E. Schlatter

The luminal K+ channel of the thick ascending limb of Henle's loop.

In vitro perfused rat thick ascending limbs of Henles loop (TAL) were used (n=260) to analyse the conductance properties of the luminal membrane applying the patch-clamp technique. Medullary (mTAL) and cortical (cTAL) tubule segments were dissected and perfused in vitro. The free end of the tubule was held and immobilized at one edge by a holding pipette kept under continuous suction. A micropositioner was used to insert a patch pipette into the lumen, and a gigaohm seal with the luminal membrane was achieved in 455 instances out of considerably more trials. In approximately 20% of all gigaohm seals recordings of single ionic channels were obtained. We have identified only one single type of K+ channel in these cell-attached and cell-excised recordings. In the cell-attached configuration with KCl or NaCl in the pipette, the channel had a conductance of 60±6 pS (n=24) and 31±7 pS (n=4) respectively. In cell-free patches with KCl either in the patch pipette or in the bath and with a Ringer-type solution (NaCl) on the opposite side the conductance was 72±4 pS (n=37) at a clamp voltage of 0 mV. The permeability was 0.33±0.02 · 10±12 cm3/s. The selectivity sequence für this channel was: K+=Rb+=NH4+=Cs+>Li+≫Na+=0; the conductance sequence was K+≫Li+≫Rb+=Cs+= NH4+=Na+=0. In excised patches Rb+, Cs+ and NH4+when present in the bath at 145 mmol/l all inhibited K+ currents out of the pipette. The channel kinetics were described by one open (9.5±1.5 ms, n=18) and by two closed (1.4±0.1 and 14±2 ms) time constants. The open probability of this channel was increased by depolarization. The channel open probability was reduced voltage dependently by Ba2+ (half maximal inhibition at 0 mV: 0.07 mmol/l) from the cytosolic side. Verapamil, diltiazem, quinine and quinidine inhibited at approximately 1 μmol/l ±0.1 mmol/l from either side. Similarly, the amino cations lidocaine, tetraethylammonium and choline inhibited at 10–100 mmol/l. The channel was downregulated in its open probability by cytosolic Ca2+ activities > 10±7 mol/l and by adenosine triphosphate ≥ 10±4 mol/l. The open probability was downregulated by decreasing cytosolic pH (2-fold by a decrease in pH by ≤ 0.2 units). The described channel differs in several properties from the K+ channels of other epithelia and of renal cells and TAL cells in culture. It appears to be responsible for K+ recycling in the TAL segment.

Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl−K+ cotransport

The macula densa cells of the juxtaglomerular apparatus probably serve as the sensor cells for the signal which leads to the appropriate tubuloglomerular feedback response. The present study reports basolateral membrane voltage (PDbl) measurements in macula densa cells. We isolated and perfused in vitro thick ascending limb segments with the glomerulus, and therefore the macula densa cells, and the early distal tubule still attached. Macula densa cells were impaled with microelectrodes under visual control. PDbl was recorded in order to examine how these cells sense changes in luminal NaCl concentrations. The addition of furosemide, a specific inhibitor of the Na+2Cl−K+ cotransporter in the thick ascending limb, to the lumen of the perfused thick ascending limb hyperpolarized PDbl from −55±5 mV to −79±4 mV (n=7). Reduction of NaCl in the lumen perfusate from 150 mmol/l to 30 mmol/l also hyperpolarized PDbl from −48±3 mV to −66±5 mV (n=4). A Cl− concentration step in the bath from 150 mmol/l to 30 mmol/l resulted in a 24±4 mV (n=4) depolarization of PDbl. This depolarization of PDbl was absent when furosemide was present during the Cl− concentration step. These data suggest that the macula densa cells sense changes in luminal NaCl concentration via coupled uptake of Na+ and Cl−. The transport pathways for NaCl transport in macula densa cells are probably identical to those in the thick ascending limb: the (Na++K+)-ATPase in the basolateral membrane drives Na+ and Cl− uptake via the luminal Na+2Cl−K+ cotransport, Cl− leaves the cell via basolateral Cl− channels and K+ recycles across the apical membrane via K+ channels. Changes in intracellular Cl− activity as a result of altered luminal NaCl uptake, and thus voltage changes of the basolateral membrane are probably the first signal in the tubuloglomerular feedback regulation.

