Properties of an inwardly rectifying K+ channel in the basolateral membrane of mouse TAL

Marc Paulais, Stéphane Lourdel, and Jacques Teulon

Institut National de la Santé et de la Recherche Médicale U.426, Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, Université Paris 7, 75018 Paris, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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We investigated the properties of K+ channels in the basolateral membrane of the cortical thick ascending limb (CTAL) using the patch-clamp technique. Approximately 34% of cell-attached patches contained an inwardly rectifying K+ channel (K+-to-Na+ permeability ratio ~22), having an inward conductance (Gin) of 44 pS and an outward conductance (Gout) of ~10 pS (Gin/Gout ~ 4). Channel activity (NPo) increased with depolarization. When the cytosolic sides of inside-out patches were exposed to an Mg2+-free medium, the channel had a Gin of 50 pS and was weakly inwardly rectifying (Gin/Gout ~ 1). Cytosolic Mg2+ reduced Gout, yielding a Gin/Gout of 3.8 at 1.3 mM Mg2+. Internal Na+ also yielded a Gin/Gout of 1.6 at 20 mM Na+. Spermine reduced NPo on inside-out membrane patches. Sensitivity to spermine at depolarizing voltages [half-maximal inhibitory concentration (Ki) = 0.2 µM] was much greater than at hyperpolarizing voltages (Ki = 26 µM). Half-inactivation by 0.5 µM spermine occurred at a clamp potential of 43 mV, with an effective valence of 1.25. A sigmoid relationship between bath pH and NPo of inside-out membrane patches was observed, with a pK of 7.6 and a Hill coefficient of 1.8. Intracellular acidification also reduced the NPo of cell-attached patches. This channel is probably a major component of K+ conductance in the CTAL basolateral membrane.

thick ascending limb of Henle's loop; basolateral membrane; potassium channel; patch clamp; mouse


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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THE THICK ASCENDING LIMB (TAL) of Henle's loop of the mammalian nephron reabsorbs ~30% of the NaCl filtered by the glomerulus and plays a crucial role in urinary concentrating ability. Basically, Na+ and Cl- ions enter the cell via a Na+-K+-2Cl- cotransporter at the apical membrane and, on the basolateral side, Cl- ions leave the cell through a Cl- conductance and a K-Cl cotransporter (Ref. 41; see also Fig. 9). In addition, a basolateral K+ conductance has been detected in the hamster medullary TAL (MTAL) (54), the rabbit cortical TAL (CTAL) (3), and the Amphiuma early distal tubule (13), the diluting segment in amphibians, and plays a key role in NaCl reabsorption by the TAL. This conductance would maintain basolateral membrane potential (VBl) difference and thus the driving force for the basolateral diffusion of Cl-. In addition, by participating in the basolateral exit of K+ ions and compensating for part of the influx of K+ ions associated with Na+/K+-ATPase activity (24), this conductance is essential for the generation of a chemical gradient of Na+ ions promoting apical Na+-K+-2Cl- cotransport activity.

The properties of ion channels underlying the basolateral potassium conductance of renal epithelial cells have been mainly studied in the proximal and cortical collecting tubules of various species (see Ref. 45). In contrast, very little is known about the conductive properties of the basolateral K+ channels in the TAL. The only patch-clamp study on this subject was restricted to channel properties in the cell-attached configuration and identified a 35-pS, inwardly rectifying K+ channel in rabbit CTAL (22).

The present study was undertaken to identify the basolateral potassium channels of mouse CTAL tubules. We were particularly interested in characterizing the conductive and regulatory properties of the channel that would allow a fruitful comparison with potassium channels described in other nephron segments and with cloned potassium channels.


    MATERIALS AND METHODS
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INTRODUCTION
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Chemicals. EDTA (disodium or dipotassium salt, when appropriate), ATP (disodium salt), and N,N'-bis[3-aminopropyl]-1,4-butanediamine tetrahydrochloride (spermine; SPM) were from Sigma-Aldrich Chimie (Saint Quentin Fallavier, France). EGTA was from Research Organics (Cleveland, OH) or from Sigma-Aldrich Chimie.

Tubule preparation. Male 15- to 20-g CD1 (Charles River France, Saint Aubin Lès Elbeuf, France) or ICR (Harlan France, Gannat, France) mice were killed by cervical dislocation. CTALs were microdissected as previously described (14). Briefly, the left kidney was first rinsed with L-15 Leibovitz medium (Eurobio, Les Ulis, France; GIBCO BRL, Life Technologies, Cergy Pontoise, France) containing 300 U/ml collagenase (300 U/ml CLS II; Worthington, Freehold, NJ). Small pieces of cortex were then incubated at 37°C for 45-75 min in this collagenase-containing medium, rinsed, and kept at 4°C until microdissection. No further enzymatic or mechanical treatment of tubules was necessary for successful seal formation.

Current recordings. Cell-attached and cell-excised, inside-out variants of the patch-clamp technique (16) were applied to the basolateral membrane of CTAL fragments. Single-channel currents were recorded with a patch-clamp amplifier (LM-EPC 7, List Electronic, Darmstadt, Germany; RK-400, Bio-Logic, Claix, France) and stored on digital audiotape (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 configuration, the clamp potential (Vc = Vbath - Vpipette) is superimposed on the cell membrane potential (Vm). In excised inside-out membrane patches, Vc = Vm. All Vc values were corrected for liquid-junction potential, as calculated by a routine of AxoScope software (Axon Instruments, Foster City, CA). The accuracy of these calculations for our experimental setup was confirmed by direct measurements utilizing a procedure described previously (30). Cations flowing from the inner to the outer face of the membrane patch are positive and shown as upward deflexions in current tracings. All experiments were conducted at room temperature.

