal potentialand permeability of ion X relative to Na+, respectively. The conductances were esti-mated in two ways, depending on the experimental protocol (see Methods §2.4).Values are means±SEM; n is the number of measurements. The pipette and bath solu-tions are reported in terms of the concentration of their principal salt (see Methods§2.2 for exact composition).

Test ion Pipettesolution

Bathsolution

n g (pS) Er (mV) PX/PNa

- 140 NaCl 140 NaCl 10 19.0±0.9 0.8±0.9 -Cl− 140 NaCl 42 NaCl 4 20.9±1.5 22.0±1.1 0.08±0.02Cl− 140 NaCl 14 NaCl 7 22.1±0.6 40.2±1.8 0.09±0.01K+ 140 NaCl 140 KCl 5 24.2±1.4 −1.3±3.6 1.2±0.2NH4

+ 140 NaCl 140 NH4Cl 6 23.7±1.8 −11.4±1.3 1.7±0.1Ca2+ 140 NaCl 100 CaCl2 4 20.4±1.3 24.4±1.9 0.28±0.03Ca2+ 100 CaCl2 140 NaCl 4 13.4±1.3 -21.0±2.9 0.35±0.06

and that the channel is a non-selective cation channel. The corre-sponding current–voltage relationships are shown in Fig. 2.

The permeability to Ca2+ was investigated in two sets of experi-ments. Recordings were first done with 100 mM CaCl2 in the pipetteand 140 mM NaCl in the bath. Openings were observed in both in-ward and outward directions, indicating that Ca2+ was permeatingthe channel (Fig. 3A). The current–voltage relationship is shown in

Fig. 3. Permeability of the channel to calcium. A, single-channel current recordingsobtained at various voltages from an inside-out patch with 100 mM CaCl2 in the pipetteand 140 mM NaCl in the bath. Vm is the applied membrane potential. Dotted lines in-dicate the closed current level. B, Current–voltage relationships under the same condi-tions (open triangles) or under the opposite ionic conditions (open circles), asindicated in the drawings. Points (means ± S.E.M. of 3-4 patches) were fitted to theGoldmann-Hodgkin-Katz equation.

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Fig. 3B. The PCa/PNa was 0.35±0.06 (n=4). The unit conductance(Table 1) was significantly lower than under control conditions(pb0.01), suggesting some kind of interaction between externalCa2+ and the channel pore. A PCa/PNa of 0.28±0.03 (n=4) was ob-served when the pipette was filled with 140 mM NaCl, and the bathcontained the 100 mM CaCl2 (Table 1 and Fig. 3B). This latter PCa/PNavalue was not significantly different from that calculated with100 mM Ca2+ in the pipette (p>0.4).

3.2. Effects of channel blockers

The effect of the classical non-selective cation channel blocker flu-fenamic acid was tested by applying the inhibitor at the inner surfaceof inside-out patches (i.e. in the bath). Fig. 4A shows the effect of flu-fenamic acid on an inside-out patch containing only one channel. Podecreased from 0.82±0.04 in the control to 0.44±0.07 (n=8) inthe presence of 0.1 mM flufenamic acid, and to 0.04±0.02 (n=5)in the presence of 0.5 mM flufenamic acid. Inhibition developed rap-idly (within a few seconds) and was reversible.

Gd3+ and amiloride were tested at a negative membrane potential(−40 mV) in the outside-out configuration to allow the molecule tobe applied to the outer surface of the membrane (i.e. in the bath).There were about twice more active channels per outside out patches(11.4±3, n=8) compared to inside-out patches (4.2±0.6, n=24).That difference is probably due to a larger membrane surface inoutside-out patches as compared to inside-out patches. Indeed, indi-vidual Po is similar in both configurations (0.64±0.1 in inside-out vs.0.66±0.04 in outside-out) and ionic conditions are similar in bothsides of the membrane. Gd3+ (10−5 M) reduced channel activity to

