Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.

Conductance and gating of epithelial Na channels from rat cortical collecting tubule. Effects of luminal Na and Li.

The behavior of individual Na channels in the apical membrane of the rat cortical collecting tubule (CCT) was studied at different concentrations of the permeant ions Na and Li. Tubules were opened to expose their luminal surfaces and bathed in K-gluconate medium to minimize tubule-to-tubule variation in cell membrane potential and intracellular Na concentration. The patch-clamp technique was used to resolve currents through individual channels. The patch-clamp pipette was filled with solutions containing variable concentrations of either NaCl or LiCl. In one series of experiments, the concentrations were changed without substitutions. In another series, the ionic strength and Cl concentration were maintained constant by partial substitution of Li with N-methyl-D-glucamine (NMDG). In cell-attached patches, both the single-channel conductance (g) and the single-channel current (i) saturated as functions of the Na or Li activity in the pipette. Without NMDG, the saturation of i was well described by Michaelis-Menten kinetics with an apparent Km of approximately 20 mM activity for Na and approximately 50 mM activity for Li. Km was independent of voltage for both ions. With substitution for Li by NMDG, the apparent Km value for Li transport through the channels increased. The values of the probability of a channel's being open (Po) varied from patch to patch, but no effect of pipette ion activity on Po could be demonstrated. A weak dependence of Po on membrane voltage was observed, with hyperpolarization increasing Po by an average of 2.3%/mV.

Regulation of the Na-K pump of the rat cortical collecting tubule by aldosterone.

Activities of Na channels and Na pumps were studied in the rat cortical collecting tubule (CCT) during manipulation of the animals' mineralocorticoid status in vivo using a low-Na diet, diuretics, or administration of exogenous aldosterone. Tubules were isolated and split open to expose the luminal membrane surface. Using the whole-cell patch-clamp technique, activities of the apical Na channels and the basolateral Na pumps were measured in principal cells as the currents inhibited by amiloride (10 microM) and ouabain (1 mM), respectively. Na channel current (INa) was not measurable in CCTs from control animals on a normal diet. INa was approximately 200 pA/cell in CCTs from animals on a low-Na diet or infused with aldosterone using osmotic minipumps. Currents attributable to the Na pump (Ipump) were similar in control animals and animals on a low-Na diet. Maximal currents were approximately 35 pA/cell in both groups, and decreased with hyperpolarization of the cell membrane. In contrast, administration of exogenous aldosterone increased Ipump fourfold. Coinfusion of aldosterone and amiloride in vivo through the minipumps did not affect the induction of INa but reduced the induction of Ipump by 80%. We conclude that the induction of channel activity in this tissue is a direct action of aldosterone, whereas the induction of pump activity may be a consequence of the increased Na traffic through the epithelial cells.

Regulation of apical K and Na channels and Na/K pumps in rat cortical collecting tubule by dietary K.

The patch-clamp technique was used to study the properties and the density of conducting K and Na channels in the apical membrane of rat cortical collecting tubule. The predominant K channel observed in cell-attached patches (SK channels) had an outward single-channel conductance (with LiCl in the pipette) of 10 pS. The inward conductance (with KCl in the pipette) was 42 pS. The channel had a high open probability that increased with depolarization. Kinetic analysis indicated the presence of a single open state and two closed states. Increasing K intake by maintaining animals on a high K diet for 12-16 d increased the number of SK channels per patch by threefold (0.7-2.0/patch) over control levels. In addition, conducting Na-selective channels, which were not observed in control animals, were seen at low density (0.5/patch). These channels had properties similar to those observed when the animals were on a low Na diet, except that the mean open probability (0.84) was higher. In other experiments, the whole-cell patch clamp technique was used to measure Na channel activity (as amiloride-sensitive current, INa) and Na pump activity (as ouabain-sensitive current, Ipump). In animals on a high K diet, INa was greater than in controls but much less than in rats on a low Na diet. Ipump was greater after K loading than in controls or Na-depleted animals. These K diet-dependent effects were not accompanied by a significant increase in plasma aldosterone concentrations. To further investigate the relationship between K channel activity and mineralocorticoids, rats were maintained on a low Na diet to increase endogenous aldosterone secretion. Under these conditions, no increase in SK channel density was observed, although there was a large increase in the number of Na channels (to 2.7/patch). Aldosterone was also administered exogenously through osmotic minipumps. As with the low Na diet, there was no change in the density of conducting SK channels, although Na channel activity was induced. These results suggest that SK channels, Na channels and Na/K pumps are regulated during changes in K intake by factors other than aldosterone.

