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Kir4.1/Kir5.1 channel forms the major K⁺ channel in the basolateral membrane of mouse renal collecting duct principal cells

¹Université Pierre et Marie Curie-Paris 6 and ²Centre National de la Recherche Scientifique, UMR7134, ³Institut National de la Santé et de la Recherche Médicale U773, Centre de Recherche Biomédicale Bichat-Beaujon (CRB3), and ⁴Université Paris 7-Denis Diderot, Paris, France; and ⁵Division of Cellular and Molecular Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan

Submitted 25 June 2007 ; accepted in final form 17 March 2008

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
K⁺ channels in the basolateral membrane of mouse cortical collecting duct (CCD) principal cells were identified with patch-clamp technique, real-time PCR, and immunohistochemistry. In cell-attached membrane patches, three K⁺ channels with conductances of 75, 40, and 20 pS were observed, but the K⁺ channel with the intermediate conductance (40 pS) predominated. In inside-out membrane patches exposed to an Mg²⁺-free medium, the current-voltage relationship of the intermediate-conductance channel was linear with a conductance of 38 pS. Addition of 1.3 mM internal Mg²⁺ had no influence on the inward conductance (G_in = 35 pS) but reduced outward conductance (G_out) to 13 pS, yielding a G_in/G_out of 3.2. The polycation spermine (6 x 10^–7 M) reduced its activity on inside-out membrane patches by 50% at a clamp potential of 60 mV. Channel activity was also dependent on intracellular pH (pH_i): a sigmoid relationship between pH_i and channel normalized current (NP_o) was observed with a pK of 7.24 and a Hill coefficient of 1.7. By real-time PCR on CCD extracts, inwardly rectifying K⁺ (Kir)4.1 and Kir5.1, but not Kir4.2, mRNAs were detected. Kir4.1 and Kir5.1 proteins cellularly colocalized with aquaporin 2 (AQP2), a specific marker of CCD principal cells, while AQP2-negative cells (i.e., intercalated cells) showed no staining. Dietary K⁺ had no influence on the properties of the intermediate-conductance channel, but a Na⁺-depleted diet increased its open probability by 25%. We conclude that the Kir4.1/Kir5.1 channel is a major component of the K⁺ conductance in the basolateral membrane of mouse CCD principal cells.

kidney; patch clamp

Despite their important functional role, and efforts to study their properties and regulatory mechanisms, little is known about the correspondence between native K⁺ channels described on the basolateral membrane of CCD principal cells (10, 42, 43, 45) and the variety of K⁺ channel genes expressed in these cells (6). From whole cell clamp studies on mouse CCD cells (21), CCD-IRK3, a 15-pS inwardly rectifying K⁺ channel cloned from the M1 mouse collecting duct cell line, which presents a high degree of homology with human inwardly rectifying K⁺ (Kir) 2.3 channel (46), is thought to be the 18-pS channel described in rat CCD principal cells (45). Using whole cell patch-clamp technique and noise analysis of K⁺ current measurements of rat CCD principal cells, Gray et al. (5) indicated that the native 30-pS K⁺ channel (45) shared some properties of members of the Kir4/Kir5 subfamily of channels. However, no comparison of the properties at the single-channel level of native K⁺ channels in the basolateral membrane of CCD principal cells with those of cloned channels has been conducted.

The purpose of the present study was to identify the K⁺ channels of the basolateral membrane of mouse CCD principal cells on the basis of the properties of cloned K⁺ channels present in CCD. We also addressed the question of their possible regulation by variations in dietary K⁺ and Na⁺.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Animal and Tissue Preparation

The experiments were conducted in accordance with the rules of the French Ministry of Agriculture under Permit No. 75-096. Male 15- to 20-g CD1 mice (Charles River Laboratories France, L'Arbresle, France) were chronically maintained on a standard (0.3% Na⁺, 0.6% K⁺) diet (SAFE, Epinay, France) with free access to tap water. When appropriate (see RESULTS), the animals were fed for 6 days before the experiment either with a high-potassium diet (8% K⁺, 0.3% Na⁺) and free access to tap water or with a low-potassium diet (0% K⁺, 0.3% Na⁺) or a low-sodium diet (0.09% Na⁺, 0.6% K⁺) and free access to deionized water. On the day of the experiment, one mouse was killed by cervical dislocation and small pieces of renal cortex were treated with collagenase (32).

Single-Channel Analysis

Patch-clamp experiments were performed on the basolateral membrane of CCD principal cells. CCDs were microdissected as previously described (26, 32), and principal cells were optically identified as flat and polygonal cells in contrast to the round and protuberant intercalated cells (5, 26).

Current recordings. Single-channel currents were recorded with a RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) and stored on either digital audiotapes (DTR-1205, Bio-Logic) or audio CDs (PDR-W839 and DRA-200, Bio-Logic). The reference electrode in the bath was an Ag/AgCl pellet. In the cell-attached configuration, the clamp potential (V_c = V_bath – V_pipette) is superimposed on the cell membrane potential (V_m). In excised inside-out membrane patches, V_c = V_m. All experiments were conducted at room temperature.

