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A Na+- and Cl−-activated K+ Channel in the Thick Ascending Limb of Mouse Kidney

This study investigates the presence and properties of Na+-activated K+ (KNa) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the KNa channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (PK/PNa ∼ 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl− activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 ± 1 mM and 3.9 ± 0.5 for internal Na+, and 35 ± 10 mM and 1.3 ± 0.25 for internal Cl−. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native KNa channels of excitable cells. This Slo2.2 type, Na+- and Cl−-activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.


I N T R O D U C T I O N
Na + -activated K + (K Na ) channels are a class of largeconductance K + channels of which the activity increases with the intracellular Na + concentration. To date, K Na channel activity has been primarily reported in excitable cells, including guinea pig cardiomyocytes (Kameyama et al., 1984), and neurons from various species (for review see Dryer, 1994). It has been suggested that K Na channels may either serve to control the resting membrane potential or be involved in the Na + -dependent slow afterhyperpolarization that follows a neuronal burst of action potentials (Dryer, 1994). They are also thought to have a protective role on guinea pig cardiomyocytes under pathological conditions, such as is chemia or hypoxia (Kameyama et al., 1984). The only report of functional K Na channels in a nonexcitable tissue of which we are aware is a very brief description of Na + -activated K + channel activity in the plasma membrane of Xenopus oocytes (Egan et al., 1992a).
The recently cloned Slo2.1 (Bhattacharjee et al., 2003) and Slo2.2 (Joiner et al., 1998;Yuan et al., 2000Yuan et al., , 2003 channels, two members of the Slo channels family, share 74% identity in the rat (Bhattacharjee et al., 2003) and display functional properties reminiscent of native K Na channels in excitable cells (Joiner et al., 1998;Bhattacharjee et al., 2003;Yuan et al., 2003): channel conductance in the 140-180 pS range, the existence of several conductance substates, and the fact of being stimulated by internal Na + . As well as being stimulated by internal Na + , Slo2.1 and Slo2.2 channels are both coactivated by internal Cl − (Bhattacharjee et al., 2003;Yuan et al., 2003), a yet unadressed property of native K Na channels. The molecular distributions of Slo2.1 and Slo2.2 channels correlate closely with the functional observation of K Na channels in excitable cells. Thus, mRNA and transcripts of both channels have been detected in the brain and, although to a lesser extent, in the heart of the mouse (Yuan et al., 2003) and rat (Bhattacharjee et al., 2003;Joiner et al., 1998). These studies also indicated a very limited distribution of channels among nonexcitable cells, as no Slo2.1 or Slo2.2 signal was detected in the spleen, lung, and liver of either rodent species. But, although Northern blot analysis also failed to demonstrate the presence of Slo2.1 mRNA in rat kidney (Bhattacharjee et al., 2003), signifi cant levels of Slo2.2 mRNA and transcripts were unexpectedly detected in whole kidneys of the rat (Joiner et al., 1998) and mouse (Yuan et al., 2003). To date, no such largeconductance, Na + -sensitive K + channels have been functionally identifi ed in renal cells.
We therefore used real-time PCR and patch-clamp techniques on microdissected tubules of mouse nephron 206 An Epithelial Slo2.2 K + Channel for molecular and functional characterization of renal K Na channels. We report here that, in addition to excitable cells and Xenopus oocytes, a Na + -activated K + channel is present in the membranes of epithelial renal cells. Moreover, this Slo2.2-type K + channel may have physiological signifi cance, since it is primarily expressed in the thick ascending limb of Henle's loop, a major site of transcellular NaCl absorption.

M AT E R I A L S A N D M E T H O D S
Chemicals EDTA (dipotassium salt) and ATP (disodium salt) were from Sigma-Aldrich. EGTA was either from Research Organics Inc. or from Sigma-Aldrich.

Animal and Tissue Preparation
The experiments were conducted according to the rules of the French Ministry of Agriculture (permit no. 75-096). Male, 15-20 g, CD1 mice (Charles River Laboratories France) were fed ad libitum with a control diet (SAFE). On the day of the experiment, one mouse was killed by cervical dislocation and small pieces of renal cortex and medulla were treated with collagenase .