Regulation of Na channels of the rat cortical collecting tubule by aldosterone.

The activity of apical membrane Na channels in the rat cortical collecting tubule was studied during manipulation of the animals' mineralocorticoid status in vivo using a low-Na diet or the diuretic furosemide. Tubules were isolated and split open to expose the luminal membrane surface. Induction of Na channel activity was studied in cell-attached patches of the split tubules. No activity was observed with control animals on a normal diet. Channel activity could be induced by putting the animals on the low-Na diet for at least 48 h. The mean number of open channels per patch (NPo) was maximal after 1 wk on low Na. Channels were also induced within 3 h after injection of furosemide (20 mg/kg body wt per d). NPo was maximal 48 h after the first injection. In both cases, increases in NPo were primarily due to increases in the number of channels per patch (N) at a constant open probability (Po). With salt depletion or furosemide injection NPo is a saturable function of aldosterone concentration with half-maximal activity at approximately 8 nM. When animals were salt repleted after 1-2 wk of salt depletion, both plasma aldosterone and NPo fell markedly within 6 h. NPo continued to decrease over the next 14 h, while plasma aldosterone rebounded partially. Channel activity may be dissociated from aldosterone concentrations under conditions of salt repletion.

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney.

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.

Functional identification of H-K-ATPase in intercalated cells of cortical collecting tubule.

A K-dependent proton extrusion mechanism was investigated by means of fluorescence techniques in rabbit cortical collecting tubules. These experiments were performed in split opened tubules from normal animals exposed to the intracellular pH (pH(i)) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. This preparation permitted the separate study of the intercalated cells (IC) from the principal cells (PC). In IC pH(i) recovery in response to an acute acid load was observed under Na-free conditions on addition of 5 mM K. This K-dependent recovery of pH(i) in the IC was only partial, but was Sch 28080 inhibitable (10(-5) M) and ouabain insensitive. This suggests the process is mediated by an H-K-adenosinetriphosphatase similar to that of gastric cells. The PC were capable of recovering from the acid load, but this Na-independent response was not blocked by the Sch 28080, suggesting some other mechanism for this result. In both cell types reintroduction of Na into the superfusate resulted in full recovery back to the initial pH(i), presumably the result of Na/H exchange.

Aldosterone and potassium secretion by the cortical collecting duct.

BACKGROUND: : Aldosterone has been implicated in the regulation of both Na and K concentrations in the plasma. Release of the hormone is known to be stimulated by high plasma K, and infusion of aldosterone lowers plasma K. However, the correlation between changes in mineralocorticoid levels and rates of K secretion is not perfect, suggesting that other factors may be involved. METHODS: : Patch-clamp recordings were made of K-channel activity in the split-open cortical collecting tubule of the rat. Estimates of channel density were made in cell-attached patches on the luminal membrane of principal cells of this segment. RESULTS: : Most of the K conductance of the apical membrane is mediated through low-conductance "SK" channels. The number of conducting SK channels is increased when animals are placed on a high-K diet. However, increasing plasma aldosterone levels by infusion of the hormone or by sodium restriction failed to change the number of active channels. CONCLUSIONS: : At least two circulating factors are required for the regulation of renal K secretion by the cortical collecting tubule. Aldosterone mainly stimulates secretion by increasing the driving force for K movement through apical channels. A second, as yet unidentified, factor increases the number of conducting K channels.

Epithelial sodium channels: characterization by using the patch-clamp technique.