Data analysis. Signals were typically low-pass filtered at 1 kHz by an eight-pole Bessel filter (LPBF-48DG; Npi electronic, Tamm, Germany) and digitized at 3 kHz by an analog-to-digital converter (Digidata 1200 or 1322A, Axon Instruments). The mean current (I) passing through N channels was used to calculate the normalized current (NP_o) according to the equation NP_o = I/i, where i is the unitary current amplitude.

Solutions. Pipettes were typically filled with a high-K⁺ solution containing (in mM) 145 KCl, 1 MgCl₂, 10 glucose, and 10 HEPES, pH 7.4 with KOH. In cell-attached patches, the bath medium contained (in mM) 140 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES, pH 7.4 with NaOH.

The intracellular side of inside-out patches was typically bathed by a high-K⁺ medium containing (in mM) 145 KCl, 10 HEPES, 10 glucose, and 2 K₂-EDTA, pH 7.4 with KOH. Channel selectivity was investigated with an internal medium containing 140 NaCl, 5 KCl, 10 HEPES, 10 glucose, and 2 K₂-EDTA, pH 7.4 with NaOH. When appropriate, the pH of this HEPES-containing solution was adjusted to pH 7–8.5 with NaOH. The effects of internal pH 6–6.5 were investigated by substituting MES for HEPES.

Channel selectivity. K⁺ permeability (P_K)/Na⁺ permeability (P_Na) was determined in inside-out membrane patches from reversal potential (E_rev) values obtained from i/V_c relationships established with opposite Na⁺ and K⁺ ion gradients across the membrane patch with the Goldman, Hodgkin, and Katz voltage equation (8):

where F, R, and T are Faraday constant, gas constant, and temperature, respectively, and subscripts o and i denote the outside and inside surfaces of the membrane patch, respectively.

Mg²⁺-dependent inward rectification. The effects of internal free Mg²⁺ on current rectification were investigated by substituting EGTA for EDTA in the high-K⁺ solution and by adding 1.5 mM Mg²⁺ to obtain 1.3 mM free Mg²⁺, on the basis of absolute stability constants for Mg²⁺, H⁺, and EGTA (33).

Data were analyzed according to the Woodhull model (see Ref. 7), where Mg²⁺ moves into the electric field of the pore, assuming a single site for Mg²⁺ binding located within the pore, with the equation

where I_Mg and I₀ are the currents in the presence and absence of Mg²⁺, respectively, is the electrical distance of the binding site from the outside of the pore, and K₀ is the inhibition constant at V_c = 0 mV.

Real-Time PCR

Tubular fragments were microdissected at 4°C in medium supplemented with RNase-free BSA (1 mg/ml) and rinsed. Pools of 20–50 microdissected tubules were then directly treated for RNA extraction (31). RT was performed with a first-strand cDNA synthesis kit for RT-PCR (Roche Diagnostics, Meylan, France). The viability of all RNA samples was ascertained by real-time PCR for the housekeeping gene cyclophilin.

The primers for the K⁺ channels studied here were as follows: ROMK sense 5'-GGGGTTACCGTTTTGT-3', antisense 5'-CATTTGGGTGTCGTCT-3'; Kir4.1 sense 5'-CCGCGATTTATCAGAGC-3', antisense 5'-AGATCCTTGAGGTAGAGGAA-3'; Kir5.1 sense 5'-ATACGGTTACCGCTGT-3', antisense 5'-TGACGAGTTTGAGGTCTT-3'; Kir4.2 sense 5'-GCAGGCCATAGCAGAG-3', antisense 5'-GTGAGCTTGTATCGCCA-3'; Kir 7.1 sense 5'-ATCTTCCCGCTAACCTAT-3', antisense 5'-CACCTTTGGAACCTCG-3'. The primers for the mouse cyclophilin sequence were as previously given (31).

Real-time PCR was performed either with a LIGHTCYCLER System (Roche Diagnostics) and the FastStart DNA Master SYBR Green 1 kit (Roche Diagnostics) as previously described (25, 31) or with a LIGHTCYCLER 480 and the LC 480 SYBR Green 1 Master kit (Roche Diagnostics), on cDNA corresponding to 0.1 mm of tubule in the presence of 4 pmol of primers per 8 µl (LIGHTCYCLER) or 5 pmol per 10 µl (LIGHTCYCLER 480) of final reaction volume. Samples were submitted to either 50 (LIGHTCYCLER) or 70 (LIGHTCYCLER 480) cycles of three temperatures (95°C for 10 s, 60°C for 12 s, 72°C for 20 s). A melting curve was then established in order to check for nonspecific PCR products.

In each experiment, a standard curve was plotted with serial dilutions (1 to 1/500) of a cDNA stock solution obtained from mouse whole kidney RNA, dilution 1 corresponding to the cDNA produced by 10 ng of RNA. For all samples, the amount of PCR product was calculated as a percentage of the RNA standard (arbitrary unit).