Data analysis. Signals were generally low-pass filtered at 500 Hz by an eight-pole Bessel filter (LPBF-48DG; NPI Electronic, Tamm, Germany) and digitized at 3 kHz with a Digidata 1200 analog-to-digital converter and AxoScope software (Axon Instruments). For analysis of conductance substates (see RESULTS), signals were filtered at 2 KHz and digitized at 10 KHz. In most cases, channel activity was quantified using software kindly provided by Prof. T. Van den Abbeele (Paris, France). The mean current (I) passing through N channels was estimated from current-amplitude histograms and used to calculate the normalized current (NPo) according to the equation NPo = I/i, where i is the unit current amplitude. In some cases (see RESULTS), NPo was measured with AxoScope software on the basis of visual differentiation between the fast and noisier K+ channel openings and the slower kinetics of 9-pS Cl- channels (14, 15).

Channel selectivity. PK/PNa, the permeabilities to K+ and Na+ ions, respectively, was determined from both cell-attached and inside-out patches. In cell-attached patches, Vm was set close to 0 mV by superfusing the tubules with a high-K solution. The shift in reversal potential (Delta Erev) was then estimated from i-Vc relationships obtained with various K+ and Na+ concentrations in the pipette while the sum of K+ and Na+ concentrations was kept constant. PK/PNa was calculated using the Goldman-Hodgkin-Katz voltage equation (17), taking the Erev in the presence of 144.8 mM K+ in the pipette (Erev[144.8K]pip) as a reference
&Dgr;E<SUB>rev</SUB><IT>=E</IT><SUB>rev</SUB>[(144.8<IT>−xK</IT>)<IT>+x</IT>Na]<SUB>pip</SUB><IT>−E</IT><SUB>rev</SUB>[144.8<IT>K</IT>]<SUB>pip</SUB>

<IT>=</IT><FR><NU><IT>RT</IT></NU><DE><IT>F</IT></DE></FR> ln <FR><NU>[<IT>x</IT>Na]<SUB>pip</SUB><IT>+</IT>[(<IT>P</IT><SUB>K</SUB><IT>/P</IT><SUB>Na</SUB>)[144.8<IT> − xK</IT>]<SUB>pip</SUB>]</NU><DE>(<IT>P</IT><SUB>K</SUB><IT>/P</IT><SUB>Na</SUB>)[144.8<IT>K</IT>]<SUB>pip</SUB></DE></FR>
where F is the Faraday constant, R is the universal gas constant, and T is absolute temperature. The use of this equation is based on the assumptions that channel anion permeability is negligible (see RESULTS) and that intracellular Na+ and K+ concentrations between cells are constant. PK/PNa on inside-out membrane patches was determined from Erev values obtained with opposite Na+ and K+ ion gradients across the membrane patch, using the equation
E<SUB>rev</SUB><IT>=</IT><FR><NU><IT>RT</IT></NU><DE><IT>F</IT></DE></FR> ln <FR><NU>[Na]<SUB>o</SUB><IT>+</IT>{(<IT>P</IT><SUB>K</SUB><IT>/P</IT><SUB>Na</SUB>)[K]<SUB>o</SUB>}</NU><DE>[Na]<SUB>i</SUB><IT> + </IT>{(<IT>P</IT><SUB>K</SUB><IT>/P</IT><SUB>Na</SUB>)[K]<SUB>i</SUB>}</DE></FR>
where o and i denote the outside and inside faces of the membrane patch, respectively.

Solutions. Tubules were bathed either in a high-Na solution containing (in mM) 140 NaCl, 4.8 KCl, 10 glucose, and 10 HEPES-NaOH, pH 7.4, or in a high-K solution containing 144.8 KCl, 10 glucose, and 10 HEPES-KOH, pH 7.4. Free Ca2+ and Mg2+ concentrations were adjusted as required (see below). Unless otherwise stated, pipettes were filled with the high-K solution. Lower K+/Na+ concentration ratios were obtained by mixing appropriate volumes of high-K and high-Na solutions, both the sum of K and Na pipette concentrations ([K+]pip + [Na+]pip) and the Cl- pipette concentration ([Cl-]pip) being kept at 144.8 mM. Higher values of [K+]pip were obtained by adding the appropriate amount of KCl to the high-K solution. No correction was made for changes in osmolarity. When necessary (see RESULTS), ion currents through 9-pS Cl- channels were minimized by using solutions in which all but 4.8 mM Cl- had been replaced by NO<UP><SUB>3</SUB><SUP>−</SUP></UP> (14). The pipette solution contained 1.2 mM free Mg2+. The bath solution typically contained 1.2 mM free Mg2+ and 1 mM free Ca2+. Various concentrations of free Mg2+ under low free Ca2+ concentrations were obtained with either EDTA or EGTA. The proportions of Mg2+ and EDTA or EGTA were calculated using the absolute stability constants for Mg2+, Ca2+, and H+ and EDTA (5) or EGTA (4, 5, 29).

Data presentation and statistics. Results are given as means ± SE for n patches. P values <0.05 (paired or unpaired t-test, when appropriate; SigmaPlot or SigmaStat, SPSS, Erkrath, Germany) were taken to represent statistically significant differences. Nonlinear regression analyses were performed with Origin software (Microcal Software, Northampton, MA). All-points amplitude histograms were constructed by TAC event detection and analysis software (Bruxton, Seattle, WA) from current traces after baseline current subtraction.