Patch-clamp experiments were performed on the basolateral membrane of tubules microdissected as previously described . For real-time PCR experiments, tubular fragments and glomeruli were microdissected at 4°C in a medium supplemented with RNase-free BSA (1 mg/ml), rinsed, and directly treated for RNA extraction (see below).

Single Channel Analysis
Current Recordings. Single-channel currents were recorded with a patch-clamp amplifi er (LM-EPC 7, List Electronic; RK-400, Bio-Logic) and stored on digital audio tapes (DTR-1205, Bio-Logic). The bath reference was 0.5 M KCl in a 4% agar bridge connected to an Ag/AgCl pellet. In the cell-attached confi guration, 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 V c values were corrected for liquid junction potentials, as calculated by a routine of the AxoScope software (Axon Instruments Inc.). All experiments were conducted at room temperature.
Data Analysis. Signals were typically low-pass fi ltered at 1 kHz by an 8-pole Bessel fi lter (LPBF-48DG; npi electronic GmbH) and digitized at 3 kHz by a Digidata 1200 A/D converter (Axon Instruments Inc.). 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.
For the analysis of channel kinetics, recordings were low-pass fi ltered at 3 kHz and digitized at 10 kHz. TAC and TACfi t softwares (Bruxton Corp.) were used for semi-automatic event detection and to fi t open and closed time distribution histograms, respectively. Open and closed time intervals covering a wide range (see RESULTS) fi ts were generated on plots of the square root of the number of observations against the logarithm of the time intervals (Colquhoun and Sigworth, 1995).
Unless otherwise stated, the maximal inward conductance (g in ) was determined by linear regression analysis of the i/V c relationship between V c = −40 mV and more hyperpolarizing voltages. When appropriate, the outward chord conductance (g out ) was defi ned as the slope of the i/V c curve when measured between the reversal potential (E rev ) and V c = +60 mV.
Channel Selectivity. P K /P Na was determined in inside-out membrane patches from E rev values obtained from i-V c relationships established with opposite Na + and K + ion gradients across the membrane patch, using the Goldman, Hodgkin and Katz voltage equation (Hille, 1992): where F, R, and T have their usual meanings, P K and P Na are the permeabilities to K + and Na + ions, respectively, and o and i denote the outside and inside faces of the membrane patch, respectively.
Real-time PCR mRNA was extracted from pools of 20-50 microdissected tubules or glomeruli, according to the technique previously described Real-time PCR data are shown as mean ± SEM from 5-10 animals and are expressed in arbitrary units relative to a whole kidney RNA standard (see MATERIALS AND METHODS). *, P < 0.05; nd, not detectable. All samples positively responded to real-time PCR for cyclophilin (in arbitrary units): Glom 0.0147 ± 0.002 (n = 5), PCT 0.0424 ± 0.006 (n = 6), PR 0.0398 ± 0.003 (n = 6), MTAL 0.0192 ± 0.003 (n = 7), CTAL 0.0195 ± 0.002 (n = 9), DCT 0.0279 ± 0.003 (n = 5), CNT 0.0312 ± 0.0026 (n = 10), CCD 0.0254 ± 0.002 (n = 10), OMCD 0.0153 ± 0.0019 (n = 7). Properties of largeconductance K + channel in situ. (A) Typical recordings of large-conductance K + channel activity in the cellattached membrane patch confi guration. The CTAL tubule was bathed in the high-Na + solution, with the high-K + solution in the pipette. Recordings were obtained from the same membrane patch at the V c values indicated to the right of each trace. C-is the current level corresponding to the closure of all K + channels. (B) Unitary current amplitude i-V c relationships in the conditions given in A (■; n = 5-8) or in the presence of 75 mM K + in the pipette (○; n = 2). Inset, voltage dependence of channel activity in the conditions given in A. Channel activity is given as NP o values normalized to that at V c = 0 mV on the same patch. Each point is the mean of fi ve to eight measurements and the SEM is shown as error bars when larger than symbol. (Elalouf et al., 1993). RT was performed using the fi rst strand cDNA synthesis kit for RT-PCR (Roche Diagnostics).
