Flow-dependent K+ secretion in the cortical collecting duct is mediated by a maxi-K channel

Craig B. Woda1, Alvina Bragin2, Thomas R. Kleyman3, and Lisa M. Satlin1

1 Division of Pediatric Nephrology, Department of Pediatrics, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029-6574; 2 Renal Division, Department of Medicine, University of Pennsylvania, Philadelphia 19104-6144; and 3 Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

K+ secretion by the cortical collecting duct (CCD) is stimulated at high flow rates. Patch-clamp analysis has identified a small-conductance secretory K+ (SK) and a high-conductance Ca2+-activated K+ (maxi-K) channel in the apical membrane of the CCD. The SK channel, encoded by ROMK, is believed to mediate baseline K+ secretion. The role of the stretch- and Ca2+-activated maxi-K channel is still uncertain. The purpose of this study was to identify the K+ channel mediating flow-dependent K+ secretion in the CCD. Segments isolated from New Zealand White rabbits were microperfused in the absence and presence of luminal tetraethylammonium (TEA) or charybdotoxin, both inhibitors of maxi-K but not SK channels, or apamin, an inhibitor of small-conductance maxi-K+ channels. Net K+ secretion and Na+ absorption were measured at varying flow rates. In the absence of TEA, net K+ secretion increased from 8.3 ± 1.0 to 23.4 ± 4.7 pmol · min-1 · mm-1 (P < 0.03) as the tubular flow rate was increased from 0.5 to 6 nl · min-1 · mm-1. Flow stimulation of net K+ secretion was blocked by luminal TEA (8.2 ± 1.2 vs. 9.9 ± 2.7 pmol · min-1 · mm-1 at 0.6 and 6 nl · min-1 · mm-1 flow rates, respectively) or charybdotoxin (6.8 ± 1.6 vs. 8.3 ± 1.6 pmol · min-1 · mm-1 at 1 and 4 nl · min-1 · mm-1 flow rates, respectively) but not by apamin. These results suggest that flow-dependent K+ secretion is mediated by a maxi-K channel, whereas baseline K+ secretion occurs through a TEA- and charybdotoxin-insensitive SK (ROMK) channel.

principal cell; intercalated cell; ROMK; epithelial sodium channels; charybdotoxin; apamin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE FINAL REGULATION OF URINARY K+ excretion is accomplished in distal nephron segments including the cortical collecting duct (CCD) (5, 12, 13, 31, 41). The CCD is a heterogeneous epithelium comprising two morphologically and functionally distinct cell populations. Principal cells mediate Na+ absorption and K+ secretion (25, 40, 49), whereas intercalated cells primarily function in H+/HCO3 transport and, under certain conditions, K+ absorption (46, 47). K+ secretion in the CCD requires basolateral Na-K-ATPase-mediated uptake of K+ into the principal cell in exchange for Na+. The high cell K+ concentration and lumen negative voltage within the CCD, generated by conductive apical Na+ entry through epithelial Na+ channels (ENaCs) and its electrogenic basolateral extrusion, favor the passive diffusion of cell K+ into the luminal fluid through apical K+-selective channels (10). The magnitude of K+ secretion is determined by its electrochemical gradient and by the permeability of the membrane to K+ (10).

Patch-clamp analysis has identified two types of apical K+ channels in the rat and rabbit CCD. The prevalence of the apical small-conductance K+ (SK) channel, its ability to conduct both K+ and Rb+, and its high open probability (Po) at the resting membrane potential (7, 10, 43, 57) suggest that this channel most likely mediates K+ secretion under baseline conditions. In contrast, the high-conductance Ca2+-activated K+ (maxi-K) channel is rarely open at physiological membrane potentials but can be activated by membrane depolarization, elevation of intracellular Ca2+ concentration ([Ca2+]i), membrane stretch, or hyposmotic stress (6, 11, 18, 22, 38, 51-53). These observations have led to the speculation that the maxi-K channel, considered not to play a major role in baseline K+ secretion in the CCD, functions in cell volume regulation (9, 18) and/or flow-mediated K+ secretion (38, 53). Although the SK channel is present solely in principal cells within the CCD, conducting maxi-K channels have been identified in both principal and intercalated cells in this segment (38, 43). The density of maxi-K channels in intercalated cells exceeds that detected in principal cells (38).