The patch-clamp technique was used to resolve currents through individual Na-selective ion channels in the apical membrane of the rat cortical collecting tubule. The channels had a single unit conductance of 5 pS under control conditions (cell-attached patches, room temperature, 140 mM NaCl in the pipette). They appeared to be highly selective for Na, as K conduction through them was not measurable in inside-out patches. The channels underwent spontaneous transitions between open and closed states, both states being long-lived. At physiological temperature (37C) the conductance increased to 9 pS and the spontaneous transitions became more rapid. In the presence of amiloride on the luminal side of the membrane, the channel flickered rapidly between open and blocked states. The size of the current transitions did not change. This channel activity was observed only in rats that were fed a low-Na diet to elevate aldosterone secretion. In addition to mineralocorticoid control, the activity of the channels in inside-out patches were modulated by the pH on the cytoplasmic side of the membrane. Alkalinization from pH 6.4 to 7.4 increased the probability of channels' being open by eightfold. Changes in Ca concentration on the cytoplasmic side of the membrane did not directly affect the channels. However, addition of ionomycin, a Ca ionophore, to the bath resulted in a decrease in channel activity measured in cell-attached patches. This suggests that high cytoplasmic Ca may indirectly down-regulate Na channels in this tissue.

Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule.

Currents through individual Na channels in the apical membrane of the rat cortical collecting tubule were resolved by using the patch-clamp technique. In cell-attached patches, the channels had a conductance of 5 pS with 140 mM NaCl in the pipet. The conductance was a saturable function of external Na, with a maximal value of about 8 pS and a half saturation at about 75 mM Na. In excised inside-out patches, the selectivity of the channels for Na over K was estimated from reversal potentials to be at least 10:1. The channels underwent spontaneous transitions between open and closed states. Both states had mean lifetimes of 3-4 sec. Amiloride (0.5 microM) added to the pipet induced more frequent closures and openings of the channels and a reduction in the mean open time. These channels are presumed to mediate Na reabsorption by this nephron segment in vivo.

Effects of cell Ca and pH on Na channels from rat cortical collecting tubule.

The patch-clamp technique was used to identify individual Na channels in the apical membrane of the rat cortical collecting tubule and to evaluate the effects of cytoplasmic Ca2+ and pH on channel activity. In excised, inside-out patches, the probability of a channels's being open (P0) increased with alkalinization of the solution bathing the cytoplasmic side of the patch. Estimates of P0 were 0.05 at pH 6.4, 0.19 at pH 6.9, and 0.41 at pH 7.4. Varying the free Ca2+ concentration of the solution bathing the cytoplasmic side of the patch had no measurable effect on P0. In cell-attached patches, addition of the Ca2+ ionophore ionomycin to the solution bathing the tubules to a final concentration of either 1 or 10 microM decreased channel activity measured as the mean number of open channels (no. open) = n X P0 where n is the number of channels in the membrane. (no. open) was significantly decreased at 3 min after addition of ionomycin and fell to less than 10% of control values after 10 min incubation. There was no fall in (no. open) either in time controls or in tubules exposed to ionomycin in the presence of low bath Ca2+ concentrations [no added Ca2+ with 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)]. The results suggest that cytoplasmic pH can directly influence channel activity. Cytoplasmic Ca2+ does not interact directly with the channels, but increased cytoplasmic Ca2+ produces a fall in channel activity through an indirect process.

Ca-activated K channels in apical membrane of mammalian CCT, and their role in K secretion.