Fig. 4. Effects of flufenamic acid, gadolinium and amiloride on channel activity. A, Singlechannel current recording obtained from an inside-out patch illustrating the effects of0.1 and 0.5 mM flufenamic acid (FA). B, Channel current recording from one outside-out patch showing the effects of 10−5 M GdCl2. Pipette and bath: 140 mMNaCl. The ap-plied membrane potential is -40 mV (A and B). The dotted line indicates the closed cur-rent level. C, Mean Po values, as a percentage of control, in the presence of flufenamicacid, amiloride or gadolinium. Bars are means ± S.E.M., and the numbers of experi-ments are indicated at the top of the bars. * indicates pb0.05 (in comparison to control).

19±4% (n=7) of control (Fig. 4B) within a few seconds in a revers-ible manner. On the other hand, amiloride (10−4 M) failed to inhibitthe channel. The activity remained 98±14% (n=4) of control in thepresence of amiloride (Fig. 4C).

3.3. Effect of internal Ca2+, pH and nucleotides

A non-selective cation channel in the basolateral membrane of theCTAL [6] is activated by internal calcium and inhibited by adenine-based nucleotides. It is also sensitive to internal pH. In contrast, thenon-selective cation channel in the apical membrane was not sensi-tive to these agents.

Fig. 5A clearly shows that decreasing the calcium concentrationfrom 10−3 to 10−9 M on the cytoplasmic face of an excised inside-out patch did not change channel activity. Mean Po was similar atthe four concentrations tested (n=3) (Fig. 5B).

Similarly, there was no change in channel activity when internalpH was changed from pH 7.2 to 6.6 (Po=96±5% of Po at pH 7.2) or7.8 (Po=106±4% of Po at pH 7.2, n=5, not shown).

Fig. 5. Effects of internal calcium on channel activity. A, Single channel current re-cordings obtained from an inside-out patch, showing that the internal Ca2+ concen-tration has no effect on channel activity. The applied membrane potential is 0 mV.Pipette: 140 mM NaCl; bath: 42 mM NaCl. The internal Ca2+ concentrations below10−5 M were produced using EGTA. The dotted line indicates the closed currentlevel. B, Mean Po values, in percent of control (10−3 M Ca2+), at various Ca2+ con-centrations. Bars are means ± S.E.M. of 3 patches for each concentration. Po (as apercentage of Po at 10−3 M Ca2+) was 97.7 ± 1.5 at 10−5 M, 101.5 ± 2.3 at10−7 M and 91.5 ± 5.6% at 10−9 M.

Fig. 6. Effects of internal nucleotides on channel activity. A, Single channel current re-cordings obtained from an inside-out patch showing that nucleotides did not alterchannel activity. ATP (1 mM), AMP (1 mM) and cGMP (0.1 mM) were perfused se-quentially onto the membrane patch between washing periods. Pipette: 140 mMNaCl solution; bath: 42 mM NaCl solution. The applied membrane potential is 0 mV.The dotted line indicates the closed current level. B, Mean Po values, as a percentageof control, in the presence of AMP, cGMP or ATP. Bars are means ± S.E.M. of 4 patchesfor each nucleotide.

Fig. 7. Effect of membrane stretch on channel activity. A, Single channel current record-ing obtained from an inside-out patch illustrating the effects of pressures of -30 and-60 mmHg within the pipette. To determine the baseline level, flufenamic acid (FA)(5.10−4 M) was applied at the end of the experiment. Pipette and bath: 140 mMNaCl. The applied membrane potential is -40 mV. The dotted line indicates the closedcurrent level. B, Mean Po values, as a percentage of control, during membrane stretch.Bars are means ± S.E.M. numbers of experiments are indicated at the top of the bars.* indicated pb0.05 (in comparison to control).

Fig. 8. Two non-selective cation channels in mouse CTAL with different permeabilitiesand regulations.