After correction for the efficiency of the PCR, the expression of gene 1 relative to that of ROMK gene in a given sample was estimated with the equation

where N₁ and N_ROMK are the numbers of cDNA copies present in the samples before amplification, E₁ and E_ROMK are the PCR efficiencies, CP₁ and CP_ROMK are the crossing points, and L₁ and L_ROMK are the respective lengths of the amplified products (25).

Immunohistochemistry

Tissues were prepared for immunohistochemical studies as previously described (17). Briefly, mice were killed by intraperitoneal injection of pentobarbital sodium. Kidneys were then rapidly removed, frozen in liquid nitrogen, and stored at –80°C until use. Kidney sections (7–9 µm) mounted on glass slides were fixed in ice-cold methanol (8 min) and incubated with either rabbit anti-rat Kir4.1 (7, 12) or rabbit anti-rat Kir5.1 (7, 11) antibodies (1:200 dilution) and then with a Cy3-conjugated goat anti-rabbit Ig (1:100 dilution) (Jackson Immunoresearch, West Grove, PA). Sections were also double-labeled with anti-Kir4.1 or anti-Kir5.1 antibody and a polyclonal antibody directed against the aquaporin-2 water channel (AQP2; kindly provided by J. Loffing, Institute of Physiology, University of Zurich, Zurich, Switzerland), the thiazide-sensitive Na⁺-Cl^– cotransporter (NCC), or the Tamm-Horsfall protein (Valbiotech, France). Binding was revealed with Cy3- or Alexa Fluor 488-conjugated FAB fragment goat anti-rabbit IgG (1:100, Molecular Probes, Eugene, OR). All tissue sections were examined by confocal laser scanning microscopy (CLSM) (Leica, Wetzlar, Germany).

Kidney sections were also double-labeled with anti-Kir4.1 and anti-AQP2 antibodies in order to quantify the number of AQP2-positive and -negative CCD cells expressing Kir4.1. Quantification of the number of labeled cells was performed on 32 separate tissue sections from 3 different kidneys. The relative fluorescence levels of Kir4.1 labeling of CCD principal cells, as identified by positive staining with anti-AQP2 antibody, and of adjacent distal convoluted tubule (DCT) cells were obtained on a single-cell basis for each digital image and measured with Zeiss CLSM Image Browser software as described elsewhere (28).

Data Presentation and Statistics

Results are given as means ± SE for n experiments. P values <0.05 (paired or unpaired t-test or Z-test on proportions, when appropriate; SIGMAPLOT, Systat Software, Erkrath, Germany) were taken to represent statistically significant differences. Nonlinear regression analyses were performed with either SIGMAPLOT or ORIGIN (MICROCAL Software, Northampton, MA) software.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Patch-clamp experiments on the basolateral membrane of CCD principal cells of mice maintained on a standard diet revealed a 40-pS K⁺ channel in 63% of the cell-attached membrane patches (52 of 83) and a 20-pS K⁺ channel in 30% of the patches (25 of 83). Throughout this study, the activity of another channel type with a conductance of 76 pS was also observed. It had an reversal potential of 56 mV, which suggested the presence of a channel with significant selectivity for K⁺, but its low occurrence (8 cell-attached patches) precluded any further analysis. In what follows, we therefore focus on the properties of the 40-pS and 20-pS K⁺ channels.

Properties of 20-pS Channel

Typical recordings of 20-pS channel activity on cell-attached membrane patches from a tubule bathed in high-NaCl medium, with the pipette filled with the high-KCl solution, are shown in Fig. 1A. Because this channel was often seen on patches containing several channel types, no confident statistical analysis of its open probability (P_o) was possible. However, on the patch shown in Fig. 1, channel activity displayed little voltage dependence, because P_o decreased only slightly from 0.8 at V_c = 0 mV to 0.6 at V_c = –90 mV. On average, channel number per patch and NP_o at V_c = 0 mV were 1.6 ± 0.4 (n = 5) and 1.23 ± 0.31 (n = 3), respectively, yielding a P_o of 0.68 ± 0.17 (n = 3). The corresponding i/V_c relationship is shown in Fig. 1B. Inward conductance (G_in) averaged 22.9 ± 0.89 pS (n = 25) and E_rev was estimated at 66.6 ± 7.3 mV (n = 10), which was consistent with the presence of a K⁺ channel.

View larger version (23K): [in this window] [in a new window]   Fig. 1. In situ properties of the 20-pS K+ channel. A: typical recordings of 20-pS K+ channel activity in the cell-attached membrane patch configuration. The cortical collecting duct (CCD) tubule was bathed in high-Na+ solution, with high-K+ solution in the pipette. Recordings were obtained from the same membrane patch on the basolateral membrane of a CCD principal cell, at the clamp potential (Vc) values given on right. C, current level corresponding to the closure of all K+ channels. Po, open probability. B: unitary current amplitude (i)/Vc relationships for the 20-pS K+ channel under the conditions given in A. Each point is the mean of at least 3 experiments, with SE shown by error bars.