A BLAST search for the base sequence that encodes for the highly conserved region of the chloride bowl (FMFRLPFA) of human, rat, and Caenorhabditis elegans Slo2 channels (Yuan et al., 2003) was performed in the mouse genome. This yielded two sequences. A 3396-bp sequence was identifi ed (GenBank/EMBL/ DDBJ accession no. XM_136252), which, when translated, showed 93% homology with the amino acid sequence of rat Slo2.1 (NM_ 198762) (Bhattacharjee et al., 2003). A 3759-bp mouse cDNA sequence was also identifi ed (AK048406), which, when translated, showed 99% homology with the 703-1238 amino acid sequence of rat Slo2.2 (NM_021853) (Yuan et al., 2003). The primers for the mouse Slo2.1 sequence were as follows: sense 5′-T A T G A T C T G-C G T T G C C C -3′ (bases 747-762) and antisense 5′-C C G A A C T T G C-A C A T C C -3′ (bases 1296-1311). The primers for the mouse Slo2.2 sequence were as follows: sense 5′

-A A A C A C A A C A G C T A C G A G -3′ (bases 188-205) and antisense 5′-G G G A A A A G C C G G A A C A -3′ (bases 555-570). The primers for the mouse cyclophilin sequence were as follows: sense 5′-A T G G C A A A T G C T G G A C C A A A -3′ (bases 339-358) and antisense 5′-G C C T T C T T T C A C C T T C C C A A A -3′ (bases 426-446).
Real-time PCR was performed on a LightCycler System (Roche Diagnostics) using the FastStart DNA Master SYBR Green 1 kit (Roche Diagnostics) on an 8-μl fi nal reaction volume as previously described (Nissant et al., 2004). In brief, measurements were performed on cDNA corresponding to either 0.1 mm of tubule or 0.2 glomerulus in the presence of either 4 pmol Slo2.2 primers/8 μl or 2 pmol Slo2.1 primers/8 μl. Samples were submitted to 50 cycles of three temperatures (Slo2.2: 95°C for 10 s, 60°C for 10 s, 72°C for 20 s; Slo2.1: 95°C for 10 s, 62°C for 10 s, 72°C for 20 s). A melting curve was then established in order to check for nonspecifi c PCR products. After 50 cycles, no DNA was detectable in blank runs.
In each experiment, a standard curve was plotted using 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 RNA. For all samples, the amount of PCR product was calculated as a percentage of the RNA standard (arbitrary unit).

Data Presentation and Statistics
Results are given as means ± SEM for n experiments. P values <0.05 (paired or unpaired t test, when appropriate; SigmaPlot, SPSS) were taken to represent statistically signifi cant differences. Nonlinear regression analyses were performed using either the SigmaPlot or the Origin (Microcal Software, Inc.) software.

Detection and Properties of a Large Conductance K + Channel in Cell-attached Membrane Patches
Using the patch-clamp technique on the basolateral membrane of collagenase-treated CTAL tubules bathed in high-NaCl solution, the activities of two types of K + channels were observed in situ. A previously described intermediate conductance K + channel  was observed in 27% (23 out of 85) of the patches. The activity of a larger conductance K + channel was also observed in ‫%21ف‬ (10 out of 85) of the patches. Both channel types were observed on the same patch in one case. Intermediate-and large-conductance K + channels were also observed on the basolateral membrane of MTAL tubules, both in 19% of the cellattached patches (6 out of 32 patches, for each channel type). In contrast, no activity of a large conductance K + channel was detected in basolateral membrane of DCT . All the data presented hereafter were obtained on patches of the basolateral membrane of CTAL tubules.