It is well established that high distal tubular flow rates stimulate net K+ secretion in the distal nephron and CCD (5, 12, 13, 23, 30, 42) and urinary K+ excretion (30). This response reflects, at least in part, an increase in delivery to and reabsorption of Na+ by principal cells (10, 44), which, in turn, increases the driving force for passive K+ efflux across the apical membrane. The SK channel has been considered to be the primary route allowing cell K+ to diffuse into the lumen down its favorable electrochemical gradient. The possibility that high urinary flow rates and hydrostatic pressure alter [Ca2+]i or the membrane stretch to which renal epithelial cells are exposed, thereby stimulating apical stretch- and Ca2+-activated maxi-K channels, has not been directly examined in the CCD.

The purpose of the present study was to test the hypothesis that stretch- and Ca2+-activated maxi-K channels mediate flow-stimulated K+ secretion in the CCD. To this end, we sought to 1) examine the effect of tetraethylammonium (TEA) and charybdotoxin, inhibitors of the maxi-K (14, 38, 45, 53) but not the SK channel (7, 53, 57), on flow-stimulated net K+ secretion in rabbit CCDs microperfused in vitro and 2) determine whether immunodetectable maxi-K channels are localized to the apical membrane of these specific nephron segments by using channel-specific antibodies.


    METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of single tubules. Adult female New Zealand White rabbits, obtained from Covance (Denver, PA), were housed in our animal care facility and given water and standard chow ad libitum. Animals were killed by intravenous injection of pentobarbital (100 mg/kg). The kidneys were removed, sliced into 2-mm coronal sections, and placed into a dish containing chilled dissection solution containing (in mM) 140 NaCl, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 D-glucose, 1 Na3 citrate, 4 lactic acid, and 6 L-alanine, pH 7.4, 290 ± 2 mosmol/kgH2O. A single tubule was studied from each animal.

Each isolated tubule was transferred immediately to a temperature- and O2/CO2-controlled specimen chamber, mounted on concentric glass pipettes, and perfused at 37°C as previously described (41). Tubules were initially perfused and bathed in Burg's solution that resembled the dissection solution except that 25 mM NaCl was replaced by NaHCO3 (41). During the 60-min equilibration period and thereafter, the perfusion chamber was continuously suffused with a gas mixture of 95% O2-5% CO2 to maintain pH of the Burg's solution at 7.4 at 37°C. The bathing solution was continuously exchanged at a rate of 0.3 ml/min by using a peristaltic pump.

Measurement of cation fluxes. Transport measurements in isolated tubules were performed in the absence of transepithelial osmotic gradients; thus water transport was assumed to be zero. Samples of tubular fluid were collected under water-saturated light mineral oil by timed filling of a precalibrated 20-nl volumetric constriction pipette. The flow rate was varied from 0.5 to 6 nl · min-1 · mm-1 by adjusting the height of a perfusate reservoir. The K+ (and Na+) concentration of perfusate and collected tubular fluid was determined by helium glow photometry, and rates of net cation transport (in pmol · min-1 · mm-1 tubular length) were calculated, as previously described (41).

For cation transport rates, three to five samples of tubular fluid were collected at each flow rate in each segment and analyzed for K+ and Na+ concentrations. The calculated ion fluxes were averaged to obtain a single mean rate of ion transport for the CCD at each flow rate. The sequence of flow rates was randomized within each group of tubules to minimize any bias induced by time-dependent changes in ion transport. The flow rate was varied at least twofold in each segment. To determine the concentration of Na+ and K+ delivered to the tubular lumen, ouabain (100 µM) was added to the bath at the conclusion of each experiment to inhibit all active transport, and three to four samples of tubular fluid were then obtained for analysis similar to that described above.

The transepithelial voltage (Vte) was measured between symmetrical calomel electrodes continuous with the perfusion pipette via a 0.16 M NaCl agarose bridge and referenced to the bath, as previously described (41). Voltages were monitored by using a high-impedance electrometer (World Precision Instruments, New Haven, CT). Readings were taken at the midpoint of each timed collection and averaged.

