Marc Corbeels
email: corbeels@cirad.fr

Rémi Cardinael

David S Powlson

Regis Chikowo

Bruno Gerard

Carbon sequestration potential through conservation agriculture in Africa has been largely overestimated Comment on: "Meta-analysis on carbon sequestration through conservation agriculture in Africa"

come

Carbon sequestration potential through conservation agriculture in Africa has been largely overestimated.

Soil organic carbon (SOC) sequestration depends on several factors, including land use, pedoclimatic conditions, topographic position and the initial SOC stock [START_REF] Post | Soil carbon sequestration and land-use change: processes and potential[END_REF][START_REF] Minasny | Soil carbon 4 per mille[END_REF]. At the plot scale, a positive SOC balance is created by increasing the input of organic matter to the soil to exceed the carbon (C) losses by mineralization, leaching and erosion or by decreasing the rate of SOC decomposition. In Africa, agricultural soils are generally known to have potential as a C sink due to previous SOC depletion (Vågen et al., 2005;Swanepoel et al., 2016). Two widely promoted crop management practices to store C in agricultural soils are conservation agriculture (CA) and agroforestry. Both practices can increase SOC through increased C inputs from higher biomass productivity and reduced C losses (through soil cover), leading to a net transfer of C from the atmosphere to the soil, thus contributing to the mitigation of climate change [START_REF] Smith | Carbon sequestration potential in European croplands has been overestimated[END_REF][START_REF] Powlson | Soil carbon sequestration to mitigate climate change: a critical re-examination to identify the true and the false[END_REF][START_REF] Griscom | Natural climate solutions[END_REF].

In their recent study published in Soil and Tillage Research: "Meta-analysis on carbon sequestration through conservation agriculture in Africa", [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF] conclude that the practice of CA in Africa can effectively contribute to mitigating global warming through SOC sequestration. [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF] claim that the SOC sequestration potential through CA for the African continent is 143 Tg C yr -1 on 160 Mha cropland (including perennial woody crops) which corresponds to about 0.90 Mg C ha -1 yr -1 .

Good estimates of the SOC sequestration potential with CA are certainly of great interest to policymakers at various levels of government in Africa regarding the nations' commitments to reduce greenhouse gas emissions by 2020. As a result, greater investments in research and innovations for the development and scaling of CA practices may be decided. However, we argue that the mitigation calculations and interpretations by [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF] are flawed and biased. [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF] evaluated datasets from a number of studies in Africa for their estimations of annual per-area SOC sequestration rates with CA practiced in annual or woody perennial cropping systems for four climatic zones (i.e. Mediterranean, Sahelian, Tropical and Equatorial, see Figure 1 and Table 1 in their study). In their analysis, the total SOC sequestration potential for Africa was then calculated from the climate-specific rates and from estimated total land areas cultivated with annual and woody perennial crops in the different countries (from FAOSTAT, http://fao.org/faostat/en/#data ), considering the major climate(s) in each country. Finally, they compared their estimate of sequestration potential with an estimated current annual SOC sequestration based on present areas of cropland under CA. They conclude that the total annual SOC sequestration potential through CA in Africa is about 93 times the current estimated figure.

Here, we challenge the excessively optimistic results of their study.

First, in contrast with their claims, the reported annual per-area SOC sequestration rates under CA in their study (see Table 1 in their paper) are high, ranging from 0.44 Mg C ha -1 yr -1 (Mediterranean climatic zone) to 1.56 Mg C ha -1 yr -1 (Equatorial climatic zone) for annual crops, and from 0.12 Mg C ha -1 yr -1 (Sahelian climatic zone) to 1.29 Mg C ha -1 yr -1 (Mediterranean climatic zone) for woody perennial crops. The resulting average rates for the whole of Africa are 0.92 and 0.70 Mg C ha -1 yr -1 for CA with annual and woody perennial crops, respectively (recalculated from Table 3 and 4 in their study). Even though [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF] refer to their analysis as a meta-analysis, their reported figures do not reveal any use of statistical tests, lacking any indicator of data variability and uncertainty of their estimates. In fact, from their paper it is not clear which, and how many studies were used for their estimates of annual per-area SOC sequestration rates. They simply list the publications they referred to but do not cite any "supplementary information" that presents the data used to derive their mean values.

We estimated average SOC sequestration rates for CA on croplands per climatic zone from published studies used in a recent literature review [START_REF] Corbeels | The 4 per 1000 goal and soil carbon storage under agroforestry and conservation agriculture systems in sub-Saharan Africa[END_REF]. Our results for the Tropical and Equatorial climate zones show rates that are 20-60% of those reported by [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF] and show high variability (Table 1). Since the review by [START_REF] Corbeels | The 4 per 1000 goal and soil carbon storage under agroforestry and conservation agriculture systems in sub-Saharan Africa[END_REF] only referred to sub-Saharan Africa (excluding South Africa), the Mediterranean region was not considered. No studies were found in [START_REF] Corbeels | The 4 per 1000 goal and soil carbon storage under agroforestry and conservation agriculture systems in sub-Saharan Africa[END_REF] for the Sahelian climatic zone. The sequestration rate of 0.5 Mg C ha -1 yr -1 for annual crops in the Sahelian region given by Gonzales-Sanchez et al ( 2029) seems extraordinarily high given the strong water limitations to crop growth in this region. Average cereal yields in this region are 1000 kg ha -1 or less (http://fao.org/faostat/en/#data). Assuming a harvest index of 0.35 and a root:shoot ratio of 0.3 (corresponding to the 0-30 cm soil layer), this represents a potential annual input of about 2700 kg dry matter ha -1 , corresponding to about 1200 kg C ha -1 . A sequestration rate of 0.5 Mg C ha -1 yr -1 would mean that 42% of the C input is converted into SOC, which is clearly not plausible. A recent study on SOC sequestration in tropical croplands found that the conversion rate of C inputs to SOC was 8.2 ± 0.8% [START_REF] Fujisaki | Soil carbon stock changes in tropical croplands are mainly driven by carbon inputs: A synthesis[END_REF]. [START_REF] Smith | Greenhouse gas mitigation in agriculture[END_REF] estimated that the annual per-area sequestration rate for no-tillage and residue management practices in warm-dry regions was about 0.10 Mg C ha -1 yr -1 with high uncertainty (range between -0.21 and 0.40 Mg C ha -1 yr -1 ). Similar results were found for sub-Saharan Africa in the meta-analysis of [START_REF] Powlson | Does conservation agriculture deliver climate change mitigation through soil carbon sequestration in tropical agro-ecosystems?[END_REF].

Table 1. Soil carbon sequestration rates (Mg C ha -1 yr -1 , average and standard deviation) in annual cropping systems under CA per climate zone (data from Corbeels et al., 2019, values larger than 4 Mg C ha -1 yr -1 or smaller than -4 Mg C ha -1 yr -1 were considered as outliers and excluded).

Climatic Zone

Soil carbon sequestration rate (Mg C ha FAOSTAT. This area include land that has recently been converted from native forest or savannah. Given the relatively high original SOC stocks under forest or savannah land, converting this land into agriculture will induce SOC losses irrespective of the type of agricultural management practices employed [START_REF] Sommer | Reducing losses but failing to sequester carbon in soilsthe case of conservation agriculture and integrated soil fertility management in the humid tropical agro-ecosystem of western Kenya[END_REF]. For example, negative SOC sequestration rates (-0.17 to -0.55 Mg C ha -1 yr -1 ) were reported in experiments in Nigeria where CA was installed following recent clearing of native vegetation [START_REF] Lal | Soil quality changes under continuous cropping for seventeen seasons of an Alfisol in western Nigeria[END_REF][START_REF] Agbede | Nutrient availability and cocoyam yield under different tillage practices[END_REF]. Thus, new croplands should have been excluded from the calculations of the SOC sequestration potential. Based on data provided by FAOSTAT, the increase of cropland area over the last ten years in Africa is estimated at about 15 to 20%.

Besides, [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF] included in their calculations the land area on which (most type of) woody perennial crops were cultivated in 2016. This land was considered as land where CA could be practiced, labelled in their study as "CA in woody crops due to ground cover". However, it seems that the annual per-area SOC sequestration rates (Table 1 in their study) were estimated from studies on agroforestry systems. In these studies, the control plot is a treeless agricultural plot having the same tillage practice as the agroforestry plot. Therefore, the SOC sequestration rates are due to the presence of trees and are not linked to CA practices.

In agroforestry systems, the soil can be tilled and is not necessarily covered by a mulch of crop residues, and tree crops can be grown in crop monoculture, as this was the case in many of the cited papers. Therefore, these rates cannot be used for woody perennial cropping systems practiced under CA. To have an estimation of the effect of CA in woody perennial cropping systems, we would need treatments in agroforestry with CA and with conventional tillage, which was not the case in the publications cited by the authors. Moreover, we found that the SOC sequestration rates were highly dependent on the type of agroforestry system [START_REF] Cardinael | Revisiting IPCC Tier 1 coefficients for soil organic and biomass carbon storage in agroforestry systems[END_REF][START_REF] Corbeels | The 4 per 1000 goal and soil carbon storage under agroforestry and conservation agriculture systems in sub-Saharan Africa[END_REF]. It is therefore not correct to group them in a single category as proposed by [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF].

Third, [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF] did not address the adoption rate of CA by farmers, supposing that all estimated cropland area (including woody perennial crops) in 2016 is easily and immediately converted to CA. This is misleading. As stated in their study, adoption of CA in 2016 covered an estimated 1.5 Mha of land, or 1.1 % of the total land area of annual crops.

A realistically achievable mitigation potential must also consider the socio-economic realities of farmers [START_REF] Smith | Carbon sequestration potential in European croplands has been overestimated[END_REF]. This consideration is crucial; it has been extensively discussed elsewhere (e.g. [START_REF] Giller | A research agenda to explore the role of conservation agriculture in African smallholder farming systems[END_REF] but was totally ignored by [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF].

Smallholder farmers in Africa often face significant technical, infrastructural or socioeconomic barriers to the adoption of CA [START_REF] Andersson | From adoption claims to understanding farmers and contexts: A literature review of Conservation Agriculture (CA) adoption among smallholder farmers in southern Africa[END_REF][START_REF] Corbeels | Understanding the impact and adoption of conservation agriculture in Africa: A multi-scale analysis[END_REF]. Therefore, it is not realistic to rely on immediate adoption of CA over millions of hectares as a major strategy to mitigate climate change [START_REF] Powlson | Does conservation agriculture deliver climate change mitigation through soil carbon sequestration in tropical agro-ecosystems?[END_REF].

Fourth, we argue that the extrapolation of the per-area SOC sequestration rates over the whole of Africa using climatic zones is simplistic, ignoring important factors of SOC sequestration.