On two patches, we were able to test for the sensitivity of the channel to internal pH (pH_i). On both patches, a decrease in pH_i from 7.4 to 6.6 had no detectable influence on channel activity. A further decrease in pH_i reduced channel NP_o, and pK values could be estimated within the range 6.1–6.4.

Properties of 40-pS K⁺ Channel in Cell-Attached Membrane Patches

Figure 2A illustrates single-channel activity of the 40-pS channel in a cell-attached membrane patch with high-NaCl medium as the bath solution and high-KCl medium as the pipette solution. From 24 patches where no other channel type was present, an average of 3.2 ± 0.38 channels per patch and a NP_o of 1.75 ± 0.23 were observed at V_c = 0 mV, yielding a P_o of 0.45 ± 0.04. Channel P_o was quite stable over the range of –100 mV to 20 mV (n = 8) (Fig. 2B, inset).

View larger version (38K): [in this window] [in a new window]   Fig. 2. In situ properties of the 40-pS K+ channel. A: typical recordings of 40-pS K+ channel activity in the cell-attached membrane patch configuration. The CCD tubule was bathed in high-Na+ solution, with high-K+ solution in the pipette. Recordings were obtained from the same membrane patch on the basolateral membrane of a CCD principal cell, at the Vc values given on right. C, current level corresponding to the closure of all K+ channels. B: i-Vc relationships for the 40-pS K+ channel under the conditions given in A () or in tubules bathed by a high-K+ bath (). Each point is the mean of at least 4 () or 3 () experiments, with SE are shown by error bars when larger than symbols. Inset: channel voltage dependence. Channel activity (Po) is plotted vs. membrane voltage (Vc). Each point is the mean of 3–7 experiments, with SE shown by error bars.

Properties of 40-pS Channel in Inside-Out Membrane Patches

We confirmed the high selectivity for potassium of the channel in cell-free patches (not shown). Keeping the high-K⁺ solution in the pipette and replacing all but 45 mM KCl by NaCl in the bath shifted the reversal potential of the i/V_c curves by 27.3 ± 0.64 mV (n = 6), which corresponds to a P_K/P_Na of 23.

Under symmetrical high-K⁺ conditions and in the absence of internal Mg²⁺, single-channel currents reversed at –0.7 ± 0.5 mV (n = 10) and the corresponding i/V_c relationship was nearly linear (Fig. 3B). Thus G_in and G_out were 37.8 ± 1.8 and 34.7 ± 1.5 pS, respectively (P = 0.009, paired t-test; n = 10), leading to a G_in-to-G_out ratio of 1.09 ± 0.03 (n = 10). The inward rectification we observed in the cell-attached configuration resulted from a voltage-dependent block of outward currents by intracellular Mg²⁺ ions. Figure 3A compares the current amplitudes of an inside-out membrane patch, the cytosolic side being exposed either to the Mg²⁺-free medium or to a medium containing 1.2 mM Mg²⁺ (1.5 mM MgCl₂ and 2 mM EGTA) at V_c = –80 and +80 mV. Addition of internal Mg²⁺ had little influence on inward currents but reduced the single-channel amplitude of outward currents. The corresponding i/V_c relationships are shown in Fig. 3B. Addition of 1.2 mM cytosolic Mg²⁺ had no significant influence on E_rev (0.2 ± 0.4 mV, n = 8; P = 0.2 vs. Mg²⁺-free medium) or on G_in (34.8 ± 1.7 pS, n = 9; P = 0.216 vs. Mg²⁺-free medium) but greatly reduced G_out [as defined by G_(chord)out between E_rev and +80 mV], which fell to 12.5 ± 1.87 pS (n = 7). The resulting G_in-to-G_out ratio was 3.17 ± 0.46 (n = 7).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Mg2+-induced inward rectification of the 40-pS K+ channel. A: single-channel recordings in a cell-excised inside-out membrane patch in the absence and presence of 1.3 mM free Mg2+ on the cytosol side at Vc = –80 and 80 mV. Dotted line, current level corresponding to the closure of all K+ channels. B: i/Vc relationships under the conditions given in A in the presence () and absence () of 1.2 mM internal Mg2+. Dashed line, least-squares fit to the data in the presence of Mg2+ using the equation given in the text, with inhibition constant K0 = 2.84 ± 0.25 mM Mg2+ and electrical distance of binding site = 24 ± 0.02 from the inside. Each point is the mean of at least 3 measurements, with SE shown by error bars when larger than symbols.