High conductance, Ca-activated K channels were studied in the apical membrane of the rat cortical collecting tubule (CCT) using the patch-clamp technique. In cell-attached patches the channels were found mainly in the closed state at the spontaneous apical membrane potential. They spent progressively more time in the open state as the pipette potential was made negative relative to the bath. In excised patches these channels had a high selectivity for K over Na and were activated by micromolar concentrations of Ca2+ on the cytoplasmic side of the membrane in a voltage-dependent manner. They had a low conductance to Rb and were blocked by Ba (1-100 microM) from the cytoplasmic side and tetraethylammonium (TEA) (0.2-1 mM) from the luminal side. Block by external TEA and small conductance to Rb were used to investigate the role of these channels in K transport by the isolated perfused rabbit CCT. Ba (2.5 mM), a well-studied blocker of apical K conductance in this segment, hyperpolarized the transepithelial voltage (VT) by 3.7 +/- 0.9 mV when added to the luminal solution of the perfused tubule. Addition of TEA (5 mM) to the luminal solution has no effect on VT. When Na transport was abolished by luminal amiloride, perfusion with 30 mM K (replacing Na) resulted in a lumen-negative VT (18-34 mV). Under these conditions, VT was reduced by 6.0 +/- 1.5 mV by 2.5 mM Ba, whereas TEA had no effect. Perfusion with 30 mM Rb (replacing Na) also caused a lumen-negative VT that was approximately 50% of that observed with 30 mM K. The apical K conductance of the perfused CCT appears to be insensitive to luminal TEA and only modestly selective for K over Rb. This conductance, at least under the conditions of our studies, is probably not mediated by the high conductance Ca-activated K channel.

Regulation of Na channels in the rat cortical collecting tubule: effects of cAMP and methyl donors.

The whole cell patch-clamp technique was used to investigate the interactions of the amiloride-sensitive Na channel of the rat cortical collecting tubule (CCT) with adenosine 3',5'-cyclic monophosphate (cAMP) and with methyl donors. The amiloride-sensitive whole cell current (INa) was measured in principal cells of dissected, split-open tubules from rats maintained either on a control diet or on a low-Na diet to increase endogenous aldosterone secretion. With Na-depleted animals, INa was highest immediately after rupture of the membrane patch and averaged 325 pA at a membrane potential of -60 mV. INa declined over 15 min to approximately 35% of the initial value. With 8-(4-chlorophenylthio)-cAMP in the pipette, INa increased within 5 min of membrane rupture and was maintained for 15 min at levels three- to fourfold higher than the corresponding control values. With Na-replete animals, INa was undetectable (< 10 pA) without cAMP. With cAMP in the pipette, INa averaged 40 pA. In cell-attached patches on tubules from Na-replete rats exposed to cAMP, single Na channels were observed with conductive and kinetic properties similar to those from Na-depleted rats but at lower density. Inclusion of the methyl donor S-adenosyl methionine to the pipette solution did not increase INa in CCTs from Na-replete rats, either in the presence or absence of cAMP. The methylation inhibitor S-adenosyl homocysteine did not affect INa in CCT from Na-depleted animals.

Regulation of apical K channels in rat cortical collecting tubule during changes in dietary K intake.

Long-term adaptation to a high-K diet is known to increase the density of conducting secretory K (SK) channels in the luminal membrane of the rat cortical collecting tubule (CCT). To examine whether these channels are involved in the short-term, day-to-day regulation of K secretion, we examined the density of K channels in animals fed a high-K diet for 6 or 48 h. CCTs were isolated and split open to provide access to the luminal membrane. Cell-attached patches were formed on principal cells with 140 mM KCl in the patch-clamp pipette. SK channels were recognized from their characteristic single-channel conductance (40-50 pS) and gating patterns. Animals fed a control diet had SK channel densities of 0.40 channels/micrometer(2). When the diet was changed for one containing 10% KCl for 6 h, the channel density increased to 1.51 channels/micrometer(2). Maintaining the animals on a high-K diet for 48 h resulted in a further increase in SK channels to 2.29 channels/micrometer(2). Animals fed a low-K diet for 5 days or longer had SK densities of 0.53 channels/micrometer(2), not significantly different from control values. The presence of conducting Na channels in the luminal membrane will also affect K secretion by the CCT by altering the electrical driving force through the K channels. The density of Na channels, measured with LiCl in the pipette, was 0. 08 for controls and 1.00 and 1.08 channels/micrometer(2) after 6 h and 48 h on a high-K diet. Plasma aldosterone increased from 15 +/- 4 ng/dl (controls ) to 36 +/- 8 and 98 +/- 23 ng/dl after 6 and 48 h of K loading, respectively. The increase in K channel density could not be reproduced by infusion of the animals with aldosterone. We conclude that regulation of the density of conducting Na and K channels may contribute to day-to-day variation in the rate of renal K secretion and to the short-term maintenance of K balance.