Western blotting of maxi-K channel protein in kidney. Mouse kidneys were homogenized in a buffer containing 250 mM sucrose, 10 mM triethanolamine, pH 7.6, and supplemented with protease inhibitors {Protease Inhibitor Cocktail Set III, final concentration 1 mM [4-(2-aminoethyl)benzenesulfonylfluoride], 0.8 µM aprotinin, 50 µM bestatin, 15 µM E-64, 20 µM leupeptin, and 10 µM pepstatin A, Calbiochem, San Diego, CA}. Aliquots of protein (100 µg) were subject to 3-15% SDS-PAGE and transferred to an Immobilon-NC membrane (Millipore, Waltham, MA). Blots were probed with a commercially available rabbit antibody directed against the alpha -subunit of the maxi-K channel (1.5 µg/ml, Alomone Labs, Jerusalem, Israel) or with nonimmune rabbit IgG (1.5 µg/ml, Jackson ImmunoResearch Labs, West Grove, PA). Bound antibody was detected following incubation with a 1:2,500 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD) by chemiluminescence (Western Blot Chemiluminescence Reagent Plus, NEN, Boston, MA).

Indirect immunofluorescence localization of maxi-K channels in the kidney. Because available antibodies directed against the maxi-K channel are raised in rabbit and can thus not be used to immunolocalize the protein in rabbit tissue, the following studies were performed in mouse kidney. To the extent that expression patterns of immunodetectable and functional ROMK are similar between rabbit and rat (7, 33, 43, 60), we considered it likely that the abundance and localization of maxi-K channels would also be similar among the various rodent species.

Adult mouse (CD-1 strain) kidney was sliced into sagittal sections and fixed in 4% paraformaldehyde prepared in 5% sucrose in PBS for 1 h. Thereafter, the tissue was incubated in 50 mM NH4Cl in 5% sucrose/PBS for 10 min to quench nuclear staining. After washing twice with 5% sucrose/PBS, the tissue was immersed in graded sucrose/PBS solutions to a final sucrose concentration of 2.3 M for cryoprotection. Blocks of tissue were embedded in OCT-compound (Sakura Finetek, Torrance, CA), diluted 1:2 with 20% sucrose, frozen, and stored at -80°C. Tissue sections were cut by using a Reichert-Jung cryostat (model 2800) and collected on Probe-on-Plus (Fisher Scientific) microscope slides.

Sections were hydrated with PBS, blocked with 10% goat serum in PBS for 1 h, and then incubated for 3 h with an anti-aquaporin-2 (AQP2) antibody raised in chicken (stock 0.26 mg/ml; generous gift from J. Wade) diluted 1:25 in PBS containing 0.1% BSA and 0.2% Triton X-100. Slides were then washed three times for 5 min with PBS and incubated in the dark for 1 h at room temperature with a 1:100 dilution of an FITC-conjugated affinity-purified donkey anti-chicken IgY/IgG (H+L) secondary antibody (Jackson ImmunoResearch Labs). Colabeling was performed by the subsequent overnight application of a 1:50 dilution of the rabbit anti-alpha -subunit maxi-K channel antibody (stock 0.3 mg/ml) at 4°C. After three successive washes in PBS, a secondary Texas red-conjugated affinity-purified goat anti-rabbit IgG (H+L; Jackson ImmunoResearch Labs) was applied at a 1:100 dilution for 1 h at room temperature in the dark. Slides were then washed three times for 5 min with PBS. Control experiments consisted of either omitting the primary or secondary antibodies or substituting the primary antibody with nonimmune chicken or rabbit antibody, as appropriate. All controls were negative for specific anti-maxi-K channel or anti-AQP2 antibody labeling.

Fluorescence microscopy was performed with a Nikon Microphot-FX microscope with a CoolSNAP digital camera (Roper Scientific).

Reagents. TEA chloride, the scorpion toxin charybdotoxin (99% purity), and the bee venom apamin (99% purity) were obtained from Sigma (St. Louis, MO). TEA, charybdotoxin, and apamin were diluted to final concentrations of 5 mM and 10 and 100 nM, respectively, in the luminal perfusate.