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K+ channel activity was observed in 34% (52 of 153) of cell-attached membrane patches of CTAL tubules bathed in the high-NaCl solution. With no applied potential, the number of active K+ channels per patch averaged 5.8 ± 0.5 (n = 52), with a mean NPo of 1.9 ± 0.4 (range 0.02-10.49; n = 40) (Fig. 1A). We found no correlation between the tip resistance of KCl-filled pipettes (4-13 MOmega ) and the number of active channels (r = 0.017).


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Fig. 1.   K+ channel activity (NPo) in cell-attached patches. A: no. of patches displaying the indicated NPo at a clamp potential (Vc) = 4 mV. Tubules were bathed in high-Na solution, with the high-K solution in the pipette. B: typical recordings of K+ channel activity in the conditions given in A. Recordings were from the same patch at the Vc values given to the right of each trace. C---, current level corresponding to closure of all K+ channels. Note the expanded scale for current amplitude obtained at +104 mV (*).

General properties of cell-attached patches. Figure 1B shows recordings of channel activity in a cell-attached patch from a tubule bathed in the high-NaCl solution, the pipette being filled with the high-KCl solution. The corresponding i-Vc relationship is shown in Fig. 2A. Under these conditions, EK across the membrane patch should be close to 0 mV, and Erev provides an estimate of Vm. Erev was 75 ± 1 mV (n = 22), in good agreement with the Vm values of -70/-80 mV reported for isolated perfused rabbit CTAL (12) and mouse MTAL (42) tubules. The i-Vc relationship for inward currents was nearly linear, single-channel Gin averaging 43.5 ± 0.6 pS (n = 22). Measurements of outward currents beyond Erev were hampered by the openings of previously described small-conductance Cl- channels (14) (not shown), but the current amplitude, measured on three occasions at 114 and 124 mV, was lower than expected for an ohmic K+ conductance (see Fig. 2A). The use of low-Cl- solutions (see MATERIALS AND METHODS) facilitated measurements of K+ currents at more depolarizing voltages (not shown) and yielded an outward chord conductance [G(chord)out], as measured between Erev and 144 mV, of 11 ± 0.6 pS (n = 3), whereas Gin was 44 ± 2 pS (n = 9), indicating inward rectification.


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Fig. 2.   K+ channel properties in cell-attached patches. A: unit current amplitude (i)-Vc relationships in conditions of Fig. 1 () or in tubules bathed in a high-K solution (). Each point is the mean of 3-22 measurements, except at Vc = 124 mV, where n = 2. SE are error bars when larger than symbols. Inset: voltage dependence of NPo in conditions given in A (). Each point is the mean of 5-14 measurements, and error bars are SE. Dotted line, nonlinear least squares fit of Boltzmann equation (see text) to the data given by maximum NPo (NPo max) = 3.75, voltage when NPo is NPo max/2 (V1/2) = -11.5 mV, and effective valence (z) = 0.7 (r = 0.846). B: relationship between channel conductance and K+ concentration in the pipette ([K+]pip). For each [K+]pip value, the conductance at Vc = 0 mV (G0 mV) was taken as the slope of the i-Vc relationship between -10 and 10 mV in tubules bathed in high-Na solution. Each point is the mean of at least 3 determinations, and SE values are shown as error bars when larger than symbols. Dotted line, nonlinear least squares fit to mean values given by maximal G0 mV [G0 mV(max)] = 58 pS, dissociation constant (Km) = 83 mM, and value of G0 mV when [K+]pip = 0 [G0 mV(0)] = 7 pS (see text). C: ionic selectivity. The shift in reversal potential (Delta Erev) was determined as described in MATERIALS AND METHODS from i-Vc relationships established in the cell-attached configuration in the presence of the indicated [K+]pip value. Each point is the mean of at least 4 determinations, and SE values are shown as error bars when larger than symbols. Dotted line through data points, nonlinear least squares fit using the Goldman-Hodgkin-Katz voltage equation (see MATERIALS AND METHODS) and a K-to-Na permeability ratio (PK/PNa) of 22.

To precisely establish inward rectification, we determined i-Vc relationships from tubules bathed in a high-K solution. With the high-K solution in the pipette, single-channel currents reversed at 2 ± 0.7 mV (P = 0.02 vs. 0 mV; n = 5), which was consistent with a fairly constant and nearly zero Vm, and Gin averaged 44 ± 1 pS (n = 5). As seen in Fig. 2A, it is clear that outward currents were smaller than inward currents. Thus G(chord)out measured between Erev and +70 mV was 11 ± 1.2 pS (P = 0.001 vs. Gin, paired t-test; n = 4). A Gin/Gout ratio of 4.94 ± 1.45 (n = 4) was obtained. Therefore, the channel we observed here belongs to the inwardly rectifying K+ channel family.

NPo of Na-bathed tubules increased with depolarization up to Vc = 44 mV (Fig. 2A, inset). The relationship between NPo and Vc was well described by the Boltzmann distribution equation (17)
NP<SUB>o</SUB><IT>=</IT><FR><NU><IT>NP</IT><SUB>o max</SUB></NU><DE>1<IT>+</IT>exp<FENCE><FR><NU><IT>−zF</IT>(<IT>V</IT><SUB>c</SUB><IT>−V</IT><SUB>1<IT>/</IT>2</SUB>)</NU><DE><IT>RT</IT></DE></FR></FENCE></DE></FR>
where NPo max is the maximum NPo, V1/2 is the voltage when NPo is NPo max/2, and z is the effective valence. Here, V1/2 = -11.5 mV and z = 0.7. NPo tended to decrease at more depolarizing voltages, but there were too few observations to permit a quantitative description of the data.

