Biomedical subjects

G Frindt

Publications and source records attributed to G Frindt.

At least 19 recordsLinked to original sources

PubMed2001

Activation of epithelial Na channels during short-term Na deprivation.

The role of epithelial Na channels in the response of the kidney to short-term Na deprivation was studied in rats. Animals were fed either a control-Na (3.9 g/kg) or a low-Na ( 3.8 mg/kg) diet for 15 h. Urinary excretion of Na (micromol/min), measured in conscious animals in metabolic cages, was 0.45 +/- 0.07 in controls and 0.04 +/- 0.01 in Na-deprived animals. Glomerular filtration rate, measured as the clearance of creatinine, was unaffected by the change in diet, suggesting that the reduced Na excretion was the result of increased Na reabsorption. K excretion (micromol/min), increased after the 15-h period of Na deprivation from 0.70 +/- 0.10 to 1.86 +/- 0.19. Thus the decrease in urine Na was compensated for, in terms of electrical charge balance, by an increase in urine K. Plasma aldosterone increased from 0.50 +/- 0.08 to 1.22 +/- 0.22 nM. Principal cells from cortical collecting tubules isolated from the animals were studied by using the patch-clamp technique. Whole cell amiloride-sensitive currents were negligible in the control group (5 +/- 4 pA/cell) but substantial in the Na-deprived group (140 +/- 28 pA/cell). The abundance of the epithelial Na channel subunits, alpha, beta, and gamma in the kidney was estimated by using immunoblots. There was no change in the overall abundance of any of the subunits after the 15-h Na deprivation. However, the apparent molecular mass of a fraction of the gamma-subunits decreased as was previously reported for long-term Na deprivation. Calculations of the rate of Na transport mediated by the Na channels indicated that activation of the channels during short-term Na deprivation could account in large part for the increased Na reabsorption under these conditions.

Aldosterone↗

PubMed2000

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.

Aldosterone↗

PubMed2000

Potassium restriction downregulates ROMK expression in rat kidney.

The ROMK family of proteins has biophysical properties and distribution within the kidney similar to those of secretory potassium channels of the distal nephron. To study the regulation of ROMK during variations in dietary potassium, we measured the abundance of ROMK protein in rat kidney by immunoblotting. Neither 2 nor 5 days of a high-potassium diet had an effect on protein abundance in the cortex or medulla. Potassium deprivation (2 or 5 days) decreased ROMK protein content in both cortical and medullary fractions, to 51 and 40% of controls, respectively. To see whether the Na-K-2Cl cotransporter is similarly affected by potassium restriction, we analyzed immunoblots by using an antibody for the rat type 1 bumetanide-sensitive cotransporter (BSC-1). Like ROMK, BSC-1 protein content was found to decrease significantly in the renal medulla of potassium-deprived rats. In the thick ascending limb of Henle's loop, a decrease in ROMK and BSC-1 could result in decreased reabsorption of NaCl, a finding associated with hypokalemia.

Animals↗

PubMed1999

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.

Animals↗

PubMed1998

Regulation of Na+ channels by luminal Na+ in rat cortical collecting tubule.

1. The idea that luminal Na+ can regulate epithelial Na+ channels was tested in the cortical collecting tubule of the rat using whole-cell and single-channel recordings. Here we report results consistent with the idea of Na+ self-inhibition. 2. Macroscopic amiloride-sensitive currents (INa) were measured by conventional whole-cell clamp. INa was a saturable function of external Na+ concentration ([Na+]o) with an apparent Km of 9 mM. Single channel currents (iNa) were measured in cell-attached patches. iNa increased with pipette Na+ concentration with an apparent Km of 48 mM. Since INa = (iNa)NPo, the different Km values imply that the channel density (N) and/or open probability (Po) increase as [Na+]o decreases. Reduction of [Na+]o after increasing intracellular Na+ concentration also increased the outward amiloride-sensitive conductance, consistent with activation of the Na+ channels. 3. The underlying mechanism was studied by changing pipette Na+ concentration while recording from cell-attached patches. No increase in NPo was observed, suggesting that the effect is not a direct interaction between [Na+]o and the channel. 4. [Na+]o was varied outside the patch-clamp pipette while recording from cell-attached patches. When amiloride was in the bath to prevent Na+ entry, no change in NPo was observed. 5. Activation of the channels by hyperpolarization was observed with 140 mM Na+o but not with 14 mM Na+o. 6. The results are consistent with the concept of self-inhibition of Na+ channels by luminal Na+. Activation of the channels by lowering [Na+]o is not additive with that achieved by hyperpolarization.

Adenine Nucleotides↗

PubMed1998

Dissociation of K channel density and ROMK mRNA in rat cortical collecting tubule during K adaptation.