Although a similarly simple approach is employed in the Tier 1 method of the Intergovernmental Panel on Climate Change [START_REF] Ipcc | Agriculture, forestry and other land use (AFOLU)[END_REF], it has clearly been shown in the broader literature that SOC sequestration depends to a large extent on soil properties [START_REF] Feller | Physical control of soil organic matter dynamics in the tropics[END_REF]Torn et al., 1997). Countries in West Africa such as Mali, Burkina Faso or Niger are mainly characterized by sandy Arenosols and Lixisols, compared to e.g. Kenya, Tanzania or Ethiopia where largely Nitisols and Vertisols are present, that have a much more clayey texture. It is generally known that the SOC sequestration potential is considerably lower in sandy soils than in clayey soils [START_REF] Chivenge | Long-term impact of reduced tillage and residue management on soil carbon stabilization: Implications for conservation agriculture on contrasting soils[END_REF]. Yet in their analysis, the basic SOC sequestration rates used for e.g. Burkina Faso are the same (or higher) than of those for Ethiopia (Table 3 and4). Digital soil maps for Africa are now available (http://soilgrids.org ), which enables to include soil factors, such as soil texture, in SOC sequestration estimates, and could have been used by [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF].

Finally, [START_REF] Gonzalez-Sanchez | Meta-analysis on carbon sequestration through conservation agriculture in Africa[END_REF] compared their estimated SOC sequestration potential (i.e. 143 Tg C yr -1 ) with an estimated (current) SOC sequestration based on the present cropland area under CA (i.e. 1.5 Tg C yr -1 ). This is not correct. A baseline including other best crop management practices that increase C input to the soil, such as fertilization, irrigation, improved crop rotations, and agroforestry, should be used. It has been estimated that 7 to 15 Tg C yr -1 can be sequestered on croplands in Africa, assuming 20% of the croplands are subjected to improved management [START_REF] Batjes | Estimation of soil carbon gains upon improved management within croplands and grasslands of Africa[END_REF].

For the reasons given in our analysis, we believe that Gonzalez-Sanchez et al. ( 2019) grossly overestimated the total SOC sequestration potential through the practice of CA in Africa.

Roughly, as a first approximation we estimate the potential at 10.8 Tg C yr -1 assuming an average per-area rate of 0.45 Mg C ha -1 yr -1 and that 20% of the current soil C-depleted (annual) croplands (estimated at 120 Mha) are cultivated with CA. It is, however, important to note that SOC stocks do not increase forever, and that annual sequestration rates decline as the soil approaches a new equilibrium, which can take from 20 to +50 years depending on climate and soil type. Hence, rates cannot be extrapolated indefinitely [START_REF] Paustian | Agricultural soils as a sink to mitigate CO2 emissions[END_REF][START_REF] Powlson | Soil carbon sequestration to mitigate climate change: a critical re-examination to identify the true and the false[END_REF][START_REF] Powlson | Limited potential of no-till agriculture for climate change mitigation[END_REF]. Lastly, it should also be mentioned that nitrous oxide (N2O) emissions could be enhanced in CA and, more generally, in practices with addition of organic amendments [START_REF] Charles | Global nitrous oxide emission factors from agricultural soils after addition of organic amendments: A meta-analysis[END_REF][START_REF] Lugato | Mitigation potential of soil carbon management overestimated by neglecting N2O emissions[END_REF][START_REF] Mei | Stimulation of N2O emission by conservation tillage management in agricultural lands: A meta-analysis[END_REF], partially offsetting the climate benefits due to increased SOC storage.

It remains critical that we determine rates of SOC sequestration through improved agricultural practices, and the role they can play in helping to meet short-to medium-term reduction targets of greenhouse gas emission. It would, however, be appropriate for Gonzalez-Sanchez et al.

(2019) to reflect on a more conservative assessment of the mitigation potential through CA in Africa. The presentation of implausible potentials leads to unrealistic expectations of climate change mitigation with improved agricultural management. There is a danger that presenting unrealistically high numbers of climate change mitigation potential through agricultural practices could have a negative impact on the necessary actions to reduce CO2 emissions from fossil fuel combustion.

On the other hand, even if CA has limited value for climate change mitigation, the practice of CAthrough crop residue mulching and crop diversification-is expected to enhance the resilience of cropping systems to climate change [START_REF] Rusinamhodzi | A meta-analysis of long-term effects of conservation agriculture on maize grain yield under rain-fed conditions[END_REF][START_REF] Steward | The adaptive capacity of maize-based conservation agriculture[END_REF]. This may bring livelihood benefits to farmers, especially in regions with increased risk of drought stress. Thus, it is more reasonable for policymakers and investors to plan promotion of CA for reasons of climate resilience benefits than for climate change mitigation.

  Sanchez et al. (2019) estimated the cropland area in 2016 based on

		-1 yr -
		1 )
	Sahel	No data
	Tropical	0.58 ± 1.06 (n = 17)
	Equatorial	0.32 ± 1.53 (n = 8)
	n denotes the number of studies
	Soil depth considered varies between 5 and 60 cm
	Second, Gonzalez-

systems to climate stress in tropical and subtropical environments: a meta-regression of yields. Agric. Ecosyst. Environ. 251, 194-202. Swanepoel, C.M., van der Laan, M., Weepener, H.L., du Preez, C.C., Annandale, J.G., 2016. Review and meta-analysis of organic matter in cultivated soils in southern Africa. Nutr.


Sébastien L'hoste

Alexi Diakov

Olga Andrini

Mathieu Genete

Laurent Pinelli

Teddy Grand

Mathilde Keck

Marc Paulais

Laurent Beck

Alexei Diakov

Christoph Korbmacher

Jacques Teulon

Stéphane Lourdel
email: stephane.lourdel@upmc.fr

Alexei Diakov

Characterization of the mouse ClC-K1/Barttin chloride channel

Keywords: Chloride channel, ClC, kidney, ClC-K1, patch-clamp NON-STANDARD ABBREVIATIONS CCD: cortical collecting duct ClC-K: kidney chloride channel CNT: connecting tubule CTAL: cortical thick ascending limb DCT: distal convoluted tubule DIDS: 4, 4′-diisothiocyanato-2, 2′-stilbene disulfonic acid disodium salt

INTRODUCTION

Two Cl -channels of the ClC family, ClC-K1 and ClC-K2 (or their human orthologs ClC-Ka and ClC-Kb, respectively), play a pivotal role in transcellular Cl -absorption in the kidney. Located on the basolateral membrane, they are present throughout the distal nephron [1][2][3], but the specific distribution of each ClC-K is still uncertain owing to the lack of isoform-specific antibodies [4]. Mutations in human genes coding for ClC-Kb and the regulatory subunit, Barttin, are responsible for salt-losing tubulopathies, namely Bartter's syndrome types 3 and 4, respectively [5][6][7]. Rare types of Bartter's syndrome can also result from compound mutations in ClC-Kb and ClC-Ka [8]. Renal function has not been investigated in knockout mice for ClC-K2 because they die very early; ClC-K1-knockout mice display impaired urine concentration and nephrogenic diabetes insipidus [9]. In accordance with pathological and physiological data, localization studies suggest that ClC-K2 is highly expressed in the cortical part of the distal nephron, and that ClC-K1 is mostly restricted to the medullary part of the renal tubule [2,3,10,11].

The functional expression of human and rat ClC-K remained controversial until their accessory subunit, Barttin, was identified. It promotes their insertion into the plasma membrane [2,12]. This discovery made it possible to identify two fundamental properties of ClC-K channels. First, the relative permeability for anions follows the sequence Cl -> Br -> NO 3 -> I -for ClC-Ka, and Cl -> Br -= NO 3 -> I -for ClC-Kb; second, the currents are enhanced by a basic external pH and high external calcium concentrations [2,12]. Interestingly, a glutamate residue that plays a critical role in the protopore gating of other ClC chloride channels is missing in ClC-K channels, where it is replaced by a valine. For this reason, it has been suggested that the two protopores in ClC-K dimers are constitutively open. Thus, the common gate (involving both subunits of the dimers) may act as the major regulator of ClC-K channels. In recent studies on rat ClC-K1, Fahlke's group reintroduced the "gating" glutamate and observed profound effects on both conductance and gating [4,13,14].

We and others have identified several basolateral chloride channels in different parts of the renal tubule, particularly in the cortical thick ascending limb (CTAL), the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD) [15][16][17][18][19][20][21][22][23]. Two of these channels, with conductances of 45 pS and 9 pS, respectively, display properties that are compatible with those of ClC-K chloride channels [24].

Nevertheless, the precise molecular identity of these channels remains elusive, because heterologous expression data are incomplete and heterogeneous. Studies of recombinant channels have involved rat and human ClC-Ks, whereas most of those of the native renal tubule have involved mouse kidney. Furthermore, there are only a few single-channel recordings available for recombinant channels, and they all derive from studies on human ClC-Ka and rat ClC-K1 [4]. In this study, we investigated the properties of recombinant mouse ClC-K1 channels expressed in the presence or absence of mouse Barttin, in an attempt to establish a coherent basis for their correspondence with native chloride channels in the mouse renal tubule.

MATERIAL AND METHODS

Molecular biology

The mouse ClC-K1 cDNA clone (image 5042736, Invitrogen, Carlsbad, CA, USA)

and mouse Barttin (a generous gift from Prof. T. J. Jentsch, MDC/FMP, Berlin, Germany)

were subcloned into pSP64-S and pVITRO2 vectors, respectively, for expression in Xenopus laevis oocytes. Capped cRNAs were synthesized in vitro from wild-type and mutant ClC-K1

and Barttin vectors using the SP6 mMessage mMachine Kit (Ambion, Austin, TX, USA). To evaluate the surface expression of ClC-K1 in Xenopus laevis oocytes, we externally tagged ClC-K1 by introducing a FLAG epitope (DYKDDDDK) between transmembrane domains L and M (beginning at position 377). This did not interfere with their ability to generate currents.

For electrophysiological recordings, HEK293 cells were cotransfected with 1 µg of a bicistronic vector pVITRO2 containing the coding sequence of mouse ClC-K1 and mouse Barttin, and 0.3 µg of a IRES vector encoding the CD8 antigen (a generous gift from Dr. J.

Barhanin, Université Nice Sophia Antipolis, Nice, France) as a transfection marker by electroporation or using X-tremeGENE9 (Roche Diagnostics, Meylan, France). Including CD8 made it possible to identify the transfected cells using anti-CD8-coated beads (Invitrogen Dynal, Oslo, Norway). Site-directed mutagenesis was performed with the Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA).

All constructs were fully sequenced.

