Renal potassium channels and their regulation.
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Animals↗
Biomedical subjects
H Sackin
Publications and source records attributed to H Sackin.
Stretch- and volume-activated channels in isolated proximal tubule cells.
Apical and basolateral channels were studied in isolated proximal tubule cells of Necturus kidney. Many of these isolated cells maintained their polarity, with clearly delineated apical and basolateral regions. A 20-pS stretch-activated (SA) cation-selective channel was identified at the apical side of these cells. This channel was permeable to Ca, K, and Na but was not significantly gated by either membrane potential or cytosolic Ca. Negative pipette pressure (15 cmH2O) increased the open probability (Po) of this channel from 0.04 +/- 0.02 to 0.26 +/- 0.08 (n = 6). Two types of Ca-independent, mechanosensitive, K-selective (SAK) channels were identified at the basolateral surface of polarized proximal tubule cells, i.e., a 30-pS long-open time (50 +/- 7 ms) channel (n = 9), and a 46-pS short-open time (1.3 +/- 0.7 ms) channel (n = 10). Pipette suction (-12 cmH2O) increased the Po of the short-open time channels from 0.008 to 0.015 and increased the Po of the long-open time channel from 0.03 to 0.19. The effect of swelling was studied with isolated cells suspended at the tip of patch pipettes. A 50% dilution of the bath doubled cell volume, hyperpolarized the membrane potential by 11 +/- 0.7 mV, and increased the Po of the basolateral SAK channels. This was followed by a spontaneous regulatory volume decrease (RVD), repolarization of the membrane potential, and a decrease in Po. In contrast, isosmotic (bath side) replacement of an impermeant anion (methanesulfonate) with a permeant anion (Cl) doubled cell volume in 5 min but without a subsequent RVD. This sustained swelling hyperpolarized the cell potential by 5.5 +/- 0.7 mV (n = 16) and increased the Po of short-open time channel by a factor of 2.3 from 0.03 +/- 0.01 to 0.07 +/- 0.02 (n = 6). The increase in Po was primarily produced by a reduction in the interburst closed time, which decreased from 142 +/- 43 ms in K methanesulfonate to 36 +/- 11 ms in KCl solutions. These results are consistent with the hypothesis that cell swelling activates Ca-independent K channels at the basolateral membrane of renal proximal tubule. Efflux of K through these channels may partially mediate renal cell volume regulation.
Animals↗
A calcium-permeable stretch-activated cation channel in renal proximal tubule.
Isolated Necturus proximal tubules were split to expose the apical membrane surface for patch clamping. When both pipette and bath solutions contained only Ca, N-methyl-D-glucamine, and methanesulfonate, inwardly directed Ca currents were observed through a stretch-activated (SA) cation channel with conductance of 18 +/- 1 pS (n = 19). The SA cation channel exhibited little discrimination among Na, K, and Ca but was at least nine times more selective for cations than anions. The channel was not significantly gated by either membrane potential or cytosolic Ca. However, application of 15 cmH2O suction to patch pipette significantly increased the mean number of open channels by a factor of 6.5, from 0.04 +/- 0.02 to 0.26 +/- 0.08 (n = 11). Ca currents through the SA cation channel were reversibly blocked by 10 microM gadolinium, which was applied to outside surface of excised patches. This is similar to gadolinium block of stretch-activated channels in Xenopus oocytes (X.-C. Yang and F. Sachs. Science Wash. DC 243: 1068-1071, 1989). A Ca-dependent, maxi-K channel (92 +/- 9 pS, n = 5) was also found at the apical membrane of the same proximal tubules. In some cases this maxi-K channel appeared to be indirectly activated by pipette suction, raising the possibility that Ca influx through the SA cation channel may regulate K efflux via the maxi-K channel. Such a process could mediate cell volume regulation and maintain electrolyte homeostasis during normal variations in Na-substrate cotransport.
Animals↗
Apical maxi K channels in intercalated cells of CCT.