The effect of fluorocitrate on urinary calcium and citrate excretion.

The renal handling of calcium and citrate was studied in dogs after the administration of fluorocitrate. The drug produced a significant increase in urinary calcium and citrate excretion. Net renal secretion of citrate occurred during the infusion of fluorocitrate since citrate clearances exceeded the glomerular filtration rate.

Low-conductance K channels in apical membrane of rat cortical collecting tubule.

Low-conductance, K-selective channels were identified in the apical membrane of the rat cortical collecting tubule (CCT) by use of the patch-clamp technique. Isolated, split tubules were bathed in K gluconate medium to depolarize the cell while keeping the intracellular K concentration high. With the patch-clamp pipette containing predominantly either Na+ or Li+ but no K, outward currents were observed through channels that had a single-channel conductance (g) of 9 pS and a probability of being open (Po) of greater than 0.9, independent of the voltage (+/- 40 mV) applied to the pipette (Vp). Similarly, only outward currents were observed when the patch was excised into high-K solution, implying a high selectivity of the channel for K+. When 1 mM BaCl2 was added to the pipette, Po decreased to 0.36 at Vp = 0; however, g was not changed but the channels flickered rapidly between open and blocked states; Po decreased as Vp was made positive, and increased as Vp was made negative. With the pipette filled with KCl + 1 mM Ba, the channels conducted K+ in both directions. The inward currents (at positive Vp were larger than the outward currents (at negative Vp) and g near Vp = 0 increased to 25 pS. When the pipette was filled with RbCl + 1 mM Ba the inward and outward currents were similar in magnitude, suggesting that the channels can conduct Rb, although not as well as K. With the tubules bathed in NaCl Ringer solution and the pipette containing KCl, inward currents were observed that could be attributed to the same pathway for K.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2(+)-dependent inhibition of sodium transport in rabbit cortical collecting tubules.

Experiments were carried out to test whether maneuvers believed to increase intracellular Ca2+ concentration [( Ca2+]cell) inhibit Na transport in cortical collecting tubules (CCTs). Unidirectional Na efflux (JNa1----b) and Na influx (JNab----1) were measured isotopically in isolated perfused renal CCTs of rabbits. The animals were either untreated or pretreated with deoxycorticosterone (DOC) for 1-3 wk. To raise [Ca2+]cell, ionomycin or quinidine were added to, or [Na] reduced in, pertubular fluid. In control DOC-pretreated CCTs JNa1----b tended to saturate as luminal Na concentration was increased, reaching 22.9 +/- 1.2 pmol.cm-1.s-1 at 145 mM. In addition, in these CCTs, in contrast to non-DOC-treated tubules, the apical cell membrane was not found to be rate limiting for Na reabsorption as neither amphotericin B nor vasopressin further enhanced JNa1----b. In non-DOC-treated CCTs 10(-6) M ionomycin inhibited JNa1----b by 44.7%. When DOC-pretreated CCTs were exposed to either 10(-6)M ionomycin or 10(-4)M quinidine, JNa1----b was inhibited by 27 and 26%, respectively, while JNab----1 remained unchanged. This ionomycin-induced inhibition was Ca dependent. Exposure of DOC-pretreated CCTs to 5 mM Na-Ringer solution (Na replaced by choline or N-methyl-D-glucamine) for 30 min reduced JNa1----b by 18-30%. The inhibition of JNa1----b caused by any of the three maneuvers was fully reversed upon addition of amphotericin B to the luminal fluid. The results are consistent with the view that a sustained increase in [Ca2+]cell reduces Na transport by inhibition of the rate of Na+ entry across the apical cell membrane.