Two-electrode voltage-clamp

Defolliculated Xenopus laevis oocytes were injected with 10 ng of ClC-K1 cRNA and 5 ng of Barttin cRNA. The oocytes were then kept at 17°C in modified Barth's solution containing (in mM): 88 NaCl, 1 KCl, 0.41 CaCl 2 , 0.32 Ca(NO 3 ) 2 , 0.82 MgSO 4 , 10 HEPES, pH 7.4, and supplemented with gentamycin (20 µg/ml). Three days after injection, twoelectrode voltage-clamp experiments were performed using a TEV-200A amplifier (Dagan, Minneapolis, MN, USA) and PClamp 9 software (Molecular Devices, Sunnyvale, CA).

Pipettes were made from borosilicate tubes (GC150T, Harvard Apparatus, Edenbridge, UK), and filled with 3 M KCl. Currents were recorded in ND96 solution containing (in mM): 96

NaCl, 2 KCl, 1.5 CaCl 2 , 1 MgCl 2 , 5 HEPES, pH 7.4. Currents were recorded in response to a voltage protocol consisting of 20 mV steps from -160 mV to +100 mV with a duration of 800 ms from a holding potential of -30 mV. The data were filtered at 300 Hz, and sampled using a Digidata 1320A analogue-to-digital converter (Molecular Devices, Sunnyvale, CA).

For anion replacement, 80 mM Cl -was replaced by equivalent amounts of Br -, I -or NO 3 -. HEPES was replaced by equimolar MES for solutions at pH 5.4-6.4, or by Tris for solutions at pH 8.4. Where appropriate, calcium acetate was added to the ND96 solution to increase the extracellular calcium concentration. The permeability ratios of various anions (X -) relative to that of Cl -(P X-/P Cl-) were calculated using the modified Goldman-Hodgkin-Katz equation [25]:

80 ) 104 ( 24 ) ( 40 2 1        E E Cl X e P P
Where E1 and E2 are the reversal potentials for Cl -and X -, respectively.

Chloride channel inhibitors were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). DPC, at a concentration of 0.5 M, and NPPB, at a concentration of 0.1 M, were dissolved in DMSO. DMSO (at the maximum concentration of 0.2% used for 10 -3 M DPC) had no effect on channel activity. following pulse protocol was used: from a holding potential of -30 mV, after a pre-pulse to +65 mV for 30 ms, voltage was stepped from -115 mV to +105 mV in 20 mV increments for 160 ms, and followed by a pulse to +105 mV.

Whole-cell recording

Single-channel recordings in outside-out patches of Xenopus laevis oocytes

Recordings in outside-out membrane patches of mClC-K1 and mBarttin expressing oocytes were performed essentially as described previously using a conventional patch-clamp technique [26,27]. Patch pipettes were pulled from borosilicate glass capillaries, and had a tip diameter of about 1-1.5 μm after fire polishing. Pipettes were filled with the KCl pipette solution (in mM: 55 KCl, 35 K-gluconate, 5 NaCl, 1.2 MgCl 2 , 2 EGTA, 10 HEPES, pH 7.28).

Seals were routinely formed in a low sodium NMDG-Cl bath solution (in mM: 95 NMDG-Cl, 1 NaCl, 4 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 HEPES, pH 7.4). In the NaCl bath solution, 95 mM NMDG-Cl was replaced by 95 mM NaCl. In the NaBr, NaI and NaNO 3 bath solutions, 95 mM NMDG-Cl was replaced by 10 mM NaCl and by 85 mM NaBr, 85 mM NaI and 85 mM NaNO 3 , respectively. After seal formation, the bath solution was changed to the NaCl, NaI, NaBr or NaNO 3 bath solutions. Single-channel current data were initially filtered at 3 kHz and sampled at 9 kHz. Current traces were re-filtered at 250 Hz to resolve the single channel current amplitude (i).

Single-channel recordings in HEK293 cells

HEK293 cells were transiently transfected using X-tremeGENE9 according to the

) / ) exp(( 1 ) ( 2 / 1 min max min k V V NPo NPo NPo V NPo     
where V is the membrane voltage, NPo min and NPo max are the minimum and maximum values of NPo, respectively, V 1/2 is the half-maximal activation voltage, and k is the slope factor of the voltage dependence. The liquid junction potentials were calculated with PClamp 9 and corrected accordingly.

Measurement of surface expression

Experiments were performed as previously described [28], using an anti-FLAG M2 monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) as the primary antibody, and a peroxidase-conjugated goat anti-mouse antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) as the secondary antibody. Chemiluminescence was quantified in a Turner TD-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA) by placing individual oocytes in 50 µl of SuperSignal Elisa Femto Maximum Sensitivity Substrate Solution (Pierce, Rockford, IL, USA).

Statistics

Results are shown as mean ± SEM. n indicates the number of experiments. Statistical significance was determined by a paired Student's t-test using SigmaStat software (SPSS, Erkrath, Germany). A value of P < 0.05 was considered to be significant.

RESULTS

Functional characterization of mClC-K1 channels at the whole-cell level in X. laevis oocytes

We expressed mouse ClC-K1 channels (mClC-K1) in X. laevis oocytes, with or without mouse Barttin (mBarttin), to characterize their conductive properties at the whole cell level. As previously reported for rat ClC-K1 (rClC-K1) [12,25,29], mClC-K1 alone induced small but significant currents, while co-expression with mBarttin markedly increased the current intensity (n = 30; Fig. 1A,B). In human Barttin, mutation of a putative prolinetyrosine (PY) motif (Y98A) further enhances hClC-K currents [2]. Likewise, mClC-K1 currents were also greater in oocytes expressing mBarttin mutated at the equivalent position (Y95A) than in oocytes expressing non-mutated Barttin (n = 30; Fig. 1A,B). mBarttin and Y95A mBarttin alone produced currents that were not different from those in non-injected oocytes (n = 30; Fig. 1A,B). There was a time-dependent increase in the mClC-K1 current at the highest positive voltages, and the corresponding current/voltage relationships showed outward rectification (Fig. 1B), which was reminiscent of hClC-Kb, rather than of hClC-Ka or rClC-K1, which display linear or slightly inwardly rectifying currents [2,12].

Using a chemiluminescence assay and taking advantage of the extracellular FLAG-tag placed on mClC-K1, we observed that mBarttin and Y95A mBarttin increased plasma membrane expression of ClC-K1 by ~177% (n = 30) and ~393% (n = 30), respectively (Fig. 2). Since surface expression correlated with channel currents (Fig. 2), the significant increase in current amplitudes of mClC-K1 channels could be ascribed to increased surface expression by mBarttin.

We then investigated the key electrophysiological properties of mClC-K1 in oocytes injected with the Y95A mBarttin mutant. We first assessed the selectivity among anions from current-voltage measurements when most of the Cl -in the bathing solution (80 mM) had been replaced by the test anion (Fig. 3A). The analysis of the data yielded P I-/P Cl-= 0.20 ± 0.05 (n = 35), P NO3-/P Cl-= 0.35 ± 0.18 (n = 11), and P Br-/P Cl-= 0.86 ± 0.14 (n = 15). This indicates a permeability sequence of Cl -> Br -> NO 3 -> I -, which is similar to that observed previously for rClC-K1, for hClC-Ka [2,12,25], and for the Cl -channel in the basolateral membrane of mouse CTAL [19].

Also consistently with previous results [2,12,25,30], we found that the currents elicited by mClC-K1/Y95A mBarttin channels decreased by 65 ± 1% (n = 6) in response to extracellular acidification (pH 6.0), and by 50 ± 1% (n = 6) in response to the removal of extracellular calcium (Fig. 3B). They increased by 55 ± 9% (n = 9) at pH 8.0 and by 76 ± 6%

(n = 6) in the presence of 5 mM of extracellular calcium (Fig. 3B). Similar results were obtained in the absence of mBarttin (Fig. 3B) (n = 11).

We investigated the effects of several classical Cl -channel blockers by adding them to the extracellular solution. At +60 mV, DIDS (100 µM) reduced mClC-K1/Y95A mBarttin currents by 53 ± 1% (n = 6) (not shown). Both DPC (1 mM) and NPPB (100 μM) blocked the channels to a lesser extent, reducing currents by 34 ± 1% (n = 6) and 42 ± 1% (n = 6), respectively (not shown). Qualitatively similar results have been obtained on rClC-K1 [25, 29 31], except for DPC, the effect of which is particularly tenuous as far as mClC-K1 is concerned.

Taking these results as a whole, mClC-K1 displays the major hallmarks of recombinant ClC-K1 channels, including Cl -> Br -> NO 3 -> I -anion selectivity, sensitivity to external pH and calcium, and increased expression at the membrane with Barttin. In addition, the anion selectivity sequence is identical to that found by Paulais et al. [19] for the 45-pS native Cl -channel, and slightly different from that found for the 9-pS channel [21,22,23].

Single-channel recordings of mClC-K1 channels in X. laevis oocytes

To investigate the single-channel properties of mClC-K1/mBarttin heterologously expressed in Xenopus laevis oocytes, we performed outside-out patch clamp experiments essentially as previously described [26,27]. Fig. 4A shows a representative single-channel current trace recorded at a holding potential of -70 mV. The corresponding binned current amplitude histogram is shown below the current trace. These data indicate that single-channel events with two different current amplitudes can be detected, as expected, for a doublebarrelled channel of the ClC ion channel family [32][33][34]. In similar recordings, single-channel current amplitude of events with "full" and "half" openings averaged -1.9 ± 0.08 pA (n = 11) and -0.98 ± 0.04 pA (n = 11), respectively.

Fig. 4B shows representative, single-channel, current traces recorded at different holding potentials in an outside-out patch from another oocyte also expressing mClC-K1/mBarttin channels. Fig. 4C shows averaged current/voltage relationships (n = 5-11) obtained from similar recordings as shown in Fig. 4B. These data demonstrate that mClC-K1/mBarttin channels has single-channel "full" and "half" conductances of 44.9 ± 1.4 pS (n = 12) and 23.9 ± 0.9 pS (n = 12), respectively. The anion selectivity of mClC-K1 expressed in oocytes, when assessed at the single-channel level, was compatible with that measured using voltage-clamp whole-cell recordings. This is illustrated by the representative single-channel current traces shown in Fig. 5 (holding potential: +50 mV). At this potential, with I -in the bath solution, no single-channel event was detected (Fig. 5A). With Br -in the bath, the "full"

and "half" single-channel current amplitudes (i Br-: 1.3 and 0.65 pA, respectively) were ~46% lower than the same currents in the presence of Cl -(i Cl-: 2.3 and 1.25 pA, respectively) (Fig. 5B). Similar observations were made in a total of 5 experiments.