High-conductance (maxi) K channels in the apical membrane of rat and rabbit cortical collecting tubules (CCT) were studied using the patch-clamp technique. Principal cells (PC) and intercalated cells (IC) were distinguished with Hoffman modulation optics in split-open tubules. IC were further identified by staining tubules with the fluorescent mitochondrial dye, rhodamine 123. Maxi-K channels were distinguished by their high conductance (greater than 80 pS) and voltage-dependent kinetics. In CCT of rats on a low-Na diet, maxi K channels were observed in 11% of the cell-attached patches on PC and 79% of patches on IC. In rats on a normal diet, the channels were seen in 23 and 79% of patches on PC and IC, respectively. In the rabbit CCT, maxi K channels were observed in 12% (4 of 32) of the patches on PC and 82% (122 of 148) of the patches on IC. The greater abundance of channels in IC was confirmed in rat CCT using the whole-cell clamp technique. Current through the maxi K channels (IK) was measured as the tetraethylammonium (TEA)-sensitive (2.5 mM) outward current in cells equilibrated with 115 mM K and 10(-5) M Ca2+ in the pipette solution. When the cell was clamped to an internal potential of +40 mV, the average IK per cell was -4 +/- 5 pA in PC and 290 +/- 90 pA in IC. Lowering cytoplasmic Ca2+ from 10(-5) M to 10(-7) M reduced IK to 32 +/- 21 pA. Neither single Na channels nor amiloride-sensitive whole-cell currents were seen in IC. Finally, maxi K channels could be activated by pipette suction (10-40 cm H2O) in either cell-attached or inside-out patches on IC from rabbit CCT. This mechanosensitivity was observed even after chelation of free Ca2+ with ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in the pipette or the bath solutions, implying that stretch activation of these channels was not mediated by increased Ca2+ entry into the cell. The IC maxi K channel may play a role in cell volume regulation or in K secretion during elevation of luminal hydrostatic pressure.
Animals↗
Whole-cell currents in rat cortical collecting tubule: low-Na diet increases amiloride-sensitive conductance.
Individual principal cells within the rat cortical collecting tubule were studied under voltage-clamp conditions using the whole-cell variation of the patch-clamp technique. Isolated tubules were split to expose the apical membrane surface and bathed in NaCl medium at 23 degrees C. When carboxyfluorescein was included in the patch pipette, the dye diffused rapidly into the cell being clamped but did not spread to neighboring cells, indicating a lack of cell-to-cell coupling. Average cell capacitance under whole-cell clamp conditions with KCl in the pipette was 18 +/- 2 pF (n = 10 cells) in rats maintained on a normal diet, consistent with that expected from morphometric measurements of cell surface area. The capacitance increased to 36 +/- 7 pF (n = 8 cells) for rats kept on a low-Na diet, indicating that cell membrane area was increased under these conditions. The amiloride-sensitive whole-cell conductance (GNa), assumed to equal the conductance through apical Na channels, was determined as the slope of the current-voltage relation near zero holding potential. GNa was 6.0 +/- 1.7 nS/cell (n = 12) for rats maintained on a low-Na diet compared with 0.06 +/- 0.08 nS/cell (n = 13) for rats kept on a normal diet. The amiloride-insensitive whole-cell conductance averaged 9.1 +/- 2.0 nS/cell, with no significant difference between low-Na and normal groups. Sodium channel density (N) was estimated from GNa, the mean open probability of the channel, and the single-channel conductance. N equals 3,000 channels/cells in rats on a low-Na diet compared with N less than 100 channels/cell for rats on a normal diet.
Amiloride↗
Regulation of renal proximal tubule basolateral potassium channels.