The tetravalent cation spermine (SPM) altered the channel activity of the 40-pS K⁺ channel. An illustration of this effect is given in Fig. 4A, where 50 µM SPM nearly abolished channel activity at V_c = +60 mV in an inside-out patch. The effects of SPM on channel activity were concentration- and voltage dependent, and Fig. 4B summarizes the results from experiments with 0.01, 1, and 50 µM SPM at V_c = –60 and +60 mV. At V_c = +60 mV, the dose-dependence curve showed an EC₅₀ of 0.6 µM SPM.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Spermine-induced 40-pS channel inhibition. A: single-channel recordings in a cell-excised inside-out membrane patch in the absence (Control) and presence of 50 µM spermine (SPM) on the cytosol side at Vc = 60 mV. C, current level corresponding to the closure of all K+ channels. Bath and pipette solutions were high-K+ media. B: dose-response relationships for the voltage-dependent effects of intracellular SPM on channel activity. Increasing SPM concentrations were applied on the cytosolic face of inside-out membrane patches, and channel activity (NPo) was measured at Vc = –60 () and 60 () mV. For each experimental condition, NPo was normalized to the value in the absence of SPM on the same membrane patch. Each point is the mean of at least 3 experiments, with SE shown by error bars.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Effects of intracellular pH (pHi) on 40-pS K+ channel activity. A: single-channel recordings from an inside-out membrane patch at the pHi values indicated on right. The patch was bathed in symmetrical high K+ and clamped at –40 mV. C, current level corresponding to the closure of all K+ channels. B: dose-response relationship of the effect of pHi on K+ channel activity under the conditions given in A. Within each experiment, NPo at a given pHi value was normalized to the NPo value at pHi = 7.5. Each point is the mean of at least 4 measurements, with SE shown by error bars when larger than symbols. The relationship between pHi and NPo is described by the equation NPo = NPo(max)/[1 + ([H+]/K) ], where NPo(max) is the maximum value of NPo, [H+] is the bath proton concentration, K = 10–pK, and nH is the Hill coefficient. The line is a nonlinear least-squares fit to the data given by NPo(max) = 138.73 ± 2.3, pK = 7.23 ± 0.1, and nH = 1.47 ± 0.08.

Expression of Kir4.1, Kir4.2, and Kir5.1 mRNAs in Mouse CCD Cells

The expression of Kir4.1, Kir4.2, and Kir5.1 mRNAs was investigated by real-time PCR on extracts of CCD segments. As indicated in MATERIALS AND METHODS, channel mRNA expression was quantitated by normalizing Kir4.1, Kir4.2, and Kir5.1 mRNA expression levels to that of the ROMK channel. Both Kir4.1 and Kir5.1 mRNAs were significantly expressed in CCD (Fig. 6). Although present at levels 25- to 100-fold lower than that of ROMK channel mRNA, Kir4.1 and Kir5.1 mRNA expression were of the same order of magnitude as that of the Kir7.1 channel (13), which has been shown to be present in the basolateral membrane of rat CCD principal cells (27). No significant Kir4.2 mRNA expression was detected in mouse CCD extracts. This negative finding could not be attributed to a lack in the effectiveness of the Kir4.2 primers because Kir4.2 mRNA was detected in proximal convoluted and straight tubules (data not shown), yielding a Kir4.2 pattern of expression consistent with results of serial analysis of gene expression (SAGE) studies in human kidney (1).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Expression of Kir4.1, Kir4.2, and Kir5.1 mRNA transcripts in mouse CCD extracts. Real-time PCR results are shown as means of 10 animals normalized to renal outer medullary K+ (ROMK) mRNA transcript expression as described in MATERIALS AND METHODS. Kir7.1 mRNA transcript expression is given for comparison (see text).

The characterization of anti-Kir4.1 and anti-Kir5.1 antibodies by Western blot analysis (immunoblotting of Kir4.1 and Kir5.1 proteins expressed in HEK293T cells and in native tissues, including kidney, and peptide competition study) and Kir4.1 peptide competition study was reported previously (7, 11, 12). Here we carried out the peptide competition assay of anti-Kir5.1 antibody, and, as illustrated in Fig. 7A, preincubation of anti-Kir5.1 antibody with an excess amount of antigenic peptide prevented the detection of protein. We also confirmed that anti-Kir4.1 and anti-Kir5.1 antibodies detected Kir4.1 and Kir5.1 proteins, respectively, in a specific manner and did not cross-react with each other (Fig. 7B).

View larger version (28K): [in this window] [in a new window]   Fig. 7. Characterization of anti-Kir4.1 and anti-Kir5.1 antibodies. A: HEK293T cells were transiently transfected with cDNA of FLAG-tagged Kir5.1, and their Triton X-100-extracted lysates were subjected to Western blot analysis (IB). The proteins were probed by anti-Kir5.1 antibody preincubated without (left) or with (right) an excess amount of antigenic peptide. B: the lysates of the cells expressing myc-tagged Kir4.1 (left lanes) or HA-tagged Kir5.1 (right lanes) were examined with anti-Kir4.1 (top) or anti-Kir5.1 (bottom) antibody. Open and filled arrowheads (top) indicate glycosylated and unglycosylated Kir4.1 proteins, respectively (see Ref. 7). Note that each antibody did not induce cross-reaction.