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ATP and AMP had no effect on the apical non-selective cationchannel (Fig. 6). Po (as a percentage of control) was 89.0±3.9% inthe presence of 1 mM ATP (n=4) and 88.5±4.1% in the presenceof 1 mM AMP (n=4). These Po values are not statistically differentfrom control (p>0.05). Cyclic GMP, which inhibits a cation channelin the apical membrane of cultured IMCD cells [16], also had no effect(Po=98.8±10.3% of control in the presence of 0.1 mM cyclic GMP;n=4; Fig. 7).

3.4. Effect of membrane stretch

Because gadolinium is a blocker of stretch-activated channels, andbecause stretch-activated non-selective cation channels have beenreported in kidney cells [8,17], we investigated the effect of stretchon the apical non-selective cation channel. During inside-out record-ings, negative pressure was applied to the membrane by controlledsuction in the pipette. Po increased from 0.63±0.10 to 0.77±0.09at a pressure of −30 mm Hg (n=3), and to 0.91±0.05 at −60 mmHg (n=5) (Fig. 7). All the channel activity recorded under these neg-ative pressures was inhibited by flufenamic acid (5.10−4 M), indicat-ing that the activated current corresponds to the NSC current (Fig. 7).

4. Discussion

We have previously described a non-selective cation channel(NSCCa channel) in the basolateral membrane of the CTAL and otherparts of the mouse nephron. It is activated by internal Ca2+ [6], inhib-ited by adenine-based nucleotides [5], sensitive to pH [18] and notpermeable to Ca2+ [4]. The NSCCa channel has properties similar tothe cloned TRPM4b channel [19,20]. The cation channel in the CTALapical membrane, which is not dependent upon internal Ca2+, nucle-otides or pH, and is permeable to Ca2+, is clearly a distinct type ofnon-selective cation channel (Fig. 8).

4.1. NSC channels in kidney

In renal preparations, apart from the NSCCa channel, the non-selective cation channels have been mostly studied in cultured renalcells and can be classified as three functional types. A cyclic GMP-inhibited cation channel has been detected in rat primary culture ofIMCD cells, which is permeable to Ca2+ [7,21], whereas the presentchannel is not sensitive to cyclic GMP. Osmotic shrinkage activates aNSC channel of about 27 pS in a mouse kidney cell line, but this chan-nel is not permeable to calcium [9,22]. It is most interesting to notethat a stretch-activated NSC channel with unit conductances ofabout 20 pS has been found in amphibian renal cells [8,10,17]. TheCTAL NSC channel is, to some extent, similar to these stretch-activated channels found in the amphibian proximal tubule andearly distal tubule, that are permeable to Ca2+ and blocked by Gd3+

[8,17]. Similar channels have been found in a number of cell types,notably in the aortic endothelium [23] and Reissner's membrane ofthe inner ear [24]. The CTAL channel could therefore be related tothese channels, but whereas they are strictly pressure-dependent, in

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our hands the CTAL cation channel was spontaneously active in theabsence of pressure.