The unit conductance of mClC-K1, as assessed in the oocyte expression system, is compatible with that of the native 45-pS Cl -channel [19].

Whole-cell recordings of mClC-K1 channels in HEK293 cells

Whole-cell current recordings were conducted in HEK293 cells, and revealed linear currents when the pipette was filled with 150 mM Cl -(n = 4) (Fig. 6A,B). The whole-cell currents were in the same range as the rat ClC-K1 currents recorded previously by Scholl et al. [13]. They were significantly increased by elevating the extracellular pH to 9.0 (n = 4), and were significantly reduced by the partial replacement of extracellular Cl -by I -(n = 4) [2,12,25,30]. In contrast to what is observed in the oocyte expression system, no time-dependent relaxation components were visible on the current traces (Fig. 6A). That the shape of the currents depended on the expression system had also been noted previously for ClC-Ka and ClC-Kb [4]. ClC-Ka also exhibits a linear current-voltage relationship with 150 mM Cl -in the pipette, while rClC-K1 is inwardly rectifying [13,14]. This difference might be attributable to the fact that rClC-K1, which is clearly voltage-dependent when transfected in cultured cells, is activated in response to hyperpolarization [13,14,35]. For mClC-K1, the current/voltage relationships (Fig. 6C) showed outward rectification in the presence of a low internal Cl - solution (35 mM) (n = 7).

These results suggest that HEK293 cells is a suitable model for studying mClC-K1

currents at the single-channel level.

Conductive properties of mClC-K1 channels at the single-channel level in HEK293 cells

Fig. 7A shows typical recordings of mClC-K1/mBarttin channel activity on cellattached membrane patches from HEK293-transfected cells bathed with physiological saline solution, with the pipette filled with a high-Cl -solution. Under these conditions, we observed highly active channels (Fig. 7A). It is apparent that most mClC-K1 channels displayed "full" opening when expressed in HEK293 cells. The current/voltage relationship displayed rather marked outward rectification, probably due to low [Cl -] i (Fig. 7B). The unit conductance of the "full" openings, measured in the linear part of the current-voltage relationship, averaged 41.9 ± 1.5 pS (n = 11). The reversal potential (-1.8 ± 1.2 mV, n =11) was close to zero. Channel activity increased at positive potentials (Fig. 7A). This was typically observed in 7 cell-attached patches, in which we investigated the voltage dependence of the NPo. The mean relationship between NPo and voltage is given in Fig. 7C. Fitting our data with a Boltzmann equation yielded a half-maximal activation voltage (V 1/2 ) of -65.6 mV. Voltage dependence was no longer observed in the inside-out configuration, suggesting that it is mediated by some intracellular factor (Fig. 7C).

To investigate the possible modulation of mClC-K1 channel gating and unit conductance by mBarttin [13,14], we performed single-channel analysis in cell-attached membrane patches using HEK293-transfected cells expressing mClC-K1 alone. Fig. 7B and These results, as a whole, show that the single-channel properties of mClC-K1/mBarttin are strikingly similar to those of the 45-pS native Cl -channel [19]. In particular, the open probability has values close to those measured in the mouse CTAL [19] when measured on HEK293 cells.

Conductive properties of V166E mClC-K1 mutant channels in HEK293 cells

We anticipated that introducing a glutamate at a position equivalent to 166 might create a protopore gate, making it easier to detect the openings of the protopores. In ClC-0, changes in the amino acid at the equivalent position 166 did not alter the protopore conductance [34]. In a first step, we performed whole-cell recordings in HEK293 cells using 150 mM Cl -in the pipette solution (Fig. 8). The V166E mutant displayed clear outward rectification under these conditions suggesting that the gating of the channel has been modified in agreement with our hypothesis.

Fig. 9A shows representative cell-attached current recordings from HEK-293 cells transfected with V166E mClC-K1/mBarttin channels. The mean current-voltage relationship was essentially linear (Fig. 8B) and reversed at -1.4 ± 2.1 mV; a unit conductance of 19.9 ± 1.3 pS was computed (n = 4). Channel activity increased at positive voltages (n = 4) (Fig. 8C).

The best fit of our data indicated that the half-maximal activation voltage of the mutant channel was shifted to more positive potentials as compared to WT mClC-K1/mBarttin (V 1/2 = 24.9 mV). Voltage dependence persisted in the cell-free mode (not shown). In contrast, two other point mutations introduced at position 166 did not have the same effect as V166E. The V166L mutation did not significantly modify the unit conductance (38.5 ± 4.0 pS, n = 3; not shown), and the V166M only moderately modified the unit conductance (38.5 and 29.5 pS, n = 2; not shown). We can therefore reasonably conclude that the V166E mutation revealed the activity of the channel protopore by introducing de novo protopore gating.

We also recorded single-channel activity from cell-attached patches from HEK-293 cells transfected with V166E mClC-K1 without mBarttin: there was no significant change in unit conductance (18.3 ± 0.7 pS; n = 4) (Fig. 9B), channel voltage dependence (n = 4) (Fig. 9C), or for half-maximal voltage activation (V 1/2 = 21.7 mV).

DISCUSSION

The apparent properties of mClC-K1 partly depend on the heterologous expression model

Fahlke & Fischer [4] have previously pointed out that there are clear kinetic and current/voltage relationships differences between ClC-K channels expressed in X. laevis oocytes and those expressed in mammalian cells. This was also observed for the mouse ClC-K1, but can be partly explained by the differing experimental conditions.

In HEK293 cells, mClC-K1/mBarttin yields linear currents with 150 mM Cl -in the pipette and outwardly rectifying currents in the presence of low Cl -(35 mM), a situation closer to that seen in the oocytes. Indeed, in X. laevis oocytes where the intracellular Cl -is around 30-50 mM, currents are outwardly rectifying, Furthermore, single-channel current amplitudes in the cell attached mode also showed outward rectification (low intracellular Cl - concentration). We can therefore assume that the discrepancy in the shapes of the current/voltage relationships obtained for whole-cell currents between heterologous expression models is due to normal, Goldman-type rectification, and is related to the level of intracellular chloride.

The time-dependent components that increase mClC-K1 currents at positive voltages in X. laevis oocytes suggest activation at depolarizing voltages, which was not observed in whole-cell recordings from HEK293 cells. However, the NP o of the mClC-K1 increased in response to depolarization in cell-attached patches from HEK293 cells, a condition in which the cell is intact (as for oocyte recordings). We may speculate that cell dialysis during wholecell recording removed some component necessary for voltage gating. Indeed, the voltagedependence that was observed in cell-attached patches also disappeared in inside-out patches.

In contrast to cell-attached recordings of HEK 293 cells, recordings from outside-out patches from oocytes always displayed very low open probability. A very low open probability has also been observed when recording ClC-Ka from cell-free patches obtained on oocytes [36]. In contrast, ClC-Ka single-channel activity recorded from patches attached to mammalian cultured cells is high (see for instance, [14]). It may be speculated that, for some unknown reason, the mClC-K1 channels experience rundown following excision. Overall, mClC-K1/Barttin expressed in HEK293 cells more faithfully reflects the native 45-pS Cl - channel.

The properties of mouse ClC-K1 are comparable to those of recombinant human ClC-Ka and rat ClC-K1

So far, only the electrophysiological properties of human and rat ClC-K channels have been investigated. Previous studies have demonstrated that recombinant hClC-Ka and rClC-K1 have identical properties at the whole-cell level in terms of anion selectivity, sensitivity to external pH and Ca 2+ , and sensitivity to chloride channels blockers as DIDS and NPPB [2,12,25,30,31,37]. However, rClC-K1 channels are active in the absence of Barttin;

whereas hClC-Ka channels need Barttin to become anion conductive at the cell surface [2,12,30]. Our findings provide evidence that mClC-K1 shares many of the properties of hClC-Ka and rClC-K1 at the whole cell level, including the anion selectivity sequence (Cl ->Br ->NO 3 ->I -), being activated at external basic pH or elevated Ca 2+ , and sensitivity to DIDS and NPPB.

We also observed that mClC-K1, like rClC-K1, is active without mBarttin. There are also small differences that concern the voltage dependence (activation by depolarization for mClC-K1, by hyperpolarization for rat ClC-K1) and unitary conductances (40 pS for mClC-K1, 33 pS for rClC-K1 and 26 pS for hClC-Ka) [13,14].

4.3.Single-channel activity in HEK293 cells reflects dimer gating of mClC-K1

ClC channels, which function as dimer complexes, are endowed with two voltage-dependent gates, the protopore ("fast") gate, and the common ("slow") gate [38]. Protopore gating requires the presence of a glutamate at equivalent position 166. Indeed, substituting glutamate for valine (or alanine) in ClC-0 abolishes most of the protopore gating, and only brief closures of the protopores are detected [34]. Since the ClC-K channels have a valine residue at position 166 rather than a glutamate, it may be anticipated that their voltage dependence is due to common gating. Accordingly, the experiments we performed in the cell-attached mode in HEK293 transfected cells showed that half openings and closings of mClC-K1 due to protopore gating were rarely detected in these recordings. As a consequence, the NP o values found in these experiments essentially reflect the activity of the common gate.

Thus, the common gate of mClC-K1 channels is moderately activated by

depolarization. It is noteworthy that the half-maximal activation voltage V 1/2 (~ -65 mV),
which is close to the resting membrane potential in renal cells, suggests that this regulation might be physiologically relevant. In sharp contrast, rClC-K1 channels are activated by hyperpolarization [13,14]. In this case, the voltage dependence is due to the fast gate, the slow gate being voltage-independent [14]. This may indicate that ClC-K regulation differs in different species. It could also indicate that the protopore gate, which according to Fahlke et al. [13,14] is activated by hyperpolarization, might contribute to the total open probability, notably under physiological circumstances that were not encountered in our experiments.

Barttin has no influence on ClC-K1 channel gating or conductance

Barttin is an accessory regulatory subunit that is essential for the functional expression of hClC-Ka and hClC-Kb, and which also increases the current generated by rClC-K1 [14].

We also observed that mClC-K1 currents were greater in the presence of Barttin. However, mClC-K1, like rClC-K1, carried currents in the absence of Barttin when expressed in heterologous expression systems. Furthermore, we observed that mClC-K1 currents were still stimulated by external calcium and inhibited by protons in the absence of Barttin.

Consequently, this must be an intrinsic property of the ClC-K, as has been reported

elsewhere [12].