The density of conducting K channels in the apical membrane of the rat cortical collecting tubule (CCT) is increased by a high-K diet. To see whether this involved increased abundance of mRNA coding for K channel protein, we measured the relative amounts of mRNA for ROMK, the clone of the gene thought to encode the secretory K channel in the CCT. Tubules were isolated and fixed for in situ hybridization with a probe based on the ROMK sequence. Radiolabeled probe associated with the tubule was quantified using densitometric analysis of the autoradiographic images of the tubules. The densitometry signal was shown to be proportional to the amount of radioactive probe in the sample and to the time of exposure of the film. The technique was able to detect an approximately twofold increase in the abundance of mRNA coding for the water channel aquaporin 3 (AQP3), in response to a 30-h dehydration period. Tubules from rats fed a normal diet or a high-K (10% KCl) diet had equal amounts of ROMK mRNA. This suggests that an increase in the abundance of mRNA does not underlie the increase in channel density observed under these conditions.

Adaptation, Physiological↗

PubMed1998

Low-NaCl diet increases H-K-ATPase in intercalated cells from rat cortical collecting duct.

Extracellular K+-dependent H+ extrusion after an acute acid load, an index of H/K exchange, was monitored in intercalated cells (ICs) from rat cortical collecting tubule (CCT) using ratiometric fluorescence imaging of the intracellular pH (pHi) indicator, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The hypothesis tested was that 12- to 14-day NaCl deprivation increases H-K-ATPase in rat ICs. The rate of H/K exchange in the low-NaCl ICs was double that of controls. In control ICs, H/K exchange was inhibited by Sch-28080 (10 microM). In the low-NaCl ICs, it was partially blocked by Sch-28080 or ouabain (1 mM). Simultaneous addition of both inhibitors abolished K-dependent pHi recovery. The induced H/K exchange observed with NaCl restriction was not due to elevated plasma aldosterone as evidenced by experiments on ICs from rats implanted with osmotic minipumps administering aldosterone such that plasma levels were comparable to those of NaCl-deficient rats. The results suggest that NaCl deficiency induces two isoforms of H-K-ATPase in ICs and that this effect is not mediated by elevated plasma aldosterone.

Aldosterone↗

PubMed1997

Characterization and regulation of H-K-ATPase in intercalated cells of rabbit cortical collecting duct.

K-dependent H+ extrusion was investigated using fluorescence techniques in rabbit cortical collecting tubules (CCTs). Experiments were performed in split-open tubules from normal animals exposed to the intracellular pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). This preparation permitted the study of individual intercalated cells (ICs). In the ICs, partial recovery of pH(i) was observed in response to an acute acid load upon readdition of 5 mM K to the superfusate. This recovery was SCH 28080-inhibitable (10(-5) M) and ouabain-insensitive suggesting the process is mediated by a gastric-type H-K ATPase. To see if H-K ATPase plays a role in acid secretion its function was evaluated under chronic metabolic acidosis (CMA) conditions. CMA was induced by replacing drinking water with 75 mM NH4Cl in 5% sucrose for 10-14 days. The SCH 28080-inhibitable K-dependent pH(i) recovery rate was three-fold higher in CMA ICs compared to controls. To determine the location of the H-K ATPase, CCTs were microperfused and individual peanut lectin binding (PNA) ICs studied. K-dependent pH(i) recovery was measured in response to an NH4Cl pulse. An apical SCH 28080-inhibited K-dependent pH(i) recovery process was observed in control and CMA ICs. Taken together these data confirm the existence of a gastric-type H-K ATPase in ICs of rabbit CCT. Based on our findings the H-K ATPase is found on the apical side of the cell and is stimulated under conditions of CMA.

Acid-Base Equilibrium↗

PubMed1997

Is the secretory K channel in the rat CCT ROMK?

The biophysical properties of low-conductance secretory K (SK) channels in the apical membrane of the rat cortical collecting tubule were examined to compare these properties with those of the cloned renal K channels of the ROMK family expressed in oocytes. At room temperature, with the tubule superfused with 140 mM K and 110 mM cation in the pipette, the inward single-channel conductance of the SK channels was 36 +/- 1 pS for K, 41 +/- 2 pS for NH4, and 22 +/- 3 pS for Tl. The reversal potential was nearly the same for K and Tl in the pipette but was shifted by -60 mV for NH4. The kinetics of the channel when K was the permeant ion could be described by a single open state (mean open time, 24 ms) and two closed states (mean closed times, 1.6 and 65 ms). The kinetics of SK changed when Tl was the permeant ion (mean open times of 6.6 ms and no long closed state) and when NH4 was the permeant ion (mean open time of 3.0 ms and a more prevalent long closed state). Thus the gating kinetics of the channel depend strongly on the nature of the conducted ion. The properties of SK channels were quite similar to those of ROMK2 expressed in Xenopus oocytes and measured under similar conditions, suggesting that these channels are identical.