View larger version (96K): [in this window] [in a new window]   Fig. 8. Immunolocalization of Kir4.1 and Kir5.1 in mouse CCD cells. A: frozen kidney sections were double-labeled with either anti-Kir4.1 or anti-Kir5.1 antibody (red), anti-Na+-Cl– (NCC) transporter, anti-Tamm-Horsfall (TH) protein, and anti-AQP2 antibody (green) and analyzed by confocal laser scanning microscopy. G, glomeruli. B: a CCD section double-labeled with anti-AQP2 antibody (green) and anti-Kir4.1 and anti-Kir5.1 antibodies (red) shows the cellular colocalization of both channel proteins in principal cells, with no staining in intercalated cells (arrowheads). C: illustrations (left) of a kidney section labeled with anti-Kir4 (red) and anti-AQP2 antibodies (green) used to quantify the number of AQP2- and Kir4.1-positive cells, AQP2-positive and Kir4.1-negative cells, or Kir4.1- and AQP2-negative cells in CCD (right). Counts were performed on 803 labeled cells from 32 tissue sections from 3 separate kidneys. Double fluorescence staining was also used to quantify the labeling intensity in CCD and distal convoluted tubule cells (see RESULTS). Scale bars, 10 µm.

A quantitative analysis of the labeling intensity of Kir4.1- and AQP2-positive cells and of Kir4.1-positive adjacent DCT cells (Fig. 8C) also revealed that the fluorescence intensity of Kir4.1 labeling per surface area in DCT cells (118.6 ± 2 fluorescence intensity arbitrary units, n = 127) was significantly (P < 0.001) greater than in AQP2-positive principal CCD cells (87.6 ± 1.9, n = 98). These data confirmed that Kir4.1 is expressed in both DCT cells and CCD principal cells and indicate that the expression of Kir4.1 predominates in the DCT.

Lack of Effect of K⁺ Diets on 40-pS K⁺ Channel

We compared the effects of low-K⁺ diets with those of high-K⁺ diets on the properties of the 40-pS K⁺ channel. After 6 days of treatment, no statistical difference between low- and high-K⁺ diets was found in the frequency of observation of the channel in the cell-attached membrane patches. Thus the channel was detected on 59.6% of active patches (28 of 46 patches containing K⁺ channels) after a low-K⁺ diet and on 39.5% of active patches (15 of 38 patches) after a high-K⁺ diet (P = 0.245, Z-test on proportions). Similarly, there was no statistically significant difference between the two groups in channel NP_o (P = 0.264), in the number of channels per patch (P = 0.433), or in the open channel probability (P = 0.331) in cell-attached membrane patches clamped at V_c = 0 mV (Table 1).

We also tested for the effects of a low-Na⁺ diet on the properties of the 40-pS K⁺ channel. As observed for the high- and low-K⁺ diets, there was no statistical difference between standard and low-Na⁺ diets in frequency of observation of the channel in cell-attached patches. The channel was observed on 63% of active patches after standard diet (see above) and on 82% of active patches (19 of 23 patches) after the low-Na⁺ diet (P = 0.091, Z-test on proportions). Also, as summarized in Table 1, there was no significant change in channel NP_o (P = 0.179) or in the number of channels per patch (P = 0.698, Mann-Whitney rank sum test) induced by low-Na⁺ conditions compared with the standard diet. In contrast, single-channel activity was affected by this maneuver, because P_o was significantly increased by 25% by the low-Na⁺ diet (P = 0.005).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study on the basolateral membrane of mouse CCD principal cells revealed the presence of three K⁺ channel types in situ, with conductance of 75, 20, and 40 pS. The 40-pS channel is the one most frequently expressed.

Molecular Candidates for Basolateral K⁺ Channels in Mouse CCD Principal Cells

The 40-pS K⁺ channel exhibited a high P_K/P_Na, little voltage dependence, an intermediate Mg²⁺-induced inward rectification, sensitivity to pH_i, insensitivity to internal ATP, and blockade by internal SPM. In addition, it exhibited strong activity in cell-excised inside-out membrane patches in the absence of internal Ca²⁺ or cyclic nucleotides. These properties rule out the possibility that this channel could be a voltage-gated K⁺ channel, a Ca²⁺-dependent K⁺ channel, or a poorly K⁺/Na⁺ discriminating cyclic nucleotide-gated K⁺ channel.

A real-time PCR study of two-pore domain K⁺ channels has revealed the expression of TWIK-1, TASK-1, TASK-2, and THIK-1 channel mRNA in the distal nephron of the human kidney (15). It is unlikely that any of these channels can be proposed as a candidate for the basolateral 40-pS K⁺ channel we describe here. First, the TASK-1 channel is only 14 pS (2). Second, TWIK-1, a 34-pS, weakly inward rectifying and ATP-insensitive K⁺ channel, appears only intracellularly located in mouse CCD cells (24), with no detectable activity in cell membranes (20). In addition, TWIK-1 is insensitive to pH_i in cell-excised inside-out membrane patches of Xenopus oocytes (14). Third, the same insensitivity to pH_i holds true for THIK-1 (35). And fourth, we failed to detect mRNA of TASK-2, a 60-pS, weakly inwardly rectifying channel (36), in mouse CCD extracts (data not shown).