Regulation and possible physiological role of the Ca2-dependent K+ channel of cortical collecting ducts of the rat

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca2+-dependent and a small Ca2+-independent K+ channel have been described. Whereas the latter most likely is responsible for the K+ secretion in this nephron segment, the function of the large-conductance K+ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K+ channel was strongly voltage- and calcium-dependent. At 3 μmol/l cytosolic Ca2+ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 μmol/l Ca2+ activity the open probability (Po) of this channel was pH-dependent. At pH 7.0 Po was decreased to 4±2% (n=9) and at pH 8.5 it was increased to 425±52% (n=9) of the control. At this low Ca2+ activity the Po of the channel was reduced by 1 mmol/l ATP to 8±4% (n=6). Cell swelling activated the large-conductance K+ channel (n=14) and hyperpolarized the membrane potential of the cells by 9±1 mV (n=23). Intracellular Ca2+ activity increased after hypotonic stress. This increase depended on the extracellular Ca2+ activity. A possible physiological function of the large-conductance K+ channel in rat CCD cells may be the reduction of the intracellular K+ concentration after cell swelling. Once this channel is activated by increases in the cytosolic Ca2+ activity it can be regulated by changes in cellular pH and ATP.

Principal cells of cortical collecting ducts of the rat are not a route of transepithelial Cl− transport

The rat cortical collecting duct (CCD) exhibits high rates of NaCl reabsorption when stimulated by mineralocorticoid and antidiuretic hormone (ADH). The present study was undertaken to determine if there is significant transcellular Cl− movement across the principal cells of the rat CCD. CCDs were dissected from kidneys of rats that had been injected with deoxycorticosterone (5 mg, i.m.) 2–9 days prior to the experiment. The ducts were perfused in vitro with identical perfusing and bathing solutions, except that 200 pmol.l−1 ADH was added to the bathing solutions. The basolateral membrane voltage (PDbl) of principal cells was −77±1 mV and the luminal membrane voltage (PD1) was −68±1 mV (mean ± SEM, n=124). Separate impalements with single-barrelled Cl−-selective microelectrodes gave an apparent intracellular Cl− activity of principal cells of 17±2 mmol.l−1. Transepithelial PD and PDbl were unaffected by luminal furosemide, hydrochlorothiazide (HCT), 4-acetamido-4-isothiocyanostilbene2,2-disulphonic acid, (SITS), or the Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB); bath addition of SITS or the Cl− channel blocker diphenylamino-2-carboxylic acid; or replacement of bath HCO3−by Cl−. The intracellular Cl− activity (acellCl) also remained unchanged with the addition of HCT, SITS or the Cl− channel blockers to either the perfusing or bathing solutions, or with replacement of the bathing solution HCO3−. With Cl− replacement in both solutions, acellCldecreased to 9 mmol.l−1, but not until after 4–6 min, indicating a very low rate of Cl− transport in these cells, even under conditions of maximal stimulation of NaCl reabsorption by mineralocorticoid plus ADH. The remaining acellClcould be attributed to interference with the Cl− selective electrodes by other cytosolic anions. We conclude that acellClof principal cells in the rat CCD is not far above passive equilibrium, and that these cells do not contribute significantly to transepithelial Cl− reabsorption, which must occur by alternative routes such as the paracellular pathway, and/or through intercalated cells.

pH dependence of K+ conductances of rat cortical collecting duct principal cells.