In summary, these studies report a voltage-gated, stretch-activated K channel at the basolateral membrane of amphibian proximal tubule. In the normal range of potential, any reduction in metabolic activity leading to membrane depolarization would reduce the open-probability of the basolateral K channel, thereby preventing excessive loss of K out of the cell. This type of voltage gating is consistent with the decrease in macroscopic K conductance observed in perfused frog tubules after cell depolarization (Messner et al., 1985). However, it does not account for the delayed increase in basolateral K conductance that accompanies Na-substrate cotransport across the apical membrane. The stretch-activation property of basolateral K channels may explain both electrolyte and volume homeostasis in the amphibian proximal tubule. Na-substrate cotransport produces a gradual increase in cell volume in several preparations (Hempling and Hare 1961; Hacking and Eddy 1981; Hudson and Schultz 1988;). Hence, the observed increase in K conductance during luminal addition of Na-cotransported substrates may be mediated by small changes in cell volume. For example, a stretch-activated K channel, stimulated by a 1% increase in cell volume, would allow K to exit the cell down its electrochemical gradient, thereby balancing the increased K uptake associated with greater Na pump activity. A number of studies have provided evidence that cell swelling increases macroscopic K conductance (Davis and Finn 1982; Germann et al. 1986; Grinstein et al. 1982; Grinstein et al. 1984; Hamill 1983; Hoffmann 1985; Lau et al. 1984; Lopes and Guggino 1987; Richards and Dawson 1986) as well as chloride channel activity (Hudson and Schultz 1988). However, the present study is the first to suggest that the swelling-induced increase in basolateral K conductance results from stretch-activated K channels. This same stretch-activation property may also be involved in the VRD that occurs during exposure of proximal tubule cells to hypotonic media (Dellesaga and Grantham 1973; Welling et al. 1985; Lopes and Guggino 1987; Kirk et al 1987). Since cell swelling undoubtably increases membrane tension (Kelly and Macklem 1988), swollen amphibian proximal tubule cells would lose K because of an increase in the open probability of stretch-activated K channels. The additional exit of bicarbonate and water would restore the cells to their original volume.
Animals↗
A stretch-activated K+ channel sensitive to cell volume.
The role of K+ channels in cell osmoregulation was investigated by using the patch-clamp technique. In cell-attached patches from Necturus proximal tubule, the short-open-time K+ channel at the basolateral membrane could be stretch-activated by pipette suction, where a negative pressure of 6 cm H2O (588.6 Pa) was sufficient to increase the open probability of the channel by a factor of 4.0 +/- 0.8 (n = 7 tubules). A 50% reduction in bath osmolarity increased cell volume by 66 +/- 10% and increased the K+-channel open probability by a factor of 5.8 +/- 1.4 (n = 7) in the same cell-attached patches that were activated by pipette suction. A kinetic analysis indicates one open state and at least two closed states for this epithelial K+ channel. Both suction and swelling shorten the longest time constant of the closed-time distribution by a factor of 3, without significant effect on either the mean open time or the shorter closed-state time constant. The similar effect of suction and swelling is consistent with the hypothesis that stretch-activated K+ channels mediate the increase in macroscopic K+ conductance that occurs during osmoregulation of amphibian proximal tubules. Calculations based on a simple model indicate that small increments in cell volume could produce statistically significant increases in K+-channel activity.
Animals↗
Regulation of renal ion channels.
Ion channels in renal epithelia are involved in maintenance of the volume and ion composition of the epithelial cells themselves and of the entire organism. The latter function depends on transepithelial ion transport, a process that often involves ion channels at the apical (luminal) and/or the basolateral (contraluminal) cell membranes. Regulation of these channels is accomplished within many different time frames, each of which can involve different molecular mechanisms of regulation. Changes in membrane voltage, intracellular ion composition, or mechanical force on the membrane mediate short-term regulation. Biosynthesis, degradation, and reversible transfer of channels to or from cytoplasmic stores are responsible for longer term regulation. Covalent modification of channel proteins can be involved in either short- or long-term regulation. In this review we outline the different models of ion channel regulation in renal epithelia and give examples that emphasize the physiological roles of these channels in specific nephron segments.
Animals↗
Basolateral potassium channels in renal proximal tubule.