Barttin also modulates ClC-K gating [14]. Fahlke et al. initially reported that rClC-K1 was activated by hyperpolarization in both the presence and the absence of Barttin [13]. Later on, however, they reported that the voltage dependence of rClC-K1 expressed alone was not voltage dependent [14]. In the absence of Barttin, V166E rClC-K1 channels were activated by membrane hyperpolarization, whereas in the presence of Barttin, mutant ClC-K1 channels were activated by membrane depolarization [13,14]. In the present study, the voltage dependence of wild-type and V166E mClC-K1 channels was not modified in the absence of Barttin.

Thus, the main effect of Barttin is to increase the number of mClC-K1 channels in the plasma membrane. However, the fact that mClC-K1, like rClC-K1, is active in the absence of Barttin raises questions about the physiological role of this accessory subunit. The observation that Barttin-knockout mice suffer from severe renal salt and water loss, and die within a few days after birth demonstrates that Barttin is strictly necessary for ClC-K function in mouse kidneys [39]. There are two possible explanations. First, in the absence of Barttin, the expression of ClC-K1 at the plasma membrane is insufficient to sustain sufficient chloride transport. A possible alternative explanation, although the two are not mutually exclusive, could be that Barttin is required for ClC-K1 to be addressed specifically to the basolateral membrane. Indeed, some mutations of BSND result in the increased expression of ClC-Kb at the apical membrane [35].

mClC-K1underlies the native 45-pS chloride channel

Several chloride channels have been identified in the basolateral membranes of microdissected mouse renal tubules by means of single-channel recordings.

The first Cl -channel we reported in mouse CTAL had a unit conductance of 45 pS, and a permeability sequence Cl -> Br -> NO 3 - [19]. No current was recorded in the presence of I -, suggesting that this anion has a blocking effect. Importantly, this channel was activated by depolarization, a property that disappeared after excision of the membrane patch.

A second Cl -channel has been detected throughout the length of the distal nephron [21][22][23]. It has a conductance of 9 pS. We have previously suggested that it is underpinned by ClC-K2 on the basis of its anion selectivity sequence and the fact that it is distributed throughout the renal tubule [24]. This remains to be directly proved, since recombinant ClC-K2 has not so far been studied at the single-channel level. Finally, we should mention that a third Cl -channel has been reported, the properties of which are incompatible with those of the ClC-Ks [20,24]

Physiological roles of ClC-K1

Initially, ClC-K1 expression was thought to be restricted to the thin ascending limb [30], where it provides a transcellular route for Cl -absorption, while Na + follows via the paracellular pathway [40]. Due to this strategic localization, ClC-K1 was believed to play a major role in urine concentration [9,40].

The current view, however, proposes that ClC-K1 is more widely distributed along the renal tubule than previously thought [2,3,12]. In particular, it is also present in the MTAL and CTAL, where we have detected its single-channel properties [19]. This segment absorbs 25% of filtered NaCl by the operation of a Na + -K + -2Cl -cotransporter at the apical membrane, and the presence of Na + -K + ATPase at the basolateral membrane. Cl -conductance is necessary for the basolateral step of Cl -absorption. ClC-K1 probably plays only a minor role, since Akzuki et al. [41] showed that there was no sodium loss in ClC-K1 -/-mice. Accordingly, two additional Cl -channels are thought to be expressed in the CTAL [24], which may compensate for the absence of ClC-K1. and Br -(i Cl-and i Br-, respectively) were determined as described in Fig. 4. No detectable single-channel current events were observed in the trace when NaI was present in the bath.

KEYWORDS

Chloride channel, ClC, kidney, ClC-K1, patch-clamp

INTRODUCTION

Two Cl -channels of the ClC family, ClC-K1 and ClC-K2 (or their human orthologs ClC-Ka and ClC-Kb, respectively), play a pivotal role in transcellular Cl -absorption in the kidney. Located on the basolateral membrane, they are present throughout the distal nephron [1][2][3], but the specific distribution of each ClC-K is still uncertain owing to the lack of isoform-specific antibodies [4]. Mutations in human genes coding for ClC-Kb and the regulatory subunit, Barttin, are responsible for salt-losing tubulopathies, namely Bartter's syndrome types 3 and 4, respectively [5][6][7]. Rare types of Bartter's syndrome can also result from compound mutations in ClC-Kb and ClC-Ka [8]. Renal function has not been investigated in knockout mice for ClC-K2 because they die very early; ClC-K1-knockout mice display impaired urine concentration and nephrogenic diabetes insipidus [9]. In accordance with pathological and physiological data, localization studies suggest that ClC-K2 is highly expressed in the cortical part of the distal nephron, and that ClC-K1 is mostly restricted to the medullary part of the renal tubule [2,3,10,11].

The functional expression of human and rat ClC-K remained controversial until their accessory subunit, Barttin, was identified. It promotes their insertion into the plasma membrane [2,12]. This discovery made it possible to identify two fundamental properties of ClC-K channels. First, the relative permeability for anions follows the sequence Cl -> Br -> NO 3 -> I -for ClC-Ka, and Cl -> Br -= NO 3 -> I -for ClC-Kb; second, the currents are enhanced by a basic external pH and high external calcium concentrations [2,12]. Interestingly, a glutamate residue that plays a critical role in the protopore gating of other ClC chloride channels is missing in ClC-K channels, where it is replaced by a valine. For this reason, it has been suggested that the two protopores in ClC-K dimers are constitutively open. Thus, the common gate (involving both subunits of the dimers) may act as the major regulator of ClC-K channels. In recent studies on rat ClC-K1, Fahlke's group reintroduced the "gating" glutamate and observed profound effects on both conductance and gating [4,13,14].

We and others have identified several basolateral chloride channels in different parts of the renal tubule, particularly in the cortical thick ascending limb (CTAL), the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD) [15][16][17][18][19][20][21][22][23]. Two of these channels, with conductances of 45 pS and 9 pS, respectively, display properties that are compatible with those of ClC-K chloride channels [24].

Nevertheless, the precise molecular identity of these channels remains elusive, because heterologous expression data are incomplete and heterogeneous. Studies of recombinant channels have involved rat and human ClC-Ks, whereas most of those of the native renal tubule have involved mouse kidney. Furthermore, there are only a few single-channel recordings available for recombinant channels, and they all derive from studies on human ClC-Ka and rat ClC-K1 [4]. In this study, we investigated the properties of recombinant mouse ClC-K1 channels expressed in the presence or absence of mouse Barttin, in an attempt to establish a coherent basis for their correspondence with native chloride channels in the mouse renal tubule.

MATERIAL AND METHODS

Mice

The animals used in this study were handled in full compliance with the French government welfare policy. This work was performed under Permit No 75-1416 from the Veterinary Department of the French Ministry of Agriculture.

Molecular biology

The mouse ClC-K1 cDNA clone (image 5042736, Invitrogen, Carlsbad, CA, USA)

and mouse Barttin (a generous gift from Prof. T. J. Jentsch, MDC/FMP, Berlin, Germany)

were subcloned into pSP64-S and pVITRO2 vectors, respectively, for expression in Xenopus laevis oocytes. Capped cRNAs were synthesized in vitro from wild-type and mutant ClC-K1 and Barttin vectors using the SP6 mMessage mMachine Kit (Ambion, Austin, TX, USA). To evaluate the surface expression of ClC-K1 in Xenopus laevis oocytes, we externally tagged ClC-K1 by introducing a FLAG epitope (DYKDDDDK) between transmembrane domains L and M (beginning at position 377). This did not interfere with their ability to generate currents.

For electrophysiological recordings, HEK293 cells were cotransfected with 1 µg of a bicistronic vector pVITRO2 containing the coding sequence of mouse ClC-K1 and mouse Barttin, and 0.3 µg of a IRES vector encoding the CD8 antigen (a generous gift from Dr. J.

Barhanin, Université Nice Sophia Antipolis, Nice, France) as a transfection marker by electroporation or using X-tremeGENE9 (Roche Diagnostics, Meylan, France). Including CD8 made it possible to identify the transfected cells using anti-CD8-coated beads (Invitrogen Dynal, Oslo, Norway). Site-directed mutagenesis was performed with the Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA).

All constructs were fully sequenced.

Two-electrode voltage-clamp

Defolliculated Xenopus laevis oocytes were injected with 10 ng of ClC-K1 cRNA and voltage protocol consisting of 20 mV steps from -160 mV to +100 mV with a duration of 800 ms from a holding potential of -30 mV. The data were filtered at 300 Hz, and sampled using a Digidata 1320A analogue-to-digital converter (Molecular Devices, Sunnyvale, CA).

For anion replacement, 80 mM Cl -was replaced by equivalent amounts of Br -, I -or NO 3 -. HEPES was replaced by equimolar MES for solutions at pH 5.4-6.4, or by Tris for solutions at pH 8.4. Where appropriate, calcium acetate was added to the ND96 solution to increase the extracellular calcium concentration. The permeability ratios of various anions (X -) relative to that of Cl -(P X-/P Cl-) were calculated using the modified Goldman-Hodgkin-Katz equation [25]:

80 ) 104 ( 24 ) ( 40 2 1        E E Cl X e P P
Where E1 and E2 are the reversal potentials for Cl -and X -, respectively. Chloride channel inhibitors were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). DPC, at a concentration of 0.5 M, and NPPB, at a concentration of 0.1 M, were dissolved in DMSO. DMSO (at the maximum concentration of 0.2% used for 10 -3 M DPC) had no effect on channel activity. following pulse protocol was used: from a holding potential of -30 mV, after a pre-pulse to +65 mV for 30 ms, voltage was stepped from -115 mV to +105 mV in 20 mV increments for 160 ms, and followed by a pulse to +105 mV.

Whole-cell recording

Single-channel recordings in outside-out patches of Xenopus laevis oocytes

Recordings in outside-out membrane patches of mClC-K1 and mBarttin expressing oocytes were performed essentially as described previously using a conventional patch-clamp technique [26,27]. Patch pipettes were pulled from borosilicate glass capillaries, and had a tip

Single-channel recordings in HEK293 cells and OMCD tubules

HEK293 cells were transiently transfected using X-tremeGENE9 according to the Recordings on mouse OMCD were done essentially as previously described [23]. Briefly, OMCD segments were microdissected from collagenase-treated kidneys, transferred into a Petri dish, and bathed in physiological saline.