Representative current tracings of large-conductance K + channel activity in situ and the corresponding i/V c relationship are shown in Fig. 2. Channel conductance averaged 82 ± 6 pS at V c = 0 mV (n = 8) and maximal inward conductance (g in ; see MATERIALS AND METHODS) reached 138 ± 7 pS (n = 8). No outward current was detected in these conditions and E rev could be only estimated using linear regression analysis of three i/V c relationships for inward currents. A value of 62 ± 8 mV was obtained, in good agreement with the activity of a K +selective channel. This was confi rmed by two measurements of ionic currents with 75 mM K + in the patch pipette (70 mM Na + subsituted for K + ) (Fig. 2 B). Under these conditions, g in was reduced to 83 pS and E rev was shifted toward V c ‫ف‬ 42 mV. This relative shift in E rev yielded a P K /P Na value >20 (for rationale see Paulais et al., 2002).
There were usually one to four active large-conductance K + channels in the cell-attached confi guration. At V c = 0 mV, an average of 1.8 ± 0.3 current levels were observed (n = 10), with a mean NP o of 0.58 ± 0.11 (n = 10). Channel activity was little affected by membrane potential difference over the range studied (−80 to +20 mV), NP o only slightly increasing in response to membrane depolarization (Fig. 2, inset).

Channel Properties in the Inside-out Confi guration
Excising patches into a high-NaCl and low-KCl medium led to greater channel activity. Under theses conditions, the large conductance K + channel was observed on 31 out of 85 patches (36.5%). Intermediate-and largeconductance K + channels were observed on the same patch in seven cases (8.2%).
Representative tracings and the corresponding i/V c curve for the large-conductance K + channel under these conditions are shown in Fig. 3 A. Channel conductance averaged 86 ± 2 pS at V c = 0 mV (n = 13), and fi tting the i/V c data with the Goldman-Hodgkin-Katz current equation (Hille, 1992) yielded a g in value of 154 ± 4 pS (n = 13) and an E rev of 62 ± 2 mV (n = 13), confi rming the K + selectivity of the channel with a P K / P Na ratio of 20 ± 3 (n = 13).
A feature common to K Na (Mistry et al., 1996;Wang et al., 1991, Dryer 1994) and rat Slo2.1 (Bhattacharjee et al., 2003) and Slo2.2 (Yuan et al., 2003) channels is the presence of numerous subconductance levels. The examples shown in Fig. 3 B illustrate the similar behavior of the large conductance K + channel in the TAL. Intermediate current levels more frequently resulted from partial channel closures from the fully open state during a burst of activity, although partial openings also arose from the closed state. Within a burst, the time spent at subconductance levels accounted for 9.8 ± 1% of the total open time (n = 7).
A continuous recording on a patch containing one active channel at V c = 0 mV is shown in Fig. 4. Typically, channel activity occurred in bursts of openings separated by closures of various duration, with no evident sign of rundown over several minutes (unpublished data). Kinetic analysis revealed that, as observed for the K Na channels of excitable cells (Kameyama et al., 1984;Veldkamp et al., 1994;Mistry et al., 1997), the time distribution of the main open state was best fi tted by the sum of two exponentials, longer openings being the most frequent ( Fig. 4; Table I). Fitting the closed times distribution required at least four exponentials (Table I).
Closures with the longest time constant were the least frequent and displayed some variability from patch to patch. Indeed, the very long and infrequent closures seen on some recordings fell outside the fi tted relationship (unpublished data).