Potassium (K+) channels in the basolateral membrane of unperfused Necturus proximal tubules were studied in both cell-attached and excised patches, after removal of the tubule basement membrane by manual dissection without collagenase. Two different K+ channels were identified on the basis of their kinetics: a short open-time K+ channel, with a mean open time less than 1 ms, and a long open-time K+ channel with a mean open time greater than 20 ms. The short open-time channel occurred more frequently than the longer channel, especially in excised patches. For inside-out excised patches with Cl- replaced by gluconate, the current-voltage relation of the short open-time K+ channel was linear over +/- 60 mV, with a K+-Na+ selectivity of 12 +/- 2 (n = 12), as calculated from the reversal potential with oppositely directed Na+ and K+ gradients. With K-Ringer in the patch pipette and Na-Ringer in the bath, the conductance of the short open-time channel was 47 +/- 2 pS (n = 15) for cell-attached patches, 26 +/- 2 pS (n = 15) for patches excised (inside out) into Na-Ringer, and 36 +/- 6 pS (n = 3) for excised patches with K-Ringer on both sides. These different conductances can be partially explained by a dependence of single-channel conductance on the K+ concentration on the interior side of the membrane. In experiments with a constant K+ gradient across excised patches, large changes in Na+ at the interior side of the membrane produced no change in single-channel conductance, arguing against a direct block of the K+ channel by Na+. Finally, the activity of the short open-time channel was voltage gated, where the mean number of open channels decreased as a linear function of basolateral membrane depolarization for potentials between -60 and 0 mV. Depolarization from -60 to -40 mV decreased the mean number of open K+ channels by 28 +/- 8% (n = 6).
Animals↗
Stretch-activated potassium channels in renal proximal tubule.
A short open-time potassium (K) channel that has previously been identified in the basolateral membrane of Necturus proximal tubule (17) is activated by membrane stretch. Application of between 12 and 20 cmH2O negative pressure to the patch pipette reversibly increases mean number of open basolateral K channels (NP0) by a factor of 5.3 +/- 2 in cell-attached patches (n = 4) and a factor of 13.7 +/- 5 in excised patches (n = 8). This stretch activation does not alter channel selectivity or conductance and depends on neither the direction of K current nor the orientation of the patch ("inside-out" vs. "outside-out"). The increase in NP0 occurs within seconds after applying negative pressure to the patch and is proportional to applied negative pressure. Stretch activation of the basolateral potassium channel may play an important role in proximal tubule cell volume regulation. For example, if swelling stretches the basolateral membrane, the resulting increase in NP0 could restore cell volume by loss of K (with an accompanying anion) followed by osmotic exit of water.
Algorithms↗
Electrophysiology of salamander proximal tubule. I. Effects of rapid cooling.
The response of the amphibian proximal tubule to a rapid decrease in temperature was studied in isolated perfused tubules of Ambystoma tigrinum. Cooling from 23 to 4 degrees C increased paracellular and cellular electrical resistances by factors of 1.7 and 3.6, respectively, but had virtually no effect on the ionic selectivity of the paracellular pathway. When lumen and bath solutions were maintained identical by rapid tubule perfusion, decreasing bath temperature from 22 to 0 degree C in 400 ms depolarized the transepithelial potential (Vte) from -3.7 +/- 0.3 to -1.1 +/- 0.2 mV and depolarized the basolateral membrane potential (Vbl) from -52 +/- 3 to -45 +/- 3 mV (n = 12). These fast depolarizations were followed by slower depolarizations of both Vte and Vbl that continued throughout the period of low temperature. Only approximately 30% of the initial slow depolarization of Vte at low temperature could be explained by changes in electrical resistance and cell membrane potential. The remaining 70% of this Vte depolarization at low temperature is consistent with equilibration of a hypertonic interspace with isotonic lumen and bath solutions. Given the anion selectivity of Ambystoma proximal tubule, the magnitude of this slow Vte depolarization implies an interspace NaCl concentration 2-5% higher than the NaCl concentration in either the lumen or bath solutions.
Ambystoma↗
Electrophysiology of salamander proximal tubule. II. Interspace NaCl concentrations and solute-coupled water transport.
The role of the paracellular interspace in solute-coupled water transport was investigated in isolated perfused salamander (Ambystoma) proximal tubules using a null-point technique to estimate interspace NaCl concentrations. Constant composition of luminal fluid was maintained by rapid (200 nl/min) perfusion of tubules 600,micron or less in length. Inhibition of active transport by a decrease in bath temperature from 22 to 0 degrees C in 400 ms produced rapid depolarizations of both the transepithelial (Vte) and basolateral (Vbl) potential, followed by slower changes in potential that occurred at low temperature. During this period, the time course of Vbl was independent of small changes in bath NaCl concentration, whereas the time course of Vte at low temperature varied from a slow depolarization to a slow repolarization depending on whether the concentration of NaCl in the bath equaled or exceeded that in the perfusate. Absence of a slow change in Vte at low temperature indicated a match between the NaCl concentration of the interspace and the test concentration of NaCl in the bath. Using this technique with 12 tubules, the normal interspace NaCl concentration appeared to be approximately 4% above the NaCl concentration of either the lumen or bath, demonstrating that the interspace of the salamander proximal tubule can function as a local hyperosmotic compartment to facilitate fluid transport between solutions of identical composition.