The pipettes made from borosilicate tubes (GC150T, Harvard Apparatus, Edenbridge, Kent) were coated with Sylgard (Dow Corning, Seneffe, Belgium), and polished just before use. The pipettes were filled with a high-Cl -solution containing (in mM): 150 NMDG-Cl, 1

MgCl2, 10 glucose, and 10 HEPES, pH 7.4. The solutions used to characterize channel properties in the inside-out configuration contained 2 mM EGTA and no calcium. To record the currents, we used a RK 400 patch-clamp amplifier (Bio-logic, Claix, France) and PClamp 9 software (Molecular Devices, Sunnyvale, CA). Currents were filtered at 300 or 500 Hz, and were sampled at 1-2 kHz using a Digidata 1322A analogue-to-digital converter (Molecular Devices, Sunnyvale, CA). The mean current (I) passing through N channels was used to calculate the normalized current (NPo) according to the equation NPo=I/i, where i is the unitary current amplitude. The relationship between NPo and voltage (V) was fitted using a Boltzmann equation:

) / ) exp(( 1 ) ( 2 / 1 min max min k V V NPo NPo NPo V NPo     
where V is the membrane voltage, NPo min and NPo max are the minimum and maximum values of NPo, respectively, V 1/2 is the half-maximal activation voltage, and k is the slope factor of the voltage dependence. The liquid junction potentials were calculated with PClamp 9 and corrected accordingly.

Measurement of surface expression

Experiments were performed as previously described [28], using an anti-FLAG M2 monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) as the primary antibody, and a peroxidase-conjugated goat anti-mouse antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) as the secondary antibody. Chemiluminescence was quantified in a Turner TD-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA) by placing individual oocytes in 50 µl of SuperSignal Elisa Femto Maximum Sensitivity Substrate Solution (Pierce, Rockford, IL, USA).

Statistics

Results are shown as mean ± SEM. n indicates the number of experiments. Statistical significance was determined by a paired Student's t-test using SigmaStat software (SPSS, Erkrath, Germany). A value of P < 0.05 was considered to be significant.

RESULTS

Functional characterization of mClC-K1 channels at the whole-cell level in X. laevis oocytes

We expressed mouse ClC-K1 channels (mClC-K1) in X. laevis oocytes, with or without mouse Barttin (mBarttin), to characterize their conductive properties at the whole cell level. As previously reported for rat ClC-K1 (rClC-K1) [12,25,29], mClC-K1 alone induced small but significant currents, while co-expression with mBarttin markedly increased the current intensity (n = 30; Fig. 1A,B). In human Barttin, mutation of a putative prolinetyrosine (PY) motif (Y98A) further enhances hClC-K currents [2]. Likewise, mClC-K1 currents were also greater in oocytes expressing mBarttin mutated at the equivalent position (Y95A) than in oocytes expressing non-mutated Barttin (n = 30; Fig. 1A,B). mBarttin and Y95A mBarttin alone produced currents that were not different from those in non-injected oocytes (n = 30; Fig. 1A,B). There was a time-dependent increase in the mClC-K1 current at the highest positive voltages, and the corresponding current/voltage relationships showed outward rectification (Fig. 1B), which was reminiscent of hClC-Kb, rather than of hClC-Ka or rClC-K1, which display linear or slightly inwardly rectifying currents [2,12].

Using a chemiluminescence assay and taking advantage of the extracellular FLAG-tag placed on mClC-K1, we observed that mBarttin and Y95A mBarttin increased plasma membrane expression of ClC-K1 by ~177% (n = 30) and ~393% (n = 30), respectively (Fig. 2). Since surface expression correlated with channel currents (Fig. 2), the significant increase in current amplitudes of mClC-K1 channels could be ascribed to increased surface expression by mBarttin.

We then investigated the key electrophysiological properties of mClC-K1 in oocytes injected with the Y95A mBarttin mutant. We first assessed the selectivity among anions from current-voltage measurements when most of the Cl -in the bathing solution (80 mM) had been replaced by the test anion (Fig. 3A). The analysis of the data yielded P I-/P Cl-= 0.20 ± 0.05 (n = 35), P NO3-/P Cl-= 0.35 ± 0.18 (n = 11), and P Br-/P Cl-= 0.86 ± 0.14 (n = 15). This indicates a permeability sequence of Cl -> Br -> NO 3 -> I -, which is similar to that observed previously for rClC-K1, for hClC-Ka [2,12,25], and for the Cl -channel in the basolateral membrane of mouse CTAL [19].

Also consistently with previous results [2,12,25,30], we found that the currents elicited by mClC-K1/Y95A mBarttin channels decreased by 65 ± 1% (n = 6) in response to extracellular acidification (pH 6.0), and by 50 ± 1% (n = 6) in response to the removal of extracellular calcium (Fig. 3B). They increased by 55 ± 9% (n = 9) at pH 8.0 and by 76 ± 6%

(n = 6) in the presence of 5 mM of extracellular calcium (Fig. 3B). Similar results were obtained in the absence of mBarttin (Fig. 3B) (n = 11).

We investigated the effects of several classical Cl -channel blockers by adding them to the extracellular solution. At +60 mV, DIDS (100 µM) reduced mClC-K1/Y95A mBarttin currents by 53 ± 1% (n = 6) (not shown). Both DPC (1 mM) and NPPB (100 μM) blocked the channels to a lesser extent, reducing currents by 34 ± 1% (n = 6) and 42 ± 1% (n = 6), respectively (not shown). Qualitatively similar results have been obtained on rClC-K1 [25, 29 31], except for DPC, the effect of which is particularly tenuous as far as mClC-K1 is concerned.

Taking these results as a whole, mClC-K1 displays the major hallmarks of recombinant ClC-K1 channels, including Cl -> Br -> NO 3 -> I -anion selectivity, sensitivity to external pH and calcium, and increased expression at the membrane with Barttin. In addition, the anion selectivity sequence is identical to that found by Paulais et al. [19] for the 45-pS native Cl -channel, and slightly different from that found for the 9-pS channel [21,22,23].

Single-channel recordings of mClC-K1 channels in X. laevis oocytes

To investigate the single-channel properties of mClC-K1/mBarttin heterologously expressed in Xenopus laevis oocytes, we performed outside-out patch clamp experiments essentially as previously described [26,27]. Fig. 4A shows a representative single-channel current trace recorded at a holding potential of -70 mV. The corresponding binned current amplitude histogram is shown below the current trace. These data indicate that single-channel events with two different current amplitudes can be detected, as expected, for a doublebarrelled channel of the ClC ion channel family [32][33][34]. In similar recordings, single-channel current amplitude of events with "full" and "half" openings averaged -1.9 ± 0.08 pA (n = 11) and -0.98 ± 0.04 pA (n = 11), respectively.

Fig. 4B shows representative, single-channel, current traces recorded at different holding potentials in an outside-out patch from another oocyte also expressing mClC-K1/mBarttin channels. Fig. 4C shows averaged current/voltage relationships (n = 5-11) obtained from similar recordings as shown in Fig. 4B. These data demonstrate that mClC-K1/mBarttin channels has single-channel "full" and "half" conductances of 44.9 ± 1.4 pS (n = 12) and 23.9 ± 0.9 pS (n = 12), respectively. The anion selectivity of mClC-K1 expressed in oocytes, when assessed at the single-channel level, was compatible with that measured using voltage-clamp whole-cell recordings. This is illustrated by the representative single-channel current traces shown in Fig. 5 (holding potential: +50 mV). At this potential, with I -in the bath solution, no single-channel event was detected (Fig. 5A). With Br -in the bath, the "full"

and "half" single-channel current amplitudes (i Br-: 1.3 and 0.65 pA, respectively) were ~46% lower than the same currents in the presence of Cl -(i Cl-: 2.3 and 1.25 pA, respectively) (Fig. 5B). Similar observations were made in a total of 5 experiments.

The unit conductance of mClC-K1, as assessed in the oocyte expression system, is compatible with that of the native 45-pS Cl -channel [19].

Whole-cell recordings of mClC-K1 channels in HEK293 cells

Whole-cell current recordings were conducted in HEK293 cells, and revealed linear currents when the pipette was filled with 150 mM Cl -(n = 4) (Fig. 6A,B). The whole-cell currents were in the same range as the rat ClC-K1 currents recorded previously by Scholl et al. [13]. They were significantly increased by elevating the extracellular pH to 9.0 (n = 4), and were significantly reduced by the partial replacement of extracellular Cl -by I -(n = 4) [2,12,25,30]. In contrast to what is observed in the oocyte expression system, no time-dependent relaxation components were visible on the current traces (Fig. 6A). That the shape of the currents depended on the expression system had also been noted previously for ClC-Ka and ClC-Kb [4]. ClC-Ka also exhibits a linear current-voltage relationship with 150 mM Cl -in the pipette, while rClC-K1 is inwardly rectifying [13,14]. This difference might be attributable to the fact that rClC-K1, which is clearly voltage-dependent when transfected in cultured cells, is activated in response to hyperpolarization [13,14,35]. For mClC-K1, the current/voltage relationships (Fig. 6C) showed outward rectification in the presence of a low internal Cl - solution (35 mM) (n = 7).

These results suggest that HEK293 cells is a suitable model for studying mClC-K1 currents at the single-channel level.

Conductive properties of mClC-K1 channels at the single-channel level in HEK293 cells

Fig. 7A shows typical recordings of mClC-K1/mBarttin channel activity on cellattached membrane patches from HEK293-transfected cells bathed with physiological saline solution, with the pipette filled with a high-Cl -solution. Under these conditions, we observed highly active channels (Fig. 7A). It is apparent that most mClC-K1 channels displayed "full" opening when expressed in HEK293 cells. The current/voltage relationship displayed rather marked outward rectification, probably due to low [Cl -] i (Fig. 7B). The unit conductance of the "full" openings, measured in the linear part of the current-voltage relationship, averaged 41.9 ± 1.5 pS (n = 11). The reversal potential (-1.8 ± 1.2 mV, n =11) was close to zero. Channel activity increased at positive potentials (Fig. 7A). This was typically observed in 7 cell-attached patches, in which we investigated the voltage dependence of the NPo. The mean relationship between NPo and voltage is given in Fig. 7C. Fitting our data with a Boltzmann equation yielded a half-maximal activation voltage (V 1/2 ) of -65.6 mV. Voltage dependence was no longer observed in the inside-out configuration, suggesting that it is mediated by some intracellular factor (Fig. 7C).

To investigate the possible modulation of mClC-K1 channel gating and unit conductance by mBarttin [13,14], we performed single-channel analysis in cell-attached membrane patches using HEK293-transfected cells expressing mClC-K1 alone. Fig. 7B and These results, as a whole, show that the single-channel properties of mClC-K1/mBarttin are strikingly similar to those of the 45-pS native Cl -channel [19]. In particular, the open probability has values close to those measured in the mouse CTAL [19] when measured on HEK293 cells.

Conductive properties of V166E mClC-K1 mutant channels in HEK293 cells

We anticipated that introducing a glutamate at a position equivalent to 166 might create a protopore gate, making it easier to detect the openings of the protopores. In ClC-0, changes in the amino acid at the equivalent position 166 did not alter the protopore conductance [34]. In a first step, we performed whole-cell recordings in HEK293 cells using 150 mM Cl -in the pipette solution (Fig. 8). The V166E mutant displayed clear outward rectification under these conditions suggesting that the gating of the channel has been modified in agreement with our hypothesis.