The effect of [K+]pip on channel properties was also studied in CTAL tubules bathed in the high-Na solution. Varying [K+]pip over the 18-400 mM range had no influence on the number of active channels per patch or on NPo (P = 0.65, 1-way ANOVA; data not shown) but shifted Erev and altered Gin. The effects of [K+]pip on Gin were quantified by taking the slope of the corresponding i-Vc relationships at 0 mV, G0 mV. Plotting of G0 mV as a function of [K+]pip revealed saturation (Fig. 2B), which was well described by the modified Michaelis-Menten equation (17) ZZZ, where G0 mV(max) is the maximal G0 mV, Km is the dissociation constant, and G0 mV(0) is the value of G0 mV when [K+]pip = 0. The best fit of experimental data points was obtained with G0 mV(max) = 58 pS, Km = 83 mM, and G0 mV(0) = 7 pS. Similar Km values were obtained at Vc of -20 and 20 mV (111 and 98 mM, respectively; not shown).

PK/PNa in cell-attached patches was determined as indicated in MATERIALS AND METHODS. The relative shift in Erev (Delta Erev) (Fig. 2C) on the variance in [Na+]pip/[K+]pip yielded a PK/PNa value of 22. Preliminary results also revealed negligible anion permeability (not shown).

Inspection of K+ channel activity recordings in cell-attached membrane patches revealed conductance substates (Fig. 3). However, because these partial openings contributed too little to the total open time and because of the low occurrence of patches containing only one active K+ channel (see Fig. 1A), the partial openings could not be discriminated from all-points amplitude histograms. We therefore manually measured the duration of these partial openings from stretches of cell-attached recordings where current due to one active channel was reached at Vc = 0 mV (Fig. 3A). Three conductance substates, S1, S2, and S3, emerged (Fig. 3A), having respective fractional amplitudes of 0.25 ± 0.03 (n = 4), 0.4 ± 0.03 (n = 6), and 0.7 ± 0.05 (n = 6) (Fig. 3B). Within a burst, S1, S2, and S3 accounted for 3.8 ± 0.6 (n = 4), 7 ± 7 (n = 6), and 9.5 ± 4.6% (n = 6) of the total open time, respectively (not shown).


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Fig. 3.   Conductance substates of cortical thick ascending limb of Henle's loop (CTAL) K+ channels. A: excerpts of single-channel recordings from the same cell-attached patch. Tubule was bathed in high-Na solution, and patch was clamped at 0 mV. C, S1, S2, S3, and O: closed level, 1st, 2nd, and 3rd subconductance levels, and level of complete opening, respectively. B: histogram of current amplitude of each conductance substate, given as the fraction of the fully open level. S1, S2, and S3 were significantly different from each other (1-way ANOVA).

Channel conductive properties in cell-excised patches. As for basolateral K+ channels from rabbit CTAL (22), the activity of basolateral K+ channels from mouse CTAL was very rapidly lost on patch excision into physiological saline. However, as with other K+ channels, the rundown of which can be partly antagonized by using a Mg2+-free bath (35), we found that CTAL K+ channel activity could be maintained for up to 20 min by carrying out patch excision in a Mg2+- and Ca2+-free solution (+5 mM of either Na2-EDTA or K2-EDTA) in ~88% of patches. Results presented below were obtained according to this procedure.

We confirmed the high PK/PNa ratio of the channel in cell-free patches. Thus, with the high-K solution kept in the pipette, the replacement of all but 39.8 mM KCl by NaCl in the bath shifted the reversal potential of the i-Vc curves by 29.5 ± 0.84 mV (n = 5) (not shown), which corresponds to a PK/PNa of 33. Channel selectivity to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was also measured in the presence of the 144.8 mM KCl solution on the cytoplasmic side of inside-out patches (not shown). The substitution of 144.8 mM NH4Cl for pipette KCl shifted Erev of i-Vc curves toward a negative value. Because inward currents could be resolved in only one of four experiments, Erev was extrapolated from the average i-Vc curves for outward currents and was estimated to be approximately -51 ± 3 mV (n = 4). This yielded a K+-NH<UP><SUB>4</SUB><SUP>+</SUP></UP> PK/PNa of ~7.3 ± 0.7.

Mechanisms underlying K+ channel inward rectification. Under symmetrical, high-K conditions, the i-Vc relationships in the absence of Mg2+ (+5 mM K2-EDTA) (Fig. 4B) were linear. The mean values from four experiments were as follows: Gin and Gout were 50.5 ± 2 and 47.9 ± 2 pS, respectively (P = 0.41, paired t-test). The Gin/Gout ratio was 1.06 ± 0.06 (n = 4). We then investigated whether inward rectification in cell-attached patches resulted from a voltage-dependent block of outward currents by intracellular Mg2+ ions, as reported in other inwardly rectifying K+ channels (32, 37). Figure 4A compares the current amplitudes of an inside-out membrane patch, with its cytosolic side exposed to either an Mg2+-free medium (no Mg2+ + 5 mM K2-EDTA) or to a medium containing 1.3 mM Mg2+ (1.5 mM MgNO3 and 2 mM EGTA), at Vc of +70 mV. Internal Mg2+ reduced the single-channel amplitude of outward currents (Fig. 4A) and had little influence on inward currents (traces not shown). The corresponding i-Vc relationships are shown in Fig. 4B. Addition of 1.3 mM cytosolic Mg2+ slightly but significantly (P < 0.01) reduced Gin (42 ± 1.7 pS, n = 4, vs. 50.5 ± 2 pS, n = 4), but its most striking effect was a major reduction in G(chord)out (measured between Erev and +70 mV), which fell to 12.2 ± 1.58 pS (n = 4). The resulting Gin/Gout ratio was 3.8 ± 0.2 (n = 4). The i-Vc relationship for outward currents obtained under these conditions was very similar to that obtained from cell-attached patches on KCl-depolarized cells (compare with Fig. 2), and indeed the Gin/Gout ratios were not statistically different (P = 0.29, unpaired t-test).