Animals↗

PubMed1997

Localization of ROMK channels in the rat kidney.

Renal potassium secretion occurs in the distal segments of the nephron through apically located secretory potassium (SK) channels. SK may correspond to the ROMK channels cloned from rat kidney. In this study, the localization of ROMK at the cellular level in the rat kidney was examined using an affinity-purified polyclonal antibody raised against a C-terminal peptide of ROMK. The specificity of the antibody was demonstrated by immunoblots of membranes of Xenopus oocytes expressing ROMK2. Immunoblots of homogenates from rat renal outer medulla and cortex revealed predominant bands of 70 to 75 kD, which were ablated by preadsorption with an excess of peptide. These bands were specific for the rat kidney. Immunolocalization studies revealed that ROMK is expressed in specific nephron segments in both the cortex and medulla. In the cortex, ROMK was found in the apical domain of the thick ascending limb of Henle's loop, the connecting tubule, and in some, but not all, cells of cortical collecting tubules. In the medulla, expression in the apical membrane of the thick ascending limbs of Henle's loop was strong, whereas outer medullary collecting ducts were weakly stained. Expression in the thick ascending limb was also heterogeneous; some cells that expressed the Na-K-Cl cotransporter were weakly stained with the anti-ROMK antibody. No staining of glomeruli, proximal tubules, or inner medullary collecting ducts was found. The localization of ROMK agrees well with the findings of SK in patch-clamp studies and supports the view that ROMK is the SK channel of the distal segments of the nephron.

Amino Acid Sequence↗

PubMed1996

Selectivity of the renal collecting duct water channel aquaporin-3.

Aquaporin-3 (AQP3) is a water channel found in the basolateral cell membrane of principal cells of the renal collecting tubule as well as in other epithelia. To examine the selectivity of AQP3, the permeability to water (Pf), urea (Pur), and glycerol (Pgly) of Xenopus oocytes injected with cRNA encoding AQP3 was measured. Oocytes injected with cRNA encoding either human or rat aquaporin-1 (AQP1) were used as controls. Although both aquaporins permit water flow across the cell membrane, only AQP3 was permeable to glycerol and urea (Pgly > Pur). The uptake of glycerol into oocytes expressing AQP3 was linear up to 165 mM. For AQP3 the Arrhenius energy of activation for Pf was 3 kcal/mol, whereas for Pgly and Pur it was >12 kcal/mol. The sulfhydryl reagent p-chloromercuriphenylsulfonate (1 mM) abolished Pf of AQP3, whereas it did not affect Pgly. In addition, phloretin (0.1 mM) inhibited Pf of AQP3 by 35%, whereas it did not alter Pgly or Pur. We conclude that water does not share the same pathway with glycerol or urea in AQP3 and that this aquaporin, therefore, forms a water-selective channel.

4-Chloromercuribenzenesulfonate↗

PubMed1996

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.

Animals↗

PubMed1996

Feedback regulation of Na channels in rat CCT. IV. Mediation by activation of protein kinase C.

The hypothesis that feedback inhibition of the apical Na+ channels in the cortical collecting tubule (CCT) is mediated by activation of a Ca(2+)-dependent protein kinase was tested using the patch-clamp technique. Na+ channel activity was monitored in cell-attached patches in principal cells of split-open rat tubules. Mean number of open channels (NPo) and single-channel current (i) were measured at 37 degrees C during continuous tubule superfusion. Phorbol 12-myristate 13-acetate (PMA; 50 nM), an activator of protein kinase C (PKC), decreased NPo to 33% of the control value. Staurosporine (200 nM), an inhibitor of PKC and of Ca(2+)-calmodulin kinase II, practically abolished the effect of PMA. Ouabain (1 mM), reduced NPo to 29% of control values and decreased i. Ouabain did not downregulate the channels in tubules exposed to staurosporine, although it still reduced i. Incubation of the tubules with 10 microM KN-62, a specific cell membrane-permeable inhibitor of Ca(2+)-calmodulin kinase II, did not interfere with the ouabain-dependent downregulation of the channels. The results support the view that the downregulation caused by ouabain involves the Ca(2+)-dependent phosphorylation of the channel itself or of proteins regulating the channel.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

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.

Aldosterone↗

PubMed1995

Feedback regulation of Na channels in rat CCT. III. Response to cAMP.