Statistics. All results are expressed as means ± SE; n equals the number of tubules or animals. Regression lines were constructed by the method of least squares. Comparisons were made by paired and unpaired t-tests as appropriate. Significance was asserted if P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of TEA on flow-stimulated net K+ secretion. As we (41) and others (5, 50) have previously reported, net K+ secretion increased significantly from 8.3 ± 1.0 to 23.4 ± 4.7 pmol · min-1 · mm-1 (P < 0.03 by linear regression analysis; n = 6-11 CCDs at each flow rate) as the tubular flow rate increased from 0.5 ± 0.1 to 6.3 ± 0.9 nl · min-1 · mm-1 (Fig. 1, solid bars). There was no significant change in Vte as flow was increased between these flow rates [-9.0 ± 0.9 to -5.0 ± 1.8 mV; P = not significant (NS)]. Note that the physiological flow rate for the distal nephron of the rabbit averages ~1 nl · min-1 · mm-1 (3).


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Fig. 1.   Flow stimulation of net K+ secretion in the cortical collecting duct (CCD): effect of luminal tetraethylammonium (TEA). Net K+ secretion (in pmol · min-1 · mm-1) was measured in each tubule at 2-3 flow rates in the presence of symmetrical Burg's solution (perf) in lumen and bath. Data were grouped into flow-rate intervals and averaged. Each data point represents the means ± SE of 6-11 tubules. In the absence of TEA (solid bars), net K+ secretion increased significantly with increasing flow rate (P < 0.05 by linear regression analysis). Luminal TEA (5 mM) inhibited the flow-stimulated increase in net K+ secretion (open bars). *P < 0.03 compared with transport in the absence of TEA at the same flow rate. #P = 0.06 compared with transport in the absence of TEA at the same flow rate.

TEA decreases the Po of the maxi-K channel in the CCD when applied to the extracellular surface of the membrane (6, 38, 45). The addition of 5 mM TEA to the luminal perfusate led to no significant change in net K+ secretion at slow flow rates <= 1.5 nl · min-1 · mm-1 (Fig. 1). However, as flow rate was increased, CCDs perfused with this agent failed to show the typical flow stimulation of net K+ secretion (Fig. 1, open bars). The rate of net K+ secretion at a flow rate of 0.6 ± 0.1 nl · min-1 · mm-1 was 8.2 ± 1.2 pmol · min-1 · mm-1 (n = 11), not significantly different from the rate of 9.9 ± 2.7 pmol · min-1 · mm-1 measured at a flow rate of 6.0 ± 0.4 nl · min-1 · mm-1 (n = 7; P = NS) (Fig. 1). The TEA-induced inhibition of net K+ secretion was not due to a reduction in the rate of net Na+ absorption. As shown in Fig. 2, luminal perfusion of CCDs with TEA tended to stimulate net Na+ absorption at flow rates >= 3 nl · min-1 · mm-1 (P = 0.07).


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Fig. 2.   Flow stimulation of net Na+ absorption in the CCD: effect of luminal TEA. Values are means ± SE. The rate of net Na+ absorption (in pmol · min-1 · mm-1), measured in the same CCDs described in Fig. 1, increased significantly (P < 0.05 by linear regression analysis) with increasing flow rate in the absence (solid bars) and presence (open bars) of luminal TEA. At flow rates >= 3 nl · min-1 · mm-1, TEA tended to stimulate net Na+ absorption (P = 0.07).

Effect of apamin and charybdotoxin on flow-stimulated net K+ secretion. Apamin inhibits a widely expressed, small-conductance maxi-K channel that is TEA insensitive (35) but does not inhibit the TEA-sensitive maxi-K channel in the CCD when studied at the single-channel level (45). Luminal perfusion with 100 nM apamin did not significantly affect the rates of net K+ secretion (Fig. 3) or Na+ absorption (Fig. 4) at a slow flow rate of 0.8 nl · min-1 · mm-1. A fourfold increase in luminal flow rate stimulated both net K+ secretion (from 4.8 ± 0.8 to 13.1 ± 1.3 pmol · min-1 · mm-1; P < 0.001) (Fig. 3) and net Na+ absorption (from 17.8 ± 1.8 to 72.4 ± 8.6 pmol · min-1 · mm-1; P < 0.01) (Fig. 4) in eight apamin-treated CCDs. The rates of net K+ secretion and Na+ absorption measured at high flow rates in the presence of apamin did not differ from those measured in a flow rate-matched group of CCDs perfused with Burg's solution alone (18.7 ± 3.6 and 57.3 ± 9.3 pmol · min-1 · mm-1, respectively, in 11 CCDs perfused at a mean rate of 4 nl · min-1 · mm-1; P > 0.2).