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Fig. 4.   Effects of internal Mg2+ and Na+ on i-Vc relationships of basolateral CTAL K+ channel. A: current traces from an inside-out membrane patch bathed in symmetrical high-K+ and the cytosolic side of which was bathed by either a medium containing 0 Mg2+ (0 Mg2++5 mM K2-EDTA; top trace) or a medium containing 1.3 mM Mg2+ (1.5 mM Mg2++2 mM EGTA; bottom trace). Clamped potential was +70 mV. C, current level corresponding to closure of all K+ channels. Also shown are corresponding all-point amplitude histograms from traces after baseline current subtraction (bottom). Solid lines are fits with a sum of 5 (0 Mg2+) or 3 (1.3 Mg2+) Gaussian components by the maximum-likelihood method. Means of components corresponding to channel opening level were 3.3 ± 0.75, 6.5 ± 0.74, 9.5 ± 0.8, 12.6 ± 0.79, and 16.5 ± 0.45 pA for 0 Mg2+ and 0.9 ± 0.21 and 1.9 ± 0.25 pA for 1.3 Mg2+. B: averaged i-Vc relationships corresponding to the conditions given in A for 0 () or 1.3 mM Mg2+ (black-triangle) or in the presence of a medium containing 0 Mg2+ (+5 mM K2-EDTA) supplemented with 20 mM Na+ on the cytosolic side of the patch (open circle ). Each point is the average of at least 3 determinations, and SE values are shown as error bars when larger than symbols. Solid line and black-triangle, best fit to respective data, as given in RESULTS.

These data were analyzed according to a model in which Mg2+ moves into the electric field of the pore, as described by Woodhull (17), which assumes a single Mg2+-binding site located within the pore, using the equation
I<SUB>Mg</SUB><IT>=</IT><FR><NU><IT>G·V</IT><SUB>c</SUB></NU><DE>1<IT>+</IT><FENCE><FR><NU>[Mg]</NU><DE><IT>K</IT><SUB>o</SUB></DE></FR><IT> · </IT>exp<FENCE><FR><NU><IT>zFV</IT><SUB>c</SUB><IT>&dgr;</IT></NU><DE><IT>RT</IT></DE></FR></FENCE></FENCE><SUP><IT>a</IT></SUP></DE></FR>
where IMg is the current in the presence of Mg2+, G is the inward conductance, z is the valence of 2, a is the number of binding sites (here, a = 1), delta  is the electrical distance of the binding site from the outside of the pore, and K0 is the half-maximal inhibitory concentration (Ki) at Vc = 0 mV. The best fit obtained with 1.3 mM Mg2+ (Fig. 4B) yielded K0 = 1.58 ± 0.25 mM Mg2+, delta  = 0.23 ± 0.02 from the inside, and G = 49.4 ± 2.42 pS.

We also investigated the possible involvement of Na+ during inward rectification. The i-Vc relationships established under symmetrical K+, with a Mg2+-free medium (+5 mM K2-EDTA) supplemented with 20 mM Na+ on the cytosolic side of the patch (Fig. 4B), showed a slight inward rectification (Gin/Gout = 1.6 ± 0.14, n = 3). An analysis of these data using the model given above, but with the assumption of two binding sites for Na+ [a = 2; (19)], Gin = 50.5 pS, and delta  = 0.23, yields an estimate of K0 of ~40 mM Na+. This indicates that intracellular Na+ made a significant contribution to inward rectification in cell-attached patches.

Intracellular SPM induces a voltage-dependent block. SPM is a naturally occurring polyamine that carries four positive charges at physiological pH and acts as a gating molecule, inducing a concentration- and voltage-dependent block of several inwardly rectifying K+ channels (6, 7, 26). We found that SPM rapidly and reversibly inhibited channel activity in cell-excised, inside-out membrane patches without affecting single-channel current amplitude (Fig. 5, insets). From Fig. 5, which compares the effects of 5 and 500 µM internal SPM on NPo at both -60 and +60 mV, it is clear that the inhibitory effect of SPM was concentration and voltage dependent. This is quantified in Fig. 6A. At both membrane potentials, increasing SPM concentration reduced NPo in a sigmoidal fashion. Fitting experimental data points with the Hill equation (see Fig. 6) revealed a channel sensitivity to SPM at positive potential (Ki = 0.2 µM) that was greater by two orders of magnitude than at negative potential (Ki = 26 µM). In contrast, Hill coefficients were similar and close to unity at both potentials (0.8 vs. 0.7, respectively). This voltage dependence was further investigated by observing the effects of 0.5 µM SPM on NPo over an extended Vc range. The results of two separate experiments are illustrated in Fig. 6B. The relationship between NPo and Vc was described by the Boltzmann distribution (Eq. 2). The best fit of data was given by NPo max = 1.0, V1/2 = 43 mV, and z = 1.25 (Fig. 6B).