The K+ channels of the principal cells of rat cortical collecting duct (CCD) are pH sensitive in excised membranes. K+ secretion is decreased with increased H+ secretion during acidosis. We examined whether the pH sensitivity of these K+ channels is present also in the intact cell and thus could explain the coupling between K+ and H+ secretion. Membrane voltages (Vm), whole-cell conductances (gc), and single-channel currents of K+ channels were recorded from freshly isolated CCD cells or isolated CCD segments with the patch-clamp method. Intracellular pH (pHi) was measured using the pH-sensitive fluorescent dye 2′-7′-bis(carboxyethyl)-5-6-carboxyfluorescein (BCECF). Acetate (20 mmol/l) had no effect on Vm, gc, or the activity of the K+ channels in these cells. Acetate, however, acidified pHi slightly by 0.17±0.04 pH units (n=19). Vm depolarized by 12±3 mV (n=26) and by 23±2 mV (n=66) and gc decreased by 26±5% (n=13) and by 55±5% (n=12) with 3–5 or 8–10% CO2, respectively. The same CO2 concentrations decreased pHi by 0.49±0.07 (n=15) and 0.73±0.11 pH units (n=12), respectively. Open probability (Po) of all four K+ channels in the intact rat CCD cells was reversibly inhibited by 8–10% CO2. pHi increased with the addition of 20 mmol/l NH4+/NH3 by a maximum of 0.64±0.08 pH units (n=33) and acidified transiently by 0.37±0.05 pH units (n=33) upon NH4+/NH3 removal. In the presence of NH4+/NH3Vm depolarized by 16±2 mV (n=66) and gc decreased by 26±7% (n=16). The activity of all four K+ channels was also strongly inhibited in the presence of NH4+/NH3. The effect of NH4+/NH3 on Vm and gc was markedly increased when the pH of the NH4+/NH3-containing solution was set to 8.5 or 9.2. From these data we conclude that cellular acidification in rat CCD principal cells down-regulates K+ conductances, thus reduces K+ secretion by direct inhibition of K+ channel activity. This pH dependence is present in all four K+ channels of the rat CCD. The inhibition of K+ channels by NH4+/NH3 is independent of changes in pHi and rather involves an effect of NH3.

Ion conductances of isolated cortical collecting duct cells.

The study of ion conductances in the intact cortical collecting duct (CCD) with the patch-clamp method is rather difficult. An optimized method to isolate CCD cells from rat kidneys using an in vivo followed by an in vitro enzyme digestion is described. Individual CCD segments were collected after this digestion and incubated in EGTA-buffered medium. This procedure resulted in single cells or cell clusters. These freshly isolated CCD cells were studied with different modifications of the patch-clamp method. Membrane voltages measured in the cell-attached-nystatin configuration were −74 ±1mV (n=13) and −68±3 mV (n=22) in cells isolated from normal and mineralocorticoid-treated rats respectively. These values and those measured with the nystatin-perforated slow-whole-cell configuration (−79 ±1mV, n=23) are comparable to those measured in principal cells of isolated CCD segments. The cells hyperpolarized after the addition of amiloride and depolarized with the addition of adiuretin to the bath. The amiloride effect was enhanced when cells were isolated from deoxycorticosterone-acetate-treated rats. The cells were strongly depolarized upon elevation of the extracellular K+-concentration and did not demonstrate a measurable Cl− conductance. A large-conductance K+ channel (174 pS, n=5, cell-attached, 145 mmol/l K+ in the pipette; 140 pS, n=12, cell-free, 3.6 mmol/l K+ in the bath) was seen. It had a very low activity on the cell, but a high open probability when excised into a solution with 1 mmol/l Ca2+ on the cytosolic side. More often a small-conductance K+ channel (36–52 pS, n=19, cell-attached; 30 pS, n=5, cell-free) with a high open probability was found on the cell. These freshly isolated cells seem to be a powerful preparation to study the properties and regulation of ion conductances of rat CCD with several electrophysiological methods. These freshly isolated CCD cells maintain the conductance properties known from principal cells of the intact CCD.