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Fig. 3.   Flow stimulation of net K+ secretion in the CCD: effect of luminal apamin and charybdotoxin. Net K+ secretion (in pmol · min-1 · mm-1) was measured in the absence of toxin and then during luminal perfusion with 100 nM apamin (n = 8) or 10 nM charybdotoxin (n = 7). Data were grouped into flow-rate intervals and averaged. Neither apamin (solid bars) nor charybdotoxin (open bars) affected the rate of net K+ secretion in CCDs perfused at slow flow rates. In the presence of apamin, net K+ secretion increased significantly with increasing flow rate. Charybdotoxin inhibited the flow-stimulated increase in net K+ secretion. Values are means ± SE. *P < 0.05 compared with transport rate at 0.8 nl · min-1 · mm-1 in the absence or presence of luminal toxin. #P < 0.05 compared with apamin at 4 nl · min-1 · mm-1.



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Fig. 4.   Flow stimulation of net Na+ absorption in the CCD: effect of luminal apamin and charybdotoxin. Net Na+ absorption (in pmol · min-1 · mm-1) was measured in the same group of CCDs described in Fig. 3. An approximately fourfold increase in flow-rate-stimulated net Na+ absorption in both apamin (solid bars)- and charybdotoxin (open bars)-treated CCDs. There was no significant difference between the transport rates measured in apamin- and charybdotoxin-treated CCDs perfused at a flow rate of 4 nl · min-1 · mm-1 (P = 0.29). Values are means ± SE. *P < 0.05 compared with transport rate at 0.8 nl · min-1 · mm-1 in the absence or presence of luminal toxin.

Luminal addition of 10 nM charybdotoxin, an inhibitor of stretch- and Ca2+-activated maxi-K channels in a variety of tissues (14, 35, 45, 53), did not significantly affect the rates of net K+ secretion (Fig. 3) or Na+ absorption (Fig. 4) in CCDs perfused at the slow flow rate of ~0.8 nl · min-1 · mm-1. However, this toxin blocked the flow-stimulated increase in net K+ secretion (Fig. 3). In seven charybdotoxin-treated tubules, the rate of net K+ secretion at 0.8 ± 0.1 nl · min-1 · mm-1 was 6.8 ± 1.6 pmol · min-1 · mm-1, a value similar (P = NS) to the rate of 8.3 ± 1.6 pmol · min-1 · mm-1 measured in the same tubules perfused at a flow rate of 3.9 ± 0.3 nl · min-1 · mm-1 (Fig. 3). Note that the latter rate of net K+ secretion was significantly less than that measured in apamin-treated CCDs perfused at identical flow rates (P < 0.05). The inhibition of flow-dependent K+ secretion was not due to a charybdotoxin-induced reduction in rate of net Na+ absorption, which increased 2.5-fold (from 18.9 ± 3.7 to 56.0 ± 12.9 pmol · min-1 · mm-1; P < 0.03) in response to the increase in luminal flow rate (Fig. 4). The rate of net Na+ absorption measured at high flow rates in the presence of charybdotoxin did not differ from that measured in a flow rate-matched group of CCDs perfused with Burg's solution alone (57.3 ± 9.3 pmol · min-1 · mm-1 in 11 CCDs perfused at a mean rate of 4 nl · min-1 · mm-1; P > 0.9). Although scorpion venom toxins modify gating of voltage-dependent Na+ channels in excitable cells (35), they have not been shown to have a direct effect on ENaC.

Western analysis of maxi-K channel protein in kidney homogenate. An immunoblot of whole mouse kidney lysate was performed to characterize the anti-maxi-K channel antibody. Polypeptides with apparent molecular masses of ~70 and 42 kDa were recognized by the antibody but not by nonimmune IgG (Fig. 5). The 70-kDa polypeptide corresponds to the reported molecular mass of the alpha -subunit of the maxi-K channel (24). The 42-kDa polypeptide may represent a proteolytic fragment of the channel protein.


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Fig. 5.   Immunoblot of homogenate of whole mouse kidney probed with rabbit antibody directed against the alpha -subunit of the maxi-K channel (lane 1) or nonimmune rabbit IgG (lane 2). One hundred micrograms of protein were loaded in each lane. Polypeptides with apparent molecular masses of 70 (arrow) and 42 kDa were recognized by the anti-maxi K channel antibody alone.