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Fig. 5.   Effects of intracellular spermine (SPM) on basolateral CTAL K+ channel activity. Traces show recording of K+ channel activity on the same cell-excised, inside-out membrane patch clamped at either -60 mV (top traces) or +60 mV (bottom traces). The patch was bathed in symmetrical high-K (144.8 mM, Cl-- and Mg2+-free+5 mM Na2-EDTA). C---, current level corresponding to closure of all K+ channels. Each trace was obtained in the absence (Control; left traces) or presence of 5 (middle traces) or 500 µM SPM (right traces) in the bath. Insets: a and b, excerpts at an expanded time scale (*) taken from the indicated sections of the traces.



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Fig. 6.   Concentration and voltage dependence of the effect of internal SPM on basolateral CTAL K+ channel activity. A: dose-response relationships for the inhibitory effect of SPM on NPo at hyperpolarizing () and depolarizing (open circle ) membrane potentials. The cytoplasmic side of cell-excised, inside-out membrane patches was exposed to the indicated SPM concentrations ([SPM]) at either Vc of -60 or +60 mV under the same conditions as in Fig. 5. Because of patch-to-patch variability, the effects of a given [SPM] on NPo were normalized to NPo for the same patch in the absence of SPM. Each value is the mean of at least 3 determinations, and SE are shown as error bars when larger than symbols. At both potentials, relationships between [SPM] and NPo were described by the equation NPo = 100/[1 + ([SPM]/Ki)nH], where Ki is the half-maximal inhibitory concentration, and nH is the Hill coefficient. The lines drawn are nonlinear, least squares fits to the respective data given by Ki = 26 ± 1 µM and nH = 0.8 ± 0.03 for hyperpolarizing voltages and Ki = 0.2 ± 0.06 µM and nH = 0.7 ± 0.1 for depolarizing voltages. B: voltage dependence of the effect of 0.5 µM SPM on NPo. Results obtained from 2 separate patches are shown (triangle  and ). Within each experiment, NPo at a given Vc value in the presence of SPM was normalized to that for the same patch in the absence of SPM (control). Dotted line, is a nonlinear least squares fit of the Boltzmann equation (see RESULTS) to the data given by NPo max = 1.0, Vo = 43 mV, and z = 1.25.

Modulation by internal pH. Varying bath pH rapidly reversibly altered the K+ channel activity in cell-excised, inside-out membrane patches (Fig. 7A). NPo was altered by intracellular pH (pHi) in a sigmoidal fashion (Fig. 7B), and fitting data points using a modified Hill equation (Fig. 7) yield an apparent pK of 7.6. A Hill coefficient of 1.8 suggested that several protons were acting cooperatively in channel regulation by pHi. The unit current amplitude through the CTAL K+ channel was not altered by pHi. i-Vc relationships at pH 6.8 (n = 3) and 7.4 (n = 5) did not show any pH-dependent change in Gin (48 ± 4 vs. 45 ± 1 pS, respectively; P = 0.32) or Gout (36 ± 0.7 vs. 37 ± 3 pS, respectively; P = 0.83).


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Fig. 7.   Effects of variations in intracellular pH (pHi) on basolateral CTAL K+-channel activity. A: single-channel recordings from a cell-excised, inside-out membrane patch at the bath pH (i.e., pHi) values indicated on the right of each trace. Patch was bathed in symmetrical high-K (Cl-- and Mg2+-free+5 mM Na2-EDTA) and clamped at Vc = -50 mV. C----, current corresponding to closure of all K+ channels. B: dose-response relationship of the effect of pHi on K+ channel activity under the same conditions as in A. Within each experiment, NPo at a given pHi value was normalized to NPo at pH = 7.4. Each point is the mean of at least 4 determinations, and SE values are shown as error bars when larger than symbols. The relationship between pHi and NPo is described by the equation NPo = NPo (max)/ [1 + ([H+]/K)nH], where NPo(max) is the maximal NPo, [H+] is the bath proton concentration, pK = -logK, and nH is the Hill coefficient. The line drawn is a nonlinear least squares fit to the data given by NPo(max) = 2.93 ± 0.09, pK = 7.6 ± 0.1, and nH = 1.8 ± 0.1.

This suggests that the basolateral CTAL K+ channel may be regulated by pHi in situ. This was tested using a previous observation that 5 mM bath NH4Cl rapidly and tonically acidifies mouse CTAL cells by 0.3-0.4 pH units (15), an approach used to demonstrate in situ the pHi sensitivity of the basolateral 9-pS Cl- channel in mouse TAL (15). Here we investigated the effects of bath NH4Cl on basolateral K+ channel activity in situ in cell-attached membrane patches, using Cl--containing solutions but under a nil Vc, thus minimizing the influence of the pH-sensitive 9-pS Cl- channel (15). In three of four attempts, K+ channel activity decreased when the tubule was exposed to 5 mM NH4Cl. This procedure led to the complete abolition of activity (Fig. 8) in two cases. In the third case, NPo fell from 1.96 to 0.87. Channel activity recovered completely when NH4Cl was removed from the bath (Fig. 8).


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Fig. 8.   Effects of intracellular acidification on K+ channel activity in situ. The trace is a continuous recording of K+ channel activity of a cell-attached membrane patch with the high-Na solution in the bath. Pipette was filled with the high-K solution, and Vc was set at 0 mV. At the period indicated by the horizontal bar, 5 mM NH4Cl was added to the superfusate. C---, current corresponding to closure of all K+ channels.