RT-PCR and immunohistochemistry data showed that several inwardly rectifying K⁺ (Kir) channels are expressed in the CCD (6). Some of these cloned channels can be eliminated as potential constituents of the 40-pS channel of mouse CCD cells. Kir7.1 channel mRNA was detected in mouse CCD in the present study, but its very low conductance of 50 fS (13) precludes its detection by single-channel patch-clamp analysis. CCD-IRK3 (46) displays a high P_o and a single-channel inward conductance of 15 pS, with much stronger inward rectification than that of the 40-pS channel we describe here.

PCR analysis revealed expression of Kir5.1 in CCD segments of rat kidney (39), and immunohistochemical studies have demonstrated that Kir4.1 and Kir5.1 proteins localize in the basolateral membrane of rat CCD principal cells (12, 40). In contrast, no Kir4.2 gene expression was found in CCD of human kidney by SAGE methods (1). Our results in mouse kidney showed the presence of both Kir4.1 and Kir5.1 mRNA, but not that of Kir4.2, in CCD extracts and the colocalization of Kir4.1 and Kir5.1 proteins in AQP2-positive, i.e., principal, CCD cells.

When expressed in heterologous expression systems, homomeric assembly of Kir4.1 subunits forms strong inwardly rectifying channels in the presence of internal Mg²⁺, with an inward conductance of 22 pS, high activity (P_o 0.9) (48), an insensitivity to changes in pH_i around physiological values with a pK of 6 (40), and sensitivity to internal SPM with a EC₅₀ of 5 x 10^–8 M (3). These properties clearly do not match those of the channel described here, which has a inward conductance of 40 pS with an intermediate inward rectification in the presence of internal Mg²⁺ (G_in/G_out = 3–4), a moderate activity (P_o 0.45), and a pK of 7.25.

On the other hand, the coexpression of Kir4.1 and Kir5.1 units leads to the formation of a 43- to 60-pS K⁺ channel (34, 49) with an intermediate inward rectification (G_in/G_out 4) and a P_o of 0.4 (49). In addition, channels formed by the Kir4.1/Kir5.1 assemblage are sensitive to pH_i changes around the physiological range, with pK values of 7.45 (49) and n_H of 2.1 (47). In the light of its properties, i.e., a conductance of 40 pS, a G_in-to-G_out ratio of 3.2, an in situ P_o of 0.45, and sensitivity to pH_i (pK = 7.24, n_H = 1.7), it is clear that the 40-pS K⁺ channel of the basolateral membrane of mouse CCD principal cells very closely resembles a heteromeric Kir4.1/Kir5.1 channel.

This conclusion may give some indications of the molecular nature of the 20-pS K⁺ channel. Thus, on the basis of its low conductance, high activity (P_o 0.7), and sensitivity to acidic pH_i values (pK of 6.1–6.4) on one hand and properties of Kir4.1 channels on the other hand, we postulate that this 20-pS K⁺ channel may be formed by homomeric Kir4.1 unit association.

Comparison with K⁺ Channels in Basolateral Membrane of Rat CCD Cells

At the time this work was initiated, K⁺ channels in the basolateral membrane of CCD principal cells of the mouse kidney were not identified. Some of the K⁺ channels previously described in the basolateral membrane of rat CCD principal cells may be similar to the 40- and 20-pS K⁺ channels we observed in mouse cells. Wang et al. (45) identified three types of K⁺ channels on the lateral membrane of rat CCD principal cells. In particular, a pH-sensitive K⁺ channel, 45 pS in cell-attached membrane patches, may resemble the 40-pS channel we describe here. However, measurements in cell-excised membrane patches under 140 mM K⁺ symmetrical conditions gave a conductance of 85 pS (42), markedly higher than the 38 pS we found for the mouse CCD K⁺ channel under similar conditions. In addition, channel activity in rat CCD cells is voltage dependent in situ, because its P_o increases from 0.2 at rest up to 0.7 with 60-mV hyperpolarization (45), whereas we observed a nearly constant P_o of 0.5 over a 120-mV range of clamp potentials in mouse CCD. Finally, the 45-pS K⁺ channel in rat CCD cells appeared to have comparatively low pH_i sensitivity, because lowering pH_i from 7.4 to 6.7 decreased P_o by 30% in cell-excised membrane patches (42), whereas we can extrapolate from our results that the same maneuver would reduce the P_o of the mouse CCD 40-pS K⁺ channel by 60%. This suggests that the basolateral 45-pS channel in rat CCD and the 40-pS channel in mouse CCD may be different channels.