Immunolocalization of maxi-K channels in the kidney. Double immunofluorescence labeling of cryosections of mouse cortex with antibodies directed against the alpha -subunit of the maxi-K channel and AQP2 revealed colocalization along the apical membranes of a discrete group of tubules, presumably collecting ducts (Fig. 6). Patch-clamp analysis has identified functional maxi-K channels in both principal (28, 38, 43, 57) and intercalated (38) cells in the CCD; thus we did not attempt to discern the identity of collecting duct cells expressing the antigen. Functional apical maxi-K channels have been identified not only in the CCD but also in the thick ascending limb of the loop of Henle (TALH) (15, 34, 56). Consistent with the latter observation, prominent labeling with the anti-maxi-K channel antibody was detected along the apical membranes of collapsed TALHs (Fig. 6).


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Fig. 6.   Indirect immunofluorescence labeling with antibodies directed against the alpha -subunit of the maxi-K channel (A; Texas red) and aquaporin-2 (AQP2) (B; FITC) in a medullary ray of the mouse renal cortex. Maxi-K channel protein is localized to the apical portions of a collapsed collecting duct (arrow in A) that coexpresses AQP2 (arrow in B). Maxi-K channel protein is also detected along the apical membranes of several collapsed thick ascending limbs (A). A section of mouse cortex labeled with nonimmune rabbit serum (C) showed no fluorescence. Bar: 20 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Flow-stimulated K+ secretion in the CCD has been proposed to arise, in part, from increased delivery to and reabsorption of luminal Na+ from the tubular lumen, a process that enhances the lumen negative potential and rate of basolateral K+ uptake, thereby increasing the driving force for passive K+ efflux across the apical membrane. Whereas the SK channel has been considered to represent the predominant channel for K+ secretion in the CCD, others have suggested that high flow rates increase luminal hydrostatic pressure and activate maxi-K channels (10, 38, 53). In support of this argument was the report by Taniguchi and Imai (53) that the flow-dependent increase in apical K+ conductance in the isolated perfused rabbit connecting tubule, estimated by measuring the deflection of Vte in response to a step increase in luminal K+ concentration, was inhibited by luminal charybdotoxin and thus likely mediated by a maxi-K channel.

To directly examine the contribution of the maxi-K channel to flow-stimulated K+ secretion in the CCD, we exploited the sensitivity of the maxi-K channel to low concentrations of luminal TEA and charybdotoxin (6, 14, 38, 45, 53). Neither the SK channel (53, 57) nor ROMK, a cDNA encoding an ATP-regulated K+ channel considered to represent the major functional subunit of the SK channel (19, 59), is inhibited by these blockers. As shown in Figs. 1 and 3, luminal perfusion of isolated CCDs with either TEA or charybdotoxin significantly inhibited flow-dependent but not baseline net K+ secretion. These data strongly suggest that the maxi-K channel mediates flow-dependent K+ secretion in the CCD. The absence of the effect of TEA and charybdotoxin on K+ secretion in CCDs perfused at physiological flow rates (~1-2 nl · min-1 · mm-1) is compatible with the proposed role of the SK channel in mediating baseline K+ secretion.

We acknowledge that other as yet unidentified TEA- and charybdotoxin-sensitive K+ channels in the CCD that contribute to K+ secretion may exist. Indeed, a growing number of cloned K+ channels have been identified at the molecular level in the CCD (4, 27, 37, 39). Among these is TWIK-1, a member of the two-pore-domain family of K+ channels (4), which has been immunolocalized to the apical membranes of intercalated cells (4). Whereas the localization of TWIK-1 suggests that it may contribute to K+ secretion, the low sensitivity of this channel to TEA (4, 27) leads us to suspect that TWIK-1 does not mediate flow-stimulated K+ secretion.

The maxi-K channel has not been considered to play a major role in K+ secretion in the CCD. When studied by patch-clamp analysis in the cell-attached configuration, this channel is characterized by a single-channel conductance of ~100 pS, a low density, and low Po (38, 43). We consider that our detection of a maxi-K channel-mediated K+ secretory flux may reflect, in part, technical differences between the conditions employed for electrophysiological analysis and those utilized in this in vitro microperfusion study. Specifically, in the present study, CCDs were perfused at 37°C in the presence of HCO3/CO2, conditions that will allow segments to transport H+/HCO3, which may, in turn, influence K+ channel activity (9, 38). Additionally, the circumferential stretch and increase in hydrostatic pressure to which CCDs are exposed when microperfused at fast flow rates, likely leading to concave deformation of the apical membrane, differ from the "convex" apical membrane deformation experienced by cells in split-open CCDs prepared for patch-clamp analysis in the cell-attached or inside-out modes. A differential activation of channel activity by convex compared with concave curvature of the membrane has been reported for the mechanosensitive TWIK-related arachidonic acid-stimulated K+ channel, TRAAK (29).