Internal ATP does not inhibit the basolateral CTAL K+ channel. ATP has no inhibitory effect on K+ channel activity (data not shown). Adding 1 mM internal ATP (Na salt) without Mg2+ for at least 1 min had no influence on channel activity of inside-out patches at Vc = -50 mV (P = 0.3, paired t-test, n = 3). No change in channel activity was seen when 5 mM internal ATP was added to the bath (n = 2).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inward rectification properties. There has been very little investigation of rectification of K+ channels in native renal tissue (45). An intermediate rectification coefficient (Gin/Gout) of 4 has been explicitly determined for the basolateral 25-pS K+ channel in the Ambystoma proximal tubule (33). A Gin/Gout ~ 4-6 can also be extrapolated from published data on proximal tubule basolateral K+ channels (1, 20, 39). The i-Vc relationship of the basolateral CTAL K+ channel also displays a Gin/Gout of ~4.5 in cell-attached patches, indicative of an intermediate-type inward rectification. This differentiates the channel from strong inward rectifiers such as Kir 2.3 (CCD-IRK3) (48) and from the weak rectifier of the apical membrane of the cortical collecting duct (CCD) (47) and Kir 1.1 (2, 18) for which a Gin/Gout of 2-2.5 can be calculated. It is worth noting that the rectification would be hardly apparent in the presence of a physiological K+ concentration: Gin would be then comparable to Gout (~10 pS).

The K0 for the Mg2+-induced rectification of the CTAL K+ channel (1.6 mM) is consistent with a weakly inwardly rectifying K+ channel. This is similar to the 2 mM for the weakly rectifying ATP-dependent K+ channel in ventricular cells (19, 31) and to the 2-29 mM for Kir 1.1 (2, 36, 43). This is considerably higher than the ~10 µM of the strong inwardly rectifying IRK1 (31, 43, 52) and slightly lower than the only published value for native intermediate inwardly rectifying renal K+ channels (K0 = 7.7 mM) (33). The electrical distance (delta ) sensed by Mg2+ (~0.2) for the CTAL K+ channel also matches that of weak inward rectifiers (~0.3) (2, 31, 36). Furthermore, the contribution of internal Na+ to inward rectification (K0 = 40 mM Na+) of the CTAL channel is very similar to the 15 mM Na+ at 40 mV (~30 mM at 0 mV) for the weakly rectifying ATP-dependent K+ channel (8, 9, 19).

On the other hand, a high sensitivity to SPM (Ki = 0.2-25 µM) distinguishes the CTAL K+ channel from weak inward rectifiers (37), which have a considerably higher Ki (~1 mM) (49, 51, 53). Still, it is 10 times higher than that for strong inward rectifiers, such as the muscarinic K+ channel in the atrial myocyte or the cloned Kir 2.1 and Kir 4.1, with a Ki in the 10 nM range (6, 51). On the other hand, the voltage dependence of the SPM block shows a z of ~1.3, yielding an delta  of ~0.3, in accordance with our results for Mg2+. This disagrees with the usual characteristics of the SPM block, as delta  values greater than unity have been reported for strong inward rectifiers (10, 27). High sensitivity to SPM usually correlates with strong Mg2+-induced inward rectification (27), but this was not the case here. Such discrepancies between the effects of SPM and Mg2+ on the CTAL channel could arise from a heterotetrameric channel structure. For instance, coexpression of Kir 1.1 (a mild inward rectifier) and Kir 4.1 (a strong inward rectifier) K+ channels in Xenopus laevis oocytes produces a channel with lower sensitivity to SPM than the Kir 4.1 homotetramer (10).

Channel regulation. The CTAL K+ channel is also sensitive to pHi. A decrease in channel activity at acid pH is observed for many renal K+ channels (45), including the K+-secreting channel (and ROMK) in the apical membrane of the CCD and the proximal convoluted tubule basolateral channel (34, 35, 47). The pK of ROMK channels was 7.0-7.2, and the channel activity peaks at pH >7.4, as a consequence of a Hill coefficient of 3. A quite different pattern was observed for the CTAL channel, with a Hill coefficient of ~2 and a pK of ~7.6. Therefore, in situ channel activity would be in the lower part of the curve, so that even slight intracellular acidification would suffice to produce a significant reduction in NPo. Our experiments indeed showed that acidifying the intracellular compartment by 0.3 pH unit was sufficient to inhibit channel activity in cell-attached patches. In view of the possible role played by this channel in NaCl reabsorption by the TAL (see below), its pH dependence is of physiological interest. Indeed, in vitro acute acidosis inhibits NaCl transport by the TAL (50). Thus, in addition to the basolateral pH-sensitive Cl- channels (15), the 45-pS K+ channel we describe here appears as another basolateral target of acidosis in the inhibition of NaCl reabsorption by the TAL.

Comparison with other K+ channels in renal basolateral membranes. The only previous patch-clamp study in the basolateral membrane of CTAL reported the presence of a 35-pS K+ channel in the rabbit (22), showing inward rectification (Gout = 7 pS). As in the mouse, activity increases with depolarization in cell-attached patches. Thus the channels in rabbit and mouse CTALs seem to be similar, but some conductive and regulatory properties were not investigated in rabbit CTAL, which limits further comparisons. Several channels described in basolateral membranes of other nephron segments share some, but not all, properties of the CTAL channel. In the basolateral membrane of rat CCD, an inwardly rectifying K+ channel has been reported, but it is highly active and voltage independent, and pH insensitive and has a slightly lower conductance (27 pS) (28, 46). A 45-pS, pH-sensitive K+ channel has also been described in cell-attached patches of rat CCD (46), but it is activated by hyperpolarization. Other inwardly rectifying K+ channels have been studied in the basolateral membrane of the proximal tubule. Although some of these channels may differ in terms of voltage dependence, they do share a number of properties: they are all inhibited by acid pH (1, 34), and their inward conductance is ~50 pS in mammalian physiological saline (21, 38, 39) and depends on the external K+ concentration, with a Km of 65-77 mM (23, 33). However, on the other hand, regarding inward rectification, the CTAL channel has a higher affinity toward Mg2+ and, unlike the PCT channel (33), exhibits high sensitivity to SPM. Second, the CTAL channel is not inhibited by ATP.