NP o under these conditions was 0.51 ± 0.12 (n = 8) at V c = 0 mV, with an average of 1.5 ± 0.2 channels per patch (n = 15). As observed in cell-attached membrane patches, single channel activity was moderately voltage dependent (Table I) As for K Na channels in guinea pig ventricular myocytes (Wang et al., 1991) and rat olfactory bulb neurons (Egan et al., 1992b), the blockade by internal Na + of outward currents fl owing through the TAL largeconductance K + channel induces an inward rectifi cation. Fig. 5 A compares single channel current amplitudes observed at V c = +60 mV and −60 mV under symmetrical 145 mM K + conditions, in the presence and absence of Na + on the inner face of the membrane patch. In the absence of Na + , ion currents were of similar amplitudes at both potentials. Adding 140 mM internal Na + had little infl uence on the amplitude of the inward current (V c = −60 mV), but reduced the amplitude of outward current (V c = +60 mV) by nearly 50%. Accordingly, the i/V c relationship in the absence of Na + was linear over the −70 to +80 mV range, with a conductance of 177 ± 19 pS (n = 3) (Fig. 5 B). Adding 140 mM internal Na + reduced g out to 93 ± 3 pS (n = 3; P = 0.001 vs. Na + -free), but did not affect g in (159 ± 8 pS; n = 4; P = 0.4), yielding a rectifi cation coeffi cient g in /g out of ‫.8.1ف‬ Dependence on Internal Na + and Cl − The internal Na + concentration also greatly affected channel activity. This was investigated in inside-out membrane patches bathed in a low-K + medium, where internal Na + was replaced by equimolar amounts of Li + , a cation that cannot substitute for Na + in activating K Na channels (see Dryer, 1994). Fig. 6 shows a continuous recording of an inside-out patch initially bathed by 140 mM internal NaCl then exposed to a Na + -free medium. With high NaCl, the activity of three large conductance K + channels was detected, yielding an NP o of 0.61. When all internal Na + was replaced by Li + (the internal Cl − being kept concentration to 145 mM), the K + channel currents were virtually abolished. The NP o fell to 0.05, and there was no change in single-channel current amplitude (Fig. 6, insets). K + channel activity was fully and rapidly restored when internal Na + was reintroduced (NP o = 0.69). The effects of internal Na + were concentration dependent, altering NP o in a sigmoidal fashion (Fig. 7 A). Fitting data points using a modifi ed Hill equation (see legend) yielded an EC 50 of 30 ± 1 mM Na + . A Hill coeffi cient n H of 3.95 ± 0.5 was found, suggesting that the cooperative action of four Na + ions is required to bring about channel activation.
Rat Slo2.1 and Slo2.2 channels are both also dependent on internal Cl − . We therefore fi rst examined the effects of internal Na + concentration after reducing the internal Cl − concentration down to 40 mM. As also illustrated in Fig. 7 A, K + channel activity was still dependent on the internal Na + concentration, but there was a clear right shift of the dose-response relationship. Under these low Cl − conditions, EC 50 was 74 ± 2.2 mM Na + and n H was 2 ± 0.13.
Only one patch with a single active channel was seen in this experimental series. This recording, obtained under low Cl − conditions, is shown in Fig. 7 B. It is reassuring to Figure 5. Na + -induced inward rectifi cation. (A) Single channel current tracings from inside-out membrane patches bathed in a symmetrical high-K + solution, at the indicated V c values. The medium bathing the cytosolic side of the patch was either a Na + -free medium (0 Na + + 5 mM K-EDTA) or a high-Na + medium (140 Na + + 5 mM K-EDTA) medium. (B) i/V c relationships for the 0 Na (■) and 140 Na (□) conditions. Each point is the average of either 2-3 (■) or 3-4 (□) measurements. When appropriate, SEM is shown as error bars when larger than symbol. observe that, as previously observed for K Na channels in excitable cells (Kameyama et al., 1984;Mistry et al., 1997), a predominant effect of reducing internal Na + was an increase in the number and duration of the intervals between bursts of activity. The concentration dependence of the effects of internal Cl − on channel activity was then examined (Fig. 8). Under 140 mM internal Na + concentration, varying internal Cl − concentration over the 0-145 mM range dose dependently affected channel activity, with EC 50 = 35 ± 9.8 mM and n H = 1.3 ± 0.25. These results constitute the fi rst demonstration of a native Na + -sensitive K + channel being coactivated by internal Cl − .