Fig. 9A shows representative cell-attached current recordings from HEK-293 cells transfected with V166E mClC-K1/mBarttin channels. The mean current-voltage relationship was essentially linear (Fig. 8B) and reversed at -1.4 ± 2.1 mV; a unit conductance of 19.9 ± 1.3 pS was computed (n = 4). Channel activity increased at positive voltages (n = 4) (Fig. 8C).

The best fit of our data indicated that the half-maximal activation voltage of the mutant channel was shifted to more positive potentials as compared to WT mClC-K1/mBarttin (V 1/2 = 24.9 mV). Voltage dependence persisted in the cell-free mode (not shown). In contrast, two other point mutations introduced at position 166 did not have the same effect as V166E. The V166L mutation did not significantly modify the unit conductance (38.5 ± 4.0 pS, n = 3; not shown), and the V166M only moderately modified the unit conductance (38.5 and 29.5 pS, n = 2; not shown). We can therefore reasonably conclude that the V166E mutation revealed the activity of the channel protopore by introducing de novo protopore gating.

We also recorded single-channel activity from cell-attached patches from HEK-293 cells transfected with V166E mClC-K1 without mBarttin: there was no significant change in unit conductance (18.3 ± 0.7 pS; n = 4) (Fig. 9B), channel voltage dependence (n = 4) (Fig. 9C), or for half-maximal voltage activation (V 1/2 = 21.7 mV).

There is a similar channel in the basolateral membranes of intercalated cells of mouse OMCD

Previous studies have characterized a chloride channel with properties similar to mClC-K1 in the basolateral membrane of mouse CTAL [19], but not in other parts of the cortical distal nephron [24]. Since immunofluorescence studies have indicated that Barttin and ClC-K1/ClC-K2 channels were present downwards in the medulla [1,2,32], we searched for Cl -channels in the intercalated cells of the outer medullary collecting duct. Using NMDG-Cl - solutions in the pipette, we were able to record a small channel with properties similar to those previously reported for the 9 pS chloride channel [21][22][23] (not shown) together with a higher-conductance channel. Representative current traces recorded at different holding potentials are shown in Fig. 9A. The unit conductance measured on cell-attached patches was 37.4 ± 4.2 pS (n = 7) (Fig. 9B). The activity of the channel was dependent on the external calcium concentration: we recorded 1.8 ± 0.2 channels per patch in the presence of 1 mM Ca 2+ (n =5) and 6.7 ± 0.9 in the presence of 5 mM Ca 2+ (n = 3, p< 0.05) in the pipette. We were unable to record any channel activity in the cell-free configuration.

DISCUSSION

The apparent properties of mClC-K1 partly depend on the heterologous expression model

Fahlke & Fischer [4] have previously pointed out that there are clear kinetic and current/voltage relationships differences between ClC-K channels expressed in X. laevis oocytes and those expressed in mammalian cells. This was also observed for the mouse ClC-K1, but can be partly explained by the differing experimental conditions.

In HEK293 cells, mClC-K1/mBarttin yields linear currents with 150 mM Cl -in the pipette and outwardly rectifying currents in the presence of low Cl -(35 mM), a situation closer to that seen in the oocytes. Indeed, in X. laevis oocytes where the intracellular Cl -is around 30-50 mM, currents are outwardly rectifying, Furthermore, single-channel current amplitudes in the cell attached mode also showed outward rectification (low intracellular Cl - concentration). We can therefore assume that the discrepancy in the shapes of the current/voltage relationships obtained for whole-cell currents between heterologous expression models is due to normal, Goldman-type rectification, and is related to the level of intracellular chloride.

The time-dependent components that increase mClC-K1 currents at positive voltages in X. laevis oocytes suggest activation at depolarizing voltages, which was not observed in whole-cell recordings from HEK293 cells. However, the NP o of the mClC-K1 increased in response to depolarization in cell-attached patches from HEK293 cells, a condition in which the cell is intact (as for oocyte recordings). We may speculate that cell dialysis during wholecell recording removed some component necessary for voltage gating. Indeed, the voltagedependence that was observed in cell-attached patches also disappeared in inside-out patches.

In contrast to cell-attached recordings of HEK 293 cells, recordings from outside-out patches from oocytes always displayed very low open probability. A very low open probability has also been observed when recording ClC-Ka from cell-free patches obtained on oocytes [36]. In contrast, ClC-Ka single-channel activity recorded from patches attached to mammalian cultured cells is high (see for instance, [14]). It may be speculated that, for some unknown reason, the mClC-K1 channels experience rundown following excision. Overall, mClC-K1/Barttin expressed in HEK293 cells more faithfully reflects the native 45-pS Cl - channel.

The properties of mouse ClC-K1 are comparable to those of recombinant human ClC-Ka and rat ClC-K1

So far, only the electrophysiological properties of human and rat ClC-K channels have been investigated. Previous studies have demonstrated that recombinant hClC-Ka and rClC-K1 have identical properties at the whole-cell level in terms of anion selectivity, sensitivity to external pH and Ca 2+ , and sensitivity to chloride channels blockers as DIDS and NPPB [2,12,25,30,31,37]. However, rClC-K1 channels are active in the absence of Barttin;

whereas hClC-Ka channels need Barttin to become anion conductive at the cell surface [2,12,30]. Our findings provide evidence that mClC-K1 shares many of the properties of hClC-Ka and rClC-K1 at the whole cell level, including the anion selectivity sequence (Cl ->Br ->NO 3 ->I -), being activated at external basic pH or elevated Ca 2+ , and sensitivity to DIDS and NPPB.

We also observed that mClC-K1, like rClC-K1, is active without mBarttin. There are also small differences that concern the voltage dependence (activation by depolarization for mClC-K1, by hyperpolarization for rat ClC-K1) and unitary conductances (40 pS for mClC-K1, 33 pS for rClC-K1 and 26 pS for hClC-Ka) [13,14].

4.3.Single-channel activity in HEK293 cells reflects dimer gating of mClC-K1

ClC channels, which function as dimer complexes, are endowed with two voltage-dependent gates, the protopore ("fast") gate, and the common ("slow") gate [38]. Protopore gating requires the presence of a glutamate at equivalent position 166. Indeed, substituting glutamate for valine (or alanine) in ClC-0 abolishes most of the protopore gating, and only brief closures of the protopores are detected [34]. Since the ClC-K channels have a valine residue at position 166 rather than a glutamate, it may be anticipated that their voltage dependence is due to common gating. Accordingly, the experiments we performed in the cell-attached mode in HEK293 transfected cells showed that half openings and closings of mClC-K1 due to protopore gating were rarely detected in these recordings. As a consequence, the NP o values found in these experiments essentially reflect the activity of the common gate.

Thus, the common gate of mClC-K1 channels is moderately activated by depolarization. It is noteworthy that the half-maximal activation voltage V 1/2 (~ -65 mV), which is close to the resting membrane potential in renal cells, suggests that this regulation might be physiologically relevant. In sharp contrast, rClC-K1 channels are activated by hyperpolarization [13,14]. In this case, the voltage dependence is due to the fast gate, the slow gate being voltage-independent [14]. This may indicate that ClC-K regulation differs in different species. It could also indicate that the protopore gate, which according to Fahlke et al. [13,14] is activated by hyperpolarization, might contribute to the total open probability, notably under physiological circumstances that were not encountered in our experiments.

Barttin has no influence on ClC-K1 channel gating or conductance

Barttin is an accessory regulatory subunit that is essential for the functional expression of hClC-Ka and hClC-Kb, and which also increases the current generated by rClC-K1 [14].

We also observed that mClC-K1 currents were greater in the presence of Barttin. However, mClC-K1, like rClC-K1, carried currents in the absence of Barttin when expressed in heterologous expression systems. Furthermore, we observed that mClC-K1 currents were still stimulated by external calcium and inhibited by protons in the absence of Barttin.

Consequently, this must be an intrinsic property of the ClC-K, as has been reported

elsewhere [12].

Barttin also modulates ClC-K gating [14]. Fahlke et al. initially reported that rClC-K1 was activated by hyperpolarization in both the presence and the absence of Barttin [13]. Later on, however, they reported that the voltage dependence of rClC-K1 expressed alone was not voltage dependent [14]. In the absence of Barttin, V166E rClC-K1 channels were activated by membrane hyperpolarization, whereas in the presence of Barttin, mutant ClC-K1 channels were activated by membrane depolarization [13,14]. In the present study, the voltage dependence of wild-type and V166E mClC-K1 channels was not modified in the absence of Barttin.

Thus, the main effect of Barttin is to increase the number of mClC-K1 channels in the plasma membrane. However, the fact that mClC-K1, like rClC-K1, is active in the absence of Barttin raises questions about the physiological role of this accessory subunit. The observation that Barttin-knockout mice suffer from severe renal salt and water loss, and die within a few days after birth demonstrates that Barttin is strictly necessary for ClC-K function in mouse kidneys [39]. There are two possible explanations. First, in the absence of Barttin, the expression of ClC-K1 at the plasma membrane is insufficient to sustain sufficient chloride transport. A possible alternative explanation, although the two are not mutually exclusive, could be that Barttin is required for ClC-K1 to be addressed specifically to the basolateral membrane. Indeed, some mutations of BSND result in the increased expression of ClC-Kb at the apical membrane [35].

Comparison with native Cl -channels in the mouse renal tubule mClC-K1underlies the native 45-pS chloride channel

Several chloride channels have been identified in the basolateral membranes of microdissected mouse renal tubules by means of single-channel recordings.

The first Cl -channel we reported in mouse CTAL had a unit conductance of 45 pS, and a permeability sequence Cl -> Br -> NO 3 - [19]. No current was recorded in the presence of I -, suggesting that this anion has a blocking effect. Importantly, this channel was activated by depolarization, a property that disappeared after excision of the membrane patch. All these properties are similar to those of mClC-K1. Thus, the 45 pS chloride channel that we have characterized in the basolateral membrane of mouse CTAL most likely corresponds to mClC-K1. In the present study, we also recorded a similar channel in the intercalated cells of the mouse OMCD, and showed that the activity of the channel was stimulated by external calcium.

A second Cl -channel has been detected throughout the length of the distal nephron [21][22][23]. It has a conductance of 9 pS. We have previously suggested that it is underpinned by ClC-K2 on the basis of its anion selectivity sequence and the fact that it is distributed throughout the renal tubule [24]. This remains to be directly proved, since recombinant ClC-K2 has not so far been studied at the single-channel level. Finally, we should mention that a third Cl -channel has been reported, the properties of which are incompatible with those of the ClC-Ks [20,24]

Physiological roles of ClC-K1

Initially, ClC-K1 expression was thought to be restricted to the thin ascending limb [30], where it provides a transcellular route for Cl -absorption, while Na + follows via the paracellular pathway [40]. Due to this strategic localization, ClC-K1 was believed to play a major role in urine concentration [9,40].