A 30-pS voltage- and ATP-independent K⁺ channel in both cell-attached and cell-excised inside-out membrane patches has also been described in rat CCD cells (18, 45). However, by its pH insensitivity and high activity in situ with a P_o = 0.8 (45), it also clearly differs from the 40-pS K⁺ channel we describe here. On the other hand, such a high P_o is reminiscent of the elevated P_o of 0.7 that we observed in situ for the 20-pS K⁺ channel. But, because we were unable to provide further information about the properties of the 20-pS K⁺ channel, the question of whether it may be related to the rat 30-pS channel remains open. The same holds true regarding the 18-pS K⁺ channel, which has also been identified on cell-excised patches from the basolateral membrane of rat CCD (43).

Our data and the data from the literature therefore indicate that basolateral membranes of mouse and rat CCD principal cells are endowed with distinct populations of K⁺ channels. The present study showed that a pH-sensitive 40-pS K⁺ channel predominated in mouse CCD cells, whereas a pH-insensitive, 30-pS K⁺ channel appeared to be the major K⁺ channel in rat CCD cells (42).

Channel Regulation After K⁺- and Na⁺-Controlled Diets

The CCD is responsible for part of the final regulation of K⁺ excretion, because K⁺ loading stimulates urinary K⁺ secretion whereas K⁺ depletion drastically reduces urinary excretion by this segment (4, 22, 44). Numerous studies have demonstrated that an adaptation to dietary K⁺ intake is, at least partially, achieved by changes in apical K⁺ conductance through modulation of the ROMK channels and of basolateral Na⁺-K⁺-ATPase pump activity in CCD principal cells (44). Regarding the latter, a high-K⁺ diet stimulates basolateral Na⁺-K⁺-ATPase activity, whereas a low-K⁺ diet reduces it (22). Because of the coupling of basolateral K⁺ conductance to Na⁺-K⁺-ATPase activity (23), parallel changes in basolateral K⁺ channel properties are expected. However, we failed to observe any change in frequencies, open probabilities, or numbers of basolateral K⁺ channels per patch of CCD principal cells after low- or high-K⁺ diets. At first glance, such a lack of effect of high-K⁺ diet on the basolateral 40-pS K⁺ channel properties rules out its role in these conditions since an increase in basolateral K⁺ conductance of CCD principal cells has been reported in DOCA-treated rabbits (37) and in rats maintained on a high-K⁺ diet (5). Nevertheless, considering the results of morphological measurements of the basolateral membrane surface of CCD principal cells of rat after chronic high-K⁺ loading (9, 38) or of rabbit after chronic DOCA treatment (41), showing a significant increase in their surface area (38), our results may appear consistent with an increase in the total number of K⁺ channels after K⁺ loading.

A low-Na⁺ diet increases Na⁺ absorption and K⁺ secretion by CCD principal cells, by stimulating apical Na⁺ channel activity and an increase in the driving force for K⁺ secretion through ROMK channels in rat (29, 30). Na⁺-K⁺-ATPase activity should be increased, partly as a result of enhanced Na⁺ entry (30). We found that keeping mice on a low-Na⁺ diet for 6 days significantly increased 40-pS K⁺ channel open probability, but not the number of active channels. This indicates that a low-Na⁺ diet did not induce any major change in channel expression and that such an adaptation of the basolateral K⁺ current is mediated via modulation of channel gating. The mechanism behind which such a regulation takes place has not been investigated here. Nevertheless, it is now established that nitric oxide (NO) and a cGMP-dependent pathway modulate the activity of the basolateral 30-pS K⁺ channel of rat CCD principal cells (18) and thus provide the link between apical Na⁺ channels and basolateral K⁺ channels. Further experiments on the regulation of the mouse 40-pS K⁺ channel are obviously needed in order to determine whether the NO pathway may be implicated in the increase in channel activity we observed under a low-Na⁺ diet.

The results of this work on basolateral membrane of CCD cells, together with previous data on cortical TAL (CTAL) (32) and DCT (17) cells, provide a new picture of the molecular nature of basolateral K⁺ channels along the mouse distal nephron. Thus our results showed that both CTAL (32) and DCT (17) tubule basolateral membranes are endowed with K⁺ channels with properties similar to those of the basolateral K⁺ channel in CCD cells, indicating that the Kir4/Kir5.1 channel may be a major component of the basolateral K⁺ conductance along the mouse distal nephron.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
S. Lachheb holds a Ph.D. fellowship from the French Ministère de la Recherche, and Marc Paulais is an Institut de la Santé et de la Recherche Médicale researcher.

	ACKNOWLEDGMENTS

 
We thank Dr J. Loffing for kindly providing us the AQP2 antibody. The English text was edited by Monika Ghosh.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Paulais, Université Pierre et Marie Curie and CNRS, Laboratoire de Physiologie et Génomique Rénales, UMR 7134, Centre de Recherches Biomédicales des Cordeliers, 15 rue de l'Ecole de Médecine, 75720 Paris Cedex 06, France (e-mail: marc.paulais{at}bhdc.jussieu.fr)

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

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

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