Inhibition by Internal Ca 2+ and the Lack of Effect of Internal ATP and pH
The activity of the large conductance K + channel was greatly dependent on the intracellular Ca 2+ concentration. As illustrated in Fig. 9 A, we observed that channel activity was greatest in a Ca 2+ -free medium ‫1ف(‬ nM free Ca 2+ ) and was reduced by increasing the free Ca 2+ concentration. At 1 μM Ca 2+ , NP o was ‫%03ف‬ of that in Ca 2+free medium (P = 0.0007; n = 4), and channel activity was further decreased by increasing free Ca 2+ concentration to 1 mM (NP o = 14 ± 3% of that in the Ca 2+ -free medium; n = 6). The dose-response curve for the inhibitory effect of internal Ca 2+ (Fig. 9 B) displayed EC 50 ‫003ف‬ nM.
Adding 2 mM Mg-ATP to the cytoplasmic side of inside-out patches for up to 1 min had no infl uence on channel activity (P = 0.49 ; n = 4). The effect of the internal pH (pH i ) on the activity of the channel was investigated by comparing single channel activities obtained at either pH i 8 or pH i 6.8 to that at pH i 7.4. In the presence of 140 mM internal Na + , no statistically signifi cant infl uence on NP o was observed by either decreasing or increasing pH i (n = 3 for each condition).

D I S C U S S I O N
Comparison with Native Na + -activated K + (K Na ) Channels of Excitable Cells The 150 pS Na + -activated K + channel in the basolateral membrane of the mouse TAL exhibits little voltage dependence, a mild Na + -induced rectifi cation, several conductance substates, and is ATP and pH independent. These functional properties are similar to those commonly reported for K Na channels of excitable cells and Xenopus oocytes (see Dryer 1994). First, the conductance of K Na channels ranges from 100 to 200 pS, and the occurrence of multiple substates has been frequently reported (Wang et al., 1991;Mistry et al., 1996). Second, internal Na + induces an inward rectifi cation of K Na channels. The intermediate rectifi cation coeffi cient (g in /g out ) of ‫2ف‬ that we observed here is close to the ratio of ‫3ف‬ which can be deduced from published data on K Na channels in guinea pig cardiomyocytes (Wang et al., 1991) and rat olfactory bulb neurons (Egan et al., 1992b). Third, K Na channel open probability in excitable cells is usually weakly (Haimann et al., 1990;Egan et al., 1992b;Koh et al., 1994) or not (Kameyama et al., 1984;Safronov and Vogel, 1996;Mistry et al., 1997) potential dependent, and very moderately pH sensitive within the 6.5-8 pH range in the presence of an elevated internal Na + concentration (Veldkamp et al., 1994). Finally, K Na channel activity in guinea pig cardiomyocytes (Kameyama et al., 1984) and in rat bulb olfactory neurons (Egan et al., 1992b) is not affected by internal ATP.
The activity of the 150 pS channel in the TAL increases with internal Na + . In the presence of 145 mM internal Cl − , the half-maximum activating Na + concentration (EC 50 ) and Hill coeffi cient (n H ) were 30 mM and 4, respectively. These values closely match the EC 50 (30-75 mM) and n H (2.4-4.6) values reported for K Na channels of excitable membrane under similar conditions Figure 6. Na + dependence of channel activity. A continuous single channel recording from an inside-out membrane patch whose internal face is exposed to either a Na + -containing medium (140 NaCl) or a Na + -free (0 NaCl + 140 LiCl) medium. Pipette and bath K + concentrations were 145 and 5 mM, respectively, and V c was set at −40 mV. C and the dotted line indicate the current level corresponding to the closure of all K + channels. Inset, a, b, and c, excerpts displayed at an expanded time scale (*) taken from the indicated sections of the trace. (Dryer, 1994). These properties provide functional identifi cation of the 150 pS K + channel of TAL basolateral membrane as a K Na channel.