Cation specificity and pharmacological properties of the Ca2+-dependent K+ channel of rat cortical collecting ducts

The luminal membrane of principal cells of rat cortical collecting duct (CCD) is dominated by a K+ conductance. Two different K+ channels are described for this membrane. K+ secretion probably occurs via a small-conductance Ca2+-independent channel. The function of the second, large-conductance Ca2+-dependent channel is unclear. This study examines properties of this channel to allow a comparison of this K+ channel with the macroscopic K+ conductance of the CCD and with similar K+ channels from other preparations. The channel is poorly active on the cell. It has a conductance of 263±11 pS (n=36, symmetrical K+ concentrations) and of 139±3 pS (n=91) with 145 mmol/l K+ on one side and 3.6 mmol/l K+ on the other side of the membrane. Its open probability is high after excision (0.71±0.03, n=85). The channel flickers rapidly between open and closed states. Its permeability in the cell-free configuration was 7.0±0.2×10−13 cm3/s (n=85). It is inhibited by several typical blockers of K+ channels such as Ba2+, tetraethylammonium, quinine, and quinidine and high concentrations of Mg2+. The Ca2+ antagonists verapamil and diltiazem also inhibit this K+ channel. As is typical for the maxi K+ channel, it is inhibited by charybdotoxin but not by apamin. The selectivity of this large-conductance K+ channel demonstrates significant differences between the permeability sequence (PK > PRb > PNH4 > PCs=PLi=PNa=Pcholine=0) and the conductance sequence (gK > gNH4 > gRb > gLi=gcholine > gCs=gNa=0). The only other cations that are significantly conducted by this channel besides K+ (gK at Vc =∞ is 279±8 pS, n=88) are NH4+(gNH4=127±22 pS, n=10) and Rb+ (gRb=36±5 pS, n=6). The K+ currents through this channel are reduced by high concentrations of choline+, Cs+, Rb+, and NH4+. These properties and the dependence of this channel on Ca2+ and voltage classify it as a “maxi” K+ channel. A possible physiological function of this channel is discussed in the accompanying paper.

Ion channels in the thick ascending limb of Henle's loop

The thick ascending limb of Henles loop (TAL) is polarized with respect to its conductances. The luminal membrane contains a K+ conductance which is made up by the synchronous operation of 60- to 80-pS K+ channels. The basolateral membrane contains a chloride conductance. This conductance corresponds most likely to a 30- to 60-pS Cl- channel present in this membrane. Our knowledge on the properties of the K+ channels of these cells has been increased rapidly by patch clamp studies: these K+ channels are inwardly rectifying. They are highly selective for K+ over Na+, Li+ and many other cations. They do not conduct Rb+, Cs+, NH+4 or other larger cations. In fact, all these three cations as well as choline, tetraethylammonium, lidocaine, verapamil, diltiazem, quinine, quinidine and Ba2+ inhibit these K+ channels. As apparent from kinetic studies the mechanisms of inhibition are different for the various blockers. The TAL K+ channels are downregulated by increasing cytosolic Ca2+ activity. Cytosolic adenosine trisphosphate (ATP) has a similar effect. This ATP inhibition is Ca2+ dependent. The affinity to ATP is augmented by increasing Ca2+. Cytosolic alkalinity increases the open probability of these channels, and cytosolic acidification has the opposite effect. This pH dependence is very marked. A change by 0.2 pH units leads to a more than twofold change in the open-channel probability. The basolateral chloride conductance reflects the properties of an outwardly rectifying 30- to 60-pS Cl- channel. This channel behaves, in many respects, like the Cl- channels of a multitude of Cl- transporting epithelia. It is characterized by two open and two closed states. It is highly selective for Cl- as compared with larger anions, and it is inhibited reversibly by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate.

Analysing ion channels with hidden Markov models.