Ambystoma↗
Rheogenic transport in the renal proximal tubule.
The electrophysiology of the renal Na-K ATPase was studied in isolated perfused amphibian proximal tubules during alterations in bath (serosal) potassium. Intracellular and extracellular ionic activity measurements permitted continuous evaluation of the Nernst potentials for Na+, K+, and Cl- across the basolateral membrane. The cell membrane and transepithelial potential differences and resistances were also determined. Return of K to the basal (serosal) solution after a 20-min incubation in K-free solution hyperpolarized the basolateral membrane to an electrical potential that was more negative than the Nernst potential for either Na, Cl, or K. This constitutes strong evidence that at least under stimulated conditions the Na-K ATPase located at the basolateral membrane of the renal proximal tubule mediates a rheogenic process which directly transfers net charge across the cell membrane. Interpretation of these data in terms of an electrical equivalent circuit permitted calculation of both the rheogenic current and the Na/K coupling ratio of the basolateral pump. During the period between 1 and 3 min after pump reactivation by return of bath K, the basolateral rheogenic current was directly proportional to the intracellular Na activity, and the pump stoichiometry transiently exceeded the coupling ratio of 3Na to 2K reported in other preparations.
Ambystoma↗
Measurement of intracellular ionic composition and activities in renal tubules.
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Amphibians↗
Isolated perfused salamander proximal tubule: methods, electrophysiology, and transport.
Techniques are presented for the isolation and perfusion of renal proximal tubules from the neotenic salamander Ambystoma tigrinum. Methods are described for a determination of normal values for fluid transport and electrophysiological parameters. Stable cellular microelectrode recordings are reported that constitute the first intracellular measurements in an isolated perfused tubule preparation. With identical solutions in lumen and bath, fluid reabsorption averaged 0.28 nl.min-1.mm-1, transepithelial potential difference averaged -4.5 mV, transepithelial resistance was 52.1 omega.cm2, and the transepithelial chloride-to-sodium transference number ratio was 3.4. The basolateral cell membrane potential difference averaged -59.6 mV, and the ratio of apical-to-basolateral cell membrane resistance was between 3.9 and 5. Viability of the isolated perfused salamander proximal tubule preparation is demonstrated by a detailed comparison of the present data with results of in vivo micropuncture experiments on both Necturus and intact Ambystoma kidneys. In addition to being an advantageous preparation for long-term intracellular recordings, the Ambystoma kidney is unique in that proximal tubules can be studied both in isolation and by conventional micropuncture.
Ambystoma↗
Isolated perfused salamander proximal tubule. II. Monovalent ion replacement and rheogenic transport.
Early proximal tubules of the salamander kidney (Ambystoma tigrinum) were isolated and perfused in vitro. Transepithelial and basolateral electrical potential differences, transepithelial resistances, and intracellular ionic activities were measured during removal of Na+, K+, or Cl- from the lumen, the bath, or both lumen and bath. The effects of these external ionic replacements are interpreted in terms of an equivalent circuit that represents the renal epithelium as a network of passive ionic resistances, ionic diffusion potentials, and active transport current sources. Results indicate that rheogenic transport across the basolateral membrane is substantially diminished by removal of Na+ from either lumen or bath or by removal of K+ from the bath. On the other hand, bilateral chloride removal produces an increase in transepithelial resistance but almost no change in the calculated rate of basolateral rheogenic transport. This suggests that the source of the basolateral rheogenic ion flux ia a Na-K-ATPae that actively transports an excess of outward Na+ over inward K+.
Animals↗
The rheogenic nature of sodium transport in leaky epithelia studied by equivalent circuit analysis.
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Ambystoma↗
Equivalent electrical circuit analysis and rheogenic pumps in epithelia.
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Animals↗