The effects of exogenous adenosine 3',5'-cyclic monophosphate (cAMP) on apical Na channels in the rat cortical collecting tubule were studied using the patch-clamp technique and fura 2 fluorescence measurements of intracellular Ca2+ (Ca2+i). When the permeant analogue, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP, 200 microM), was added to the superfusate during recording from cell-attached patches, both the mean number of open channels (NPo) and the single-channel current (i) decreased within 3 min. When the superfusate also contained amiloride (10 microM), there was no effect of CPT-cAMP on either NPo or i. When CPT-cAMP was added to the bath before formation of the patch, the density of conducting channels was increased from 10 +/- 2 to 37 +/- 6 per patch, as estimated by analysis of channel-induced noise. This suggests that cAMP increases open-channel density in the regions of the apical membrane outside the patch but not within the patch. Channels already active in the patch before stimulation with the nucleotide are subject to feedback inhibition secondary to increased Na entry into the cell. CPT-cAMP increased Ca2+i from 104 to 198 nM. This increase in Ca2+i was abolished by benzamil (0.5 microM) or by low extracellular Ca2+. The cAMP-dependent reduction in NPo was still observed in Ca(2+)-free medium, indicating that a rise in Ca2+i was not essential for the feedback response. The decrease in NPo was attenuated, however, when cAMP was added in the absence of Ca2+ and in the presence of ouabain (1 mM) in the superfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1995

Aquaporin-3 water channel localization and regulation in rat kidney.

The aquaporins are a family of water channels expressed in several water-transporting tissues, including the kidney. We have used a peptide-derived, affinity-purified polyclonal antibody to aquaporin-3 (AQP-3) to investigate its localization and regulation in the kidney. Immunoblotting experiments showed expression in both renal cortex and medulla, with greatest expression in the base of the inner medulla. Subcellular fractionation of membranes, using progressively higher centrifugation speeds, revealed that AQP-3 is present predominantly in the 4,000 and 17,000 g pellets and, in contrast to AQP-2, is virtually absent in the high-speed (200,000 g) pellet that contains small intracellular vesicles. Immunocytochemistry and immunofluorescence studies revealed that labeling is restricted to the cortical, outer medullary, and inner medullary collecting ducts. Within the collecting duct, principal cells were labeled, whereas intercalated cells were unlabeled. Consistent with previous immunofluorescence studies (K. Ishibashi, S. Sasaki, K. Fushimi, S. Uchida, M. Kuwahara, H. Saito, T. Furukawa, K. Nakajima, Y. Yamaguchi, T. Gojobori, and F. Marumo. Proc. Natl. Acad. Sci. USA 91: 6269-6273, 1994; T. Ma, A. Frigeri, H. Hasegawa, and A. S. Verkman. J. Biol. Chem. 269: 21845-21849, 1994), the labeling was confined to the basolateral domain. Immunoelectron microscopy, using the immunogold technique in ultrathin cryosections, demonstrated a predominant labeling of the basolateral plasma membranes. In contrast to previous findings with AQP-2, there was only limited AQP-3 labeling of intracellular vesicles, suggesting that this water channel is not regulated acutely through vesicular trafficking. Immunoblotting studies revealed that thirsting of rats for 48 h approximately doubled the amount of AQP-3 protein in the inner medulla. These studies are consistent with a role for AQP-3 in osmotically driven water absorption across the collecting duct epithelium and suggest that the expression of AQP-3 is regulated on a long-term basis.

Amino Acid Sequence↗

PubMed1994

Cloning and expression of AQP3, a water channel from the medullary collecting duct of rat kidney.

The terminal part of the inner medullary collecting duct exhibits a high degree of water permeability that is independent of increased intracellular cAMP and not accounted for by the activity of the known renal epithelial water channels CHIP28 (28-kDa channel-forming integral protein) and WCH-CD (collecting duct water channel protein). Starting with rat kidney papilla mRNA, reverse transcription PCR was performed with degenerate primers assuming that the putative channel would be a member of the major intrinsic protein (MIP) family of proteins. A cDNA fragment was identified and used to screen a rat kidney cDNA library. A 1.9-kb cDNA clone was isolated. The open reading frame of 876 bp coded for a protein of 292 amino acids (M(r), 31,431). Aquaporin 3 (AQP3; 31.4-kDa water channel protein) is a newly discovered member of the MIP family. Northern blot analysis showed a single transcript for AQP3 of approximately 1.9 kb present in the renal medulla, predominantly in the inner medulla. With in situ hybridization, abundant message was found in the cells of the medullary collecting ducts. Injection of the complementary RNA of AQP3 into Xenopus oocytes markedly increased the osmotic water permeability. This permeability had an energy of activation of 3.0 kcal/mol (1 cal = 4.184 J), it was fully blocked by 1 mM p-chloromercuriphenylsulfonate, and this inhibition was reversed by 5 mM dithiothreitol. cAMP did not increase this water permeability. AQP3 did not permit passage of monovalent ions (Na, K, Cl); however, it is slightly permeable to urea. The present study demonstrates the existence of an additional water channel, AQP3, in epithelial cells of the medullary collecting duct.

Amino Acid Sequence↗

PubMed1994

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.

Adrenalectomy↗