Ion channel current amplitudes (μ) and open probabilities (Po) have been analysed so far by defining a 50% threshold to distinguish between open and closed states of the channels. With this standard method (SM) it is very difficult or even impossible to analyse channels of different size in one membrane patch correctly. A stochastical model, named the hidden Markov model (HMM), separates between observation noise and the stochastic process of opening and closing of ion channels. The HMM allows the independent analysis of μ, Po, and mean dwell times (τ) of different channels in one membrane patch, without defining threshold levels. Using this method errors in the analysis are not summarized like in the SM because all different analysing procedures (e. g. filtering, setting of threshold, fitting processes) are done in one step. Two different K+ channels in excised basolateral membranes of the cortical collecting duct of rat (CCD) were analysed by the SM and the HMM. The μ value of the intermediate-conductance K+ channel (i-K+) was 3.9±0.1 pA (SM) and 3.8±0.2 pA (HMM) for 11 observations. The Po value of this channel was 10.2±4.2% (SM) and 10.1±4.0% (HMM). The mean τ values were 5.4±0.6 ms for the open state and 9.6±2.2 ms and 145±21 ms for the closed states (SM) and 7.8±1.1 ms, 7.7±0.9 ms and 148±24 ms (HMM), respectively. For seven small-conductance K+ (s-K+) channels, which were found in the same membrane patches as the i-K+, an accurate analysis of Po and τ was not possible with the SM. The μ value was 1.0±0.1 (SM), 0.9±0.1 (HMM) pA. Po was 16.6±4.6%, the open τ value was 11.1±2.8 ms, and the closed τ value was 34.9±8.5 ms. The HMM allows the analysis of single-channel currents, Po, and mean τ values when different or more than one ion channel(s) are colocalized in one membrane patch. Where analysis with the SM was possible results did not significantly differ from those obtained with the HMM. Thus for this kind of analysis the method of setting a 50% threshold appears justified.

Effect of NH4+/NH3 on cytosolic pH and the K+ channels of freshly isolated cells from the thick ascending limb of Henle's loop.

The conductance properties of the luminal membrane of cells from the thick ascending limb of Henles loop of rat kidney (TAL) are dominated by K+. In excised membrane patches the luminal K+ channel is regulated by pH changes on the cytosolic side. To examine this pH regulation in intact cells of freshly isolated TAL segments we measured the membrane voltage (Vm) in slow-whole-cell (SWC) recordings and the open probability (Po) of K+ channels in the cell-attached nystatin (CAN) configuration, where channel activity and part of Vm can be recorded. The pipette solution contained K+ 125 mmol/l and Cl− 32 mmol/l. Intracellular pH was determined by 2′,7′ bis(2-carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) fluorescence. pH changes were induced by the addition of 10 mmol/l NH4+/NH3 to the bath. In the presence of NH4+/NH3 intracellular pH acidified by 0.53±0.11 units (n=7). Inhibition of the Na+2Cl− K+ cotransporter by furosemide (0.1 mmol/l) reversed this effect and led to a transient alkalinisation by 0.62±0.14 units (n=7). In SWC experiments Vm of TAL cells was -72±1 mV (n=70). NH4+/NH3 depolarised Vm by 22±2 mV (n=25). In 11 SWC experiments furosemide (0.1 mmol/l) attenuated the depolarising effect of NH4+ from 24±3 mV to 7±3 mV. Under control conditions the single-channel conductance of TAL K+ channels in CAN experiments was 66±5 pS and the reversal voltage for K+ currents was 70±2 mV (n=35). The Po of K+ channels in CAN patches was reduced by NH4+/NH3 from 0.45±0.15 to 0.09±0.07 (n=7). NH4+/NH3 exposure depolarised the zero current voltage of the permeabilised patches by-9.7±3.6 mV (n=5). The results show that TAL K+ channels are regulated by cytosolic pH in the intact cell. The cytosolic pH is acidified by NH4+/NH3 exposure at concentrations which are physiologically relevant because Na+2Cl−K+(NH4+) cotransporter-mediated import of NH4+ exceeds the rate of NH3 diffusion into the TAL. K+ channels are inhibited by this acidification and the cells depolarise. In the presence of furosemide TAL cells alkalinise proving that NH4+ uptake occurs by the Na+2Cl−K+ cotransporter. The findings that, in the presence of NH4+/NH3 and furosemide, Vm is not completely repolarised and that K+ channels are not activated suggest that the respective K+ channels may in addition to their pH regulation be inhibited directly by NH4+/NH3.