4.2. Molecular identity of the apical NSC channel

It has been proposed that TRP proteins may be molecular determi-nants of a large number of native NSC currents. On the basis of struc-tural data, the TRP-related channels have been divided into threemain subfamilies: TRPC (canonical TRP channels), TRPV (vaniloidgroup of TRP channels), and TRPM (which are homologous to the ar-chetypal melastatin). Among these channels, TRPV5 and TRPM6,which play key roles in Ca2+ andMg2+ renal absorption, respectively,are located exclusively in the apical membranes of the distal convo-luted tubule or connecting tubule [25–27], and are highly selectivefor divalent cations [28], precluding their identification with the pre-sent channel. Most of the information about other TRPs in the kidneyconcerns the rat. One study failed to detect TRPC4, –C5 or –C7 pro-teins in the whole kidney [29], and localized TRPC1, –C3 and –C6 ex-clusively in the apical membranes of the rat collecting duct [29]. Thepresence of the TRPV4 protein has been demonstrated in the rat kid-ney, including in CTAL, by immunohistochemical studies, but exclu-sively on the basolateral membrane [30]. Moreover, its permeabilityfor calcium (PCa/PNa≈6–10), as measured in heterologous expressionsystems [31], is not compatible with those of the present channel. Inaddition to TRPM6, which is exclusively expressed in the DCT [26],TRPM7 mRNA is present in the (rat) kidney, including in CTAL [32].Several characteristics of TRPM7 are compatible with those reportedfor the apical CTAL channel, including its permeability ratios (PK/PNa=1.1, PCa/PNa=0.34 [33]), the fact it is blockaded by gadolinium[34] and flufenamic acid [35], and is insensitive to internal calcium[33]. TRPM7 is probably also insensitive to internal ATP [36]. Pioneerstudies reported a TRPM7 unit conductance of 40–100 pS associatedwith a steep outward rectification [33,37], which is not compatiblewith those of the present channel, but more recently a linear conduc-tance of 20 pS has been reported in HEK cells [38]. Interestingly, theauthors of this study reported that an endogenous 20 pS TRPM7channel was directly activated by membrane expansion in human ep-ithelial HeLa cells, in a manner independent of exocytosis, a propertysimilar to what we observed in our inside-out patches subjected topressure [39]. Taken together, this evidence suggests that TRPM7could be a possible substrate for the Ca2+-permeable NSC channel de-scribed here.

Interestingly, it was shown that the whole-cell current induced byTRPM7 expression in HEK-293 cells is reduced by the presence of ex-ternal Ca2+ [34]. The reduction of single channel conductance fromthe apical channel in the presence of high concentration of externalCa2+ would similarly lead to a reduction in whole-cell current. Varia-tion of single channel conductance under different Ca2+ concentra-tions was previously observed for other channels, in particular forthe nicotinic acetylcholine receptor that is, similarly to the apicalchannel, a Ca2+- permeable non-selective cation channel [40–42].High concentration of external Ca2+ (40 mM), in replacement ofNa+, reduced the single channel conductance of the nicotinic recep-tor from 30 pS to 10 pS. This was attributed to a competition of Na+

and Ca2+ for a binding site with Ca2+ ions binding more tightly[41]. The apical channel might be subject to the same phenomenonwith a binding site at the outside part of the pore, since high concen-tration of Ca2+ at the inside of the channel did not impact singlechannel conductance.

4.3. Physiological implications of the apical NSC channel

TAL is involved in the absorption of salt to regulate urine and diva-lent cations concentration (see [43] for review). While the mecha-nisms involved in Na+, K+, and Cl− absorption have been welldescribed, Ca2+ absorption in this nephron segment remains poorly

documented. A trans-cellular pathway has been proposed [44], inwhich the apical CNS channel might be involved since it is the onlyknown Ca2+-permeable channel in the TAL. However, the currentview is that Ca2+ reabsorption across CTAL is essentially passive andproceeds via the paracellular pathway [44]. This means that Ca2+ per-meable channels might rather be involved in Ca2+ signaling. Indeed,NaCl absorption in the thick ascending limb, which is stimulated bycyclic AMP, is also down-modulated by several hormones and para-crine agents that stimulate the PLC/calcium pathway, such as extracel-lular ATP/UTP, angiotensin 2 and bradykinin, which trigger Ca2+ entry[11,45–47]. TRP channels showing either high or relatively low per-meability to calcium are known to be implicated in Ca2+ signaling ina variety of tissues [48]. In addition, TRPM7 has been shown to medi-ate cell growth, survival and proliferation (see [49] for review). Be-cause TRPM7 is ubiquitous, it might play a same role in cells,independently of the tissues where they occur. Further investigationis needed to clarify the possible function of this channel in the TAL,the first reported to be permeable to both monovalent and divalentcations in the renal tubule.

Acknowledgements

Wewould like to thank Dr. Van Den Abbeele (Paris, France) for theloan of a pressure monitor and Monika Ghosh for editing the manu-script. Romain Guinamard, Stéphane Lourdel and Jacques Teulon areemployed by the French Ministry of Education and Research. MarcPaulais is employed by the CNRS.

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