The current view, however, proposes that ClC-K1 is more widely distributed along the renal tubule than previously thought [2,3,12]. In particular, it is also present in the MTAL and CTAL, where we have detected its single-channel properties [19]. This segment absorbs 25% of filtered NaCl by the operation of a Na + -K + -2Cl -cotransporter at the apical membrane, and the presence of Na + -K + ATPase at the basolateral membrane. Cl -conductance is necessary for the basolateral step of Cl -absorption. ClC-K1 probably plays only a minor role, since Akzuki et al. [41] showed that there was no sodium loss in ClC-K1 -/-mice. Accordingly, two additional Cl -channels are thought to be expressed in the CTAL [24], which may compensate for the absence of ClC-K1. and Br -(i Cl-and i Br-, respectively) were determined as described in Fig. 4. No detectable single-channel current events were observed in the trace when NaI was present in the bath.

  HEK293 cells were transiently transfected by electroporation, and plated on 35-mm plastic Petri culture dishes. 20 h after transfection, the Petri dishes were mounted on the inverted stage of a microscope. The bath solution contained (in mM): 140 NaCl, 2 CaCl 2 , 1 MgCl 2 , 22 Sucrose, 10 HEPES, pH 7.4. HEPES was replaced by TRIS for solutions at pH 9.0. For the chloride substitution experiments, 140 NaCl was replaced by 140 NaI. The pipette solution contained (in mM): 150 NMDG-Cl, 1 MgCl 2 , 2 EGTA, 1 Mg-ATP, 10 HEPES, pH 7.4 or 33 CsCl, 100 NaGluconate, 1 MgCl 2 2 EGTA, 1 Mg-ATP, and 10 HEPES, pH 7.0. Standard whole-cell recordings were performed using an Axopatch 200B (Molecular Devices, Sunnyvale, CA) and PClamp 9 software (Molecular Devices, Sunnyvale, CA). The bath was earthed via an agar 3 M agar bridge. Patch-clamp pipettes had resistances of 2-3 MΩ. The

  Manufacturer's instructions (Roche Diagnostics, Meylan, France). Recordings were performed two days after transfection. The cells were initially bathed in physiological saline containing (in mM): 140 NaCl, 5 KCl, 1 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES, pH 7.4. The pipettes made from borosilicate tubes (GC150T, Harvard Apparatus, Edenbridge, Kent) were coated with Sylgard (Dow Corning, Seneffe, Belgium), and polished just before use. The pipettes were filled with a high-Cl -solution containing (in mM): 150 NMDG-Cl, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4. The solutions used to characterize channel properties in the inside-out configuration contained 2 mM EGTA and no calcium. To record the currents, we used a RK 400 patch-clamp amplifier (Bio-logic, Claix, France) and PClamp 9 software (Molecular Devices, Sunnyvale, CA). Currents were filtered at 300 or 500 Hz, and were sampled at 1-2 kHz using a Digidata 1322A analogue-to-digital converter (Molecular Devices, Sunnyvale, CA). The mean current (I) passing through N channels was used to calculate the normalized current (NPo) according to the equation NPo=I/i, where i is the unitary current amplitude. The relationship between NPo and voltage (V) was fitted using a Boltzmann equation:

Fig. 7B also

  Fig. 7B also shows the linear current/voltage relationship obtained in the inside-out configuration when high-Cl -solutions were present on both sides of the membrane patch. The

Fig

  Fig 7C show that neither the unit conductance (38.1 ± 1.4 pS; n = 4) nor the voltage

Fig. 1 .

 1 Fig. 1. Activation of mClC-K1 channels by mBarttin. A, steady-state current-voltage

Fig. 2 .

 2 Fig. 2. Increase in the surface expression of mClC-K1 channels produced by mBarttin.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Electrophysiological properties of mClC-K1/Y95A mBarttin channels at the whole-

Fig. 5 .

 5 Fig. 5. mClC-K1/mBarttin channel anion selectivity determined at the single-channel level

Fig. 6 .

 6 Fig. 6. Whole-cell recordings of mClC-K1/mBarttin in HEK293-transfected cells. A,

Fig. 7 .

 7 Fig. 7. mClC-K1 single-channel activity in HEK293-transfected cells. A, representative

Fig. 8 .

 8 Fig. 8. Whole-cell recordings of V166E mClC-K1/mBarttin in HEK293-transfected cells. A, representative, original, whole-cell recordings. The pipette was filled with 150 mM Cl -. Cells were bathed in a solution at pH 7.4 or or in a solution in which 140 mM extracellular Cl -had been replaced by I -. B, steady-state current-voltage relationships under the same conditions as described in panel A (pH 7.4, n = 12; I -, n = 14).

Fig. 9 .

 9 Fig. 9. V166E mClC-K1 single-channel activity in cell-attached membrane patches from

  HEK293 cells were transiently transfected by electroporation, and plated on 35-mm plastic culture dishes. 20 h after transfection, the Petri dishes were mounted on the inverted stage of a microscope. The bath solution contained (in mM): 140 NaCl, 2 CaCl 2 , 1 MgCl 2 , 22 Sucrose, 10 HEPES, pH 7.4. HEPES was replaced by TRIS for solutions at pH 9.0. For the chloride substitution experiments, 140 NaCl was replaced by 140 NaI. The pipette solution contained (in mM): 150 NMDG-Cl, 1 MgCl 2 , 2 EGTA, 1 Mg-ATP, 10 HEPES, pH 7.4 or 33 CsCl, 100 NaGluconate, 1 MgCl 2 2 EGTA, 1 Mg-ATP, and 10 HEPES, pH 7.0. Standard whole-cell recordings were performed using an Axopatch 200B (Molecular Devices, Sunnyvale, CA) and PClamp 9 software (Molecular Devices, Sunnyvale, CA). The bath was earthed via an agar 3 M agar bridge. Patch-clamp pipettes had resistances of 2-3 MΩ. The

  diameter of about 1-1.5 μm after fire polishing. Pipettes were filled with the KCl pipette solution (in mM: 55 KCl, 35 K-gluconate, 5 NaCl, 1.2 MgCl 2 , 2 EGTA, 10 HEPES, pH 7.28). Seals were routinely formed in a low sodium NMDG-Cl bath solution (in mM: 95 NMDG-Cl, 1 NaCl, 4 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 HEPES, pH 7.4). In the NaCl bath solution, 95 mM NMDG-Cl was replaced by 95 mM NaCl. In the NaBr, NaI and NaNO 3 bath solutions, 95 mM NMDG-Cl was replaced by 10 mM NaCl and by 85 mM NaBr, 85 mM NaI and 85 mM NaNO 3 , respectively. After seal formation, the bath solution was changed to the NaCl, NaI, NaBr or NaNO 3 bath solutions. Single-channel current data were initially filtered at 3 kHz and sampled at 9 kHz. Current traces were re-filtered at 250 Hz to resolve the single channel current amplitude (i).

  Manufacturer's instructions (Roche Diagnostics, Meylan, France). Recordings were performed two days after transfection. The cells were initially bathed in physiological saline containing (in mM): 140 NaCl, 5 KCl, 1 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES, pH 7.4.

Fig. 7B also

  Fig. 7B also shows the linear current/voltage relationship obtained in the inside-out configuration when high-Cl -solutions were present on both sides of the membrane patch. The

Fig

  Fig 7C show that neither the unit conductance (38.1 ± 1.4 pS; n = 4) nor the voltage

Fig. 1 .

 1 Fig. 1. Activation of mClC-K1 channels by mBarttin. A, steady-state current-voltage

Fig. 2 .

 2 Fig. 2. Increase in the surface expression of mClC-K1 channels produced by mBarttin.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Electrophysiological properties of mClC-K1/Y95A mBarttin channels at the whole-

Fig. 5 .

 5 Fig. 5. mClC-K1/mBarttin channel anion selectivity determined at the single-channel level

Fig. 6 .

 6 Fig. 6. Whole-cell recordings of mClC-K1/mBarttin in HEK293-transfected cells. A,

Fig. 7 .

 7 Fig. 7. mClC-K1 single-channel activity in HEK293-transfected cells. A, representative

Fig. 8 .

 8 Fig. 8. Whole-cell recordings of V166E mClC-K1/mBarttin in HEK293-transfected cells. A, representative, original, whole-cell recordings. The pipette was filled with 150 mM Cl -. Cells were bathed in a solution at pH 7.4 or or in a solution in which 140 mM extracellular Cl -had been replaced by I -. B, steady-state current-voltage relationships under the same conditions as described in panel A (pH 7.4, n = 12; I -, n = 14).

Fig. 9 .

 9 Fig. 9. V166E mClC-K1 single-channel activity in cell-attached membrane patches from

Fig. 9 .

 9 Fig. 9. Single-channel recordings from cell-attached membrane patches from intercalated

  Figure 1 Click here to download high resolution image

ACKNOWLEDGMENTS

This work was supported by grants from the Agence Nationale de la Recherche (ANR) (ANR07-PHYSIO-008-2 and BLAN 2010-111201), and by a grant from the BHFZ (Bayerisch-Französisches Hochschulzentrum; FK-13/09). S. L'Hoste and O. Andrini hold ANR postdoctoral fellowships, and M. Keck, T. Grand and L. Pinelli PhD fellowships from the Ministère de l'Enseignement Supérieur et de la Recherche. The English text was edited by M. Ghosh. NON-STANDARD ABBREVIATIONS CCD: cortical collecting duct ClC-K: kidney chloride channel CNT: connecting tubule CTAL: cortical thick ascending limb DCT: distal convoluted tubule DIDS: 4,4′-diisothiocyanato-2,2′-stilbene disulfonic acid disodium salt DPC: 2-(phenylamino) benzoic acid NPPB: 5-nitro-2-(3-phenylpropylamino) benzoic acid OMCD: outer medullary collecting duct ACKNOWLEDGMENTS This work was supported by grants from the Agence Nationale de la Recherche (ANR) (ANR07-PHYSIO-008-2 and BLAN 2010-111201), and by a grant from the BHFZ (Bayerisch-Französisches Hochschulzentrum; FK-13/09). S. L'Hoste and O. Andrini hold ANR postdoctoral fellowships, and M. Keck, T. Grand and L. Pinelli PhD fellowships from the Ministère de l'Enseignement Supérieur de la Recherche. The English text was edited by M. Ghosh.