We did not address the nature of the molecular entity underlying the basolateral K+ channel in the CTAL, but functional properties allow us to rule out the Kir 2, Kir 6, and Kir 7 channels families, as well as the tandem of P domains in the weak inward rectifier K+ channel and related channels (see Refs. 25 and 37 for reviews). Comparative studies will probably help to ascertain the molecular nature of the channel.

K+ conductance in the CTAL basolateral membrane. Studies of microperfused isolated tubules have given rise to conflicting interpretations on the issue of basolateral K+ conductance in CTAL. Greger and Schlatter (11) initially found that the basolateral membrane of rabbit CTAL was not conductive to K+ and postulated a basolateral K+ exit through a K+-Cl- cotransporter (see Fig. 9). However, subsequent studies reported a K+-conductive pathway in the basolateral membranes of hamster and rabbit TAL (3, 54) and those of the Amphiuma diluting segment (13). The previous patch-clamp study in rabbit CTAL (22) and the present work in the mouse clearly establish that the basolateral membrane of CTAL cells is endowed with K+ channels.


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Fig. 9.   Basic model of NaCl reabsorption by the TAL epithelium. Dotted lines indicate passive ion movements down electrochemical gradients. Vm, membrane potential.

We found an average of six channels per patch displaying K+ channel activity. The dependence of channel conductance on external K+ concentration yielded a unitary conductance of ~10 pS for the K+ channel under physiological conditions (5 mM external K+). This corresponds to a maximal K+ conductance of ~60 pS/membrane patch. In previous studies, we observed an average of 20 Cl- channels of 9 pS/patch under similar experimental conditions (14, 30), giving a Cl- conductance of 180 pS. Although this is probably an underestimate, as it does not take into account the contribution of the less common 45-pS Cl- channel in this membrane (40), Cl- conductance does seem to be greater than K+ conductance by a factor of at least three. This is in qualitative agreement with macroscopic electrophysiological data, which show that Cl- conductance predominates over K+ conductance in the basolateral membrane (13, 54). Thus it does not contradict the fact that the basolateral electromotive force is primarily determined by Cl- conductance, as required for the observed positive transepithelial voltage difference (Fig. 9).

A depolarization-activated K+ channel showing noticeable activity at resting membrane potential may serve two purposes. First, it may help maintain VBl above ECl (see Fig. 9) to provide a continuous driving force for basolateral conductive Cl- exit. On stimulation of overall NaCl reabsorption, the opening of basolateral Cl- channels allows Cl- efflux but, in turn, tends to decrease VBl toward ECl and thus dissipate the driving force for Cl- exit. The decrease in VBl could be the signal upmodulating the depolarization-activated 45-pS K+ channel, the resulting increase in VBl restoring the driving force for basolateral Cl- efflux.

Second, because continuous activity of the basolateral Na+/K+-ATPase is mandatory to transcellular NaCl reabsorption by the TAL, basolateral K+ recycling is necessary to avoid the intracellular accumulation of K+. In proximal tubular cells, this role is devoted to basolateral ATP-sensitive K+ channels (44), the cell ATP content being the link between the pump and activities of the these channels. In the CTAL, the 45-pS K+ channel might have a similar role, but because this channel is insensitive to ATP, another factor would have to link Na+/K+-ATPase activity and basolateral K+ conductance in the CTAL. For instance, an increase in apical Na+ entry into CCD cells stimulates both pump rate and nitric oxide production, nitric oxide then activating an ATP-insensitive, low-conductance basolateral K+ channel (45).

The K+ channel was present in ~30% of the patches, but there were usually several channels per patch. This suggests that K+ channels are arranged in clusters. Alternatively, this would also be consistent with different cell types in TAL tubules, as suggested for the diluting segment of the Amphiuma (13) and for hamster MTAL (54). In their study, Yoshitomi et al. (54) reported the presence of a first cell type with high basolateral conductance of K+ and Cl- and then a second with low conductances of these ions. In this respect, it is worth mentioning that we occasionally recorded Cl- channels in patches with no K+ channel activity, and, conversely, some patches contained K+ channels, but no Cl- channel (not shown). This finding does not support the hypothesis of two cell types, although it could be argued that the K+ channel linked to the Cl- channel may not be the same as the K+ channel reported here but another, hitherto unidentified, K+ channel.


    ACKNOWLEDGEMENTS

We thank Stéphanie Nelson for technical assistance and Nadège Hurson, Séverine Layre, and Lydie René-Corail for secretarial work. The English text was corrected by Monika Ghosh.


    FOOTNOTES

This study was supported by Contrat Prisme Institut National de la Santé et de la Recherche Médicale. S. Lourdel is a recipient of a research studentship from the Ministère de la Recherche.

Address for reprint requests and other correspondence: M. Paulais, INSERM U.426, Faculté de Médecine Xavier Bichat, 16 Rue Henri Huchard, B. P. 416, 75870 Paris Cedex 18, France (E-mail: paulais{at}bichat.inserm.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.

First published November 27, 2001;10.1152/ajprenal.00238.2001

Received 1 August 2001; accepted in final form 23 November 2001.


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DISCUSSION
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