Comparison with Slo2 Channels
To date, two paralogues, Slo2.1 and Slo2.2, have been shown to encode for Na + -and Cl − -activated, largeconductance K + channel (Bhattacharjee et al, 2003;Joiner et al, 1998;Yuan et al, 2000Yuan et al, , 2003. Using real-time PCR, Slo2.2 mRNA, but not that of Slo2.1, was detected in the TAL. This strongly suggests that Slo2.2 underlies the TAL K Na channel. The heterologous expression of Slo2.1 and Slo2.2 yields K + channels with very similar conductive properties (Joiner et al., 1998;Bhattacharjee et al., 2003;Yuan et al., 2000Yuan et al., , 2003. Both genes encode for 140-180 pS K + channels with subconductance levels that are coactivated by internal levels of both Na + and Cl − . Several functional observations confi rm that Slo2.2 underlies the K Na channel in the TAL. First, the Na + -dependent activation of TAL K Na channel also depends on the internal Cl − , and reducing the internal Cl − from 140 to 40 mM increased the EC 50 for Na + from 30 to 74 mM (Fig. 7). These values are of the same order of magnitude as those reported for Slo2.2 channels under similar conditions (Yuan et al., 2003). Furthermore, Slo2.1  Fig. 6 or in the presence of 40 mM internal Cl − (□). Under both conditions, the NP o at a given internal Na + concentration was normalized to the NP o obtained in the presence of 140 mM NaCl. Each point is the mean of at least three experiments, and the SEM is shown as error bars when larger than symbols. The relationships between the Na + concentration and NP o were described by the equation NP o = 1/[1 + ([Na + ]/ K) nH ], where [Na + ] is the internal Na + concentration, K is the half-maximum activating concentration (EC 50 ), and n H is the Hill coeffi cient. (B) Typical recordings of channel activity with the low-K + solution in the bath, and with the high-K + solution in the pipette. Recordings were obtained from the same membrane patch at the internal Na + concentrations indicated to the right of each trace. Bath Cl − concentration was maintained at 40 mM. C-is the current level corresponding to the closure of all K + channels. displays a signifi cant activity in a 35 mM Cl − and Na +free medium (Bhattacharjee et al., 2003), whereas Slo2.2 is inactive under these conditions (Bhattacharjee et al., 2003;Yuan et al., 2003). Similar ionic conditions (0 Na + and 40 mM Cl − , Fig. 7) abolished TAL K Na channel activity. Also, the EC 50 for Cl − (35 mM, Fig. 8) we observed in the presence of an elevated internal Na + concentration is of the same order of magnitude as that reported for Slo2.2 channel ‫02ف(‬ mM Cl − ) under similar conditions (Yuan et al., 2003).
Second, Slo2.1 is inhibited by internal ATP (Bhattacharjee et al., 2003), possibly via the presence of a consensus ATP binding motif in the COOH-terminal tail of its sequence. There is no such ATP binding motif in the Slo2.2 sequences of the rat (Bhattacharjee et al., 2003) and mouse (present study). Although the possi-bility that ATP could inhibit rat Slo2.2 by ATP was not tested for (Bhattacharjee et al., 2003), the fact that ATP had no effect on the TAL K Na channel clearly shows that this channel is not of a Slo2.1 type.
Finally, an elevation in the cytosolic free Ca 2+ concentration inhibits rat Slo2.2 (Joiner et al., 1998). Our observation that K Na channel activity in the TAL also decreased in response to an increase in the cytosolic free Ca 2+ is also consistent with the presence of a Slo2.2 channel.

Physiological Role of a K Na Channel in the TAL Basolateral Membrane
The TAL transcellularly reabsorbs 15-20% of the fi ltered NaCl load. Basically, NaCl enters TAL cells via an apical Na + -K + -2Cl − cotransporter driven by the basolateral Na + /K + ATPase and, on the basolateral side, Cl − exits through chloride channels (Fig. 10). Basolateral K + channels fi rst maintain the membrane potential difference and thus the driving force for basolateral Cl − diffusion and, second, provide a major pathway for the effl ux of K + ions entered into the cell by Na + /K + ATPase activity. We  and others (Hurst et al., 1992;Gu and Wang, 2002) have previously demonstrated the presence of an ‫54ف‬ pS K + channel in this membrane. This channel is observed in ‫%03ف‬ of cell-attached patches, with an average NP o of 1.9 and a conductance of ‫01ف‬ pS under physiological conditions . This makes it possible to calculate a mean conductance of ‫6ف‬ pS per membrane patch. The K Na channel was observed here in ‫%21ف‬ of the patches, with an average NP o of 0.6. Assuming a conductance of ‫54ف‬ pS under physiological conditions (Safronov and Vogel, 1996), one may calculate a conductance of ‫3ف‬ pS per membrane patch. Thus, the 150 pS K + channel would account for ‫%33ف‬ of the basolateral Figure 9. The activity of the K Na channel in the CTAL is reduced by intracellular Ca 2+ . (A) Single channel recordings from a cellexcised inside-out membrane patch exposed to the indicated intracellular Ca 2+ concentration ([Ca 2+ ] i ). Recordings were made with a high-K + pipette solution and a high-Na + bath solution, and V c was set at 0 mV. C indicates the current level corresponding to the closure of all K + channels. (B) Summary of results obtained in the conditions given in A. Each point is the mean ± SEM of 4-6 experiments. NP o is given as the channel open probability normalized to that in 1 nM Ca 2+ (0 Ca 2+ + 2 EGTA). membrane K + conductance. But it should be pointed out that a signifi cant fraction of K Na channels may be inactive under resting conditions, since the channel frequency of observations increased from 12% to 35% after patch excision into a Na + -rich medium. This may indicate that this channel is in a position to more signifi cantly contribute to the K + conductance of the TAL basolateral membrane.
In situ the TAL K Na channel has an NP o of ‫.6.0ف‬ This observation is somewhat surprising since the Na + and Cl − sensitivities we measured in cell-free patches indicate that the channel should be nearly inactive in situ. Thus, resting intracellular Na + and Cl − concentrations in the TAL are ‫51ف‬ mM (Rajerison et al., 1986;Lu et al., 1996) and ‫53ف‬ mM (Greger et al., 1983), respectively. According to the Na + dependence relationship in the presence of 40 mM Cl − in cell-free patches, channel activity is very low under these conditions. Furthermore, these later experiments were performed under Ca 2+free conditions, whereas we observed that the activity of the channel was inhibited when the cytosolic free Ca 2+ concentration was increased to a value near resting free Ca 2+ concentration in the mouse TAL (Paulais et al., 1996). This further indicates that the TAL K Na channel sensitivities to internal Na + and Cl − we measured in cellexcised membrane patches are indeed underestimated, as reported for the Na + sensitivity of K Na channel in excitable cells (Haimann et al., 1992;Rodrigo, 1993), and suggests that they may be subject to modulation. Further studies are obviously needed to identify these modulators in order to provide a more precise picture of the conditions under which it is activated, and hence its role in the reabsorption of NaCl in the TAL.
The interior of TAL cells has to cope with large fl uxes of Na + and Cl − . According to the TAL transport scheme, stimulating apical Na + and Cl − infl ux is expected to produce a rapid increase in the Na + /K + ATPase-mediated Na + effl ux, and a parallel increase in basolateral K + conductance. Accordingly, apical and basolateral membranes must be functionally linked in order to maintain ionic reabsorption while preventing major perturbations in cellular ionic content and volume (Schultz and Lapointe, 2000). The nature of this functional linkage (or cross-talk) has been debated (Schultz and Lapointe, 2000). Numerous studies in frog skin and in amphibian and mammalian nephrons have provided evidence for the involvement of several factors, including membrane potential, cell volume, and intracellular Ca 2+ , pH, and ATP (Schultz and Lapointe, 2000). We therefore propose that the basolateral K Na channel may be involved in membrane cross-talk in the TAL by means of its sensitivity to intracellular Na + and Cl − concentrations. In this case, an elevation in intracellular Na + and Cl − concentrations would be expected to activate the K Na channel, leading to a coupling between increased Na + /K + ATPase activity and basolateral recycling of K + .
In conclusion, the present study provides the fi rst evidence of the presence of a K Na channel in an epithelial cell membrane. So far, K Na channels were thought to be present primarily in the membranes of excitable cells (Dryer, 1994). Our fi ndings, together with the observation of K Na channels in Xenopus oocytes (Egan et al., 1992a), may indicate that these channels have hitherto unsuspected functional roles.