Among the likely signals mediating the activation of the maxi-K channel at high flow rates are membrane depolarization, elevation of [Ca2+]i, and/or membrane stretch. Fast tubular flow rates stimulate net Na+ absorption in the CCD in the presence (Fig. 2) and absence (44) of K+ secretion and would be expected to result in membrane depolarization (17). We have recently shown that an acute increase in luminal fluid flow rate transiently increases [Ca2+]i from ~100 to 250 nM in both principal and intercalated cells in the rabbit CCD (58). Although this peak elevation in [Ca2+]i is within range of that considered necessary to activate maxi-K channels in the CCD (>100 nM) (38) and connecting tubule (500 nM) (53), the concentration of Ca2+ achieved in the immediate vicinity of the channel may be much greater. A flow-dependent increase in [Ca2+]i may reflect inhibition of basolateral Na+/Ca2+ exchange as cell Na+ entry increases at high flow rates (1, 2, 8). Alternatively, an increase in luminal fluid flow rate may stimulate apical Ca2+ entry through stretch-activated Ca2+-permeable channels as has been proposed to occur in the isolated perfused rabbit connecting tubule (54). Note that membrane depolarization and Ca2+ can independently activate maxi-K channels and may lead to additive effects (20, 21).

It is well established that the apical maxi-K channel in the distal nephron is stretch activated. Application of negative pressure to the patch-clamp pipette increases the mean number of open channels in apical cell-attached patches of TALH (52), connecting tubule (53), and CCD (38). The relationship between membrane stretch and [Ca2+]i in maxi-K activation has been explored by patch-clamp analysis in both the cell-attached and excised inside-out configuration after chelation of free Ca2+ with EGTA added to either the pipette or bath solutions, respectively. Whereas Pacha et al. (38) showed that the mechanosensitivity of the maxi-K channel in rat CCD was retained under both of these conditions, suggesting that stretch activation of the maxi-K channel was not mediated by an increase in [Ca2+]i, Taniguchi and Imai (53) reported that the maxi-K channel in rabbit connecting tubule was not stretch activated in the absence of ambient Ca2+. Whether the discrepant results reflect interspecies differences in inhibitor sensitivity or the presence of different isoforms of the maxi-K channel in CCD and connecting tubule remains to be determined.

Additional support for cell stretch as a signal for apical channel activation in CCDs perfused at high flow rates is suggested by our recent observation that amiloride-sensitive Na+ currents in oocytes expressing alpha -, beta -, and gamma -mENaC increased sixfold in response to increasing the rate of flow of a Na+/K+ chloride bath, superfusing the oocytes from 0 to 6 ml/min, a maneuver that presumably results in some mechanical deformation of the oocyte membrane (44). In contrast, Ba2+-sensitive K+ currents in oocytes injected with ROMK1 increased only twofold in response to an identical maneuver (44).

The presence of immunodetectable maxi-K channel protein along the apical membranes of both CCD and TALH (Fig. 6) is consistent with functional expression of a ~70- to 100-pS K+ channel in both of these nephron segments (15, 34, 56). Two subunits of the maxi-K channel have been cloned. Coexpression of the pore-forming alpha - and regulatory beta -subunits of the maxi-K channel in Xenopus laevis oocytes increases the Ca2+, voltage, and charybdotoxin sensitivity of the channel compared with that observed when the alpha -subunit is expressed alone (16, 32). Several alternate transcripts of the alpha -subunit have been reported in different species (26, 36, 55). Two transcripts of the alpha -subunit (rbslo 1 and 2) have been identified in rabbit kidney (36). Rbslo is expressed in glomeruli, thin limb of Henle, TALH, and cortical and medullary collecting ducts but is rarely detected in proximal tubule (36). Whether the identity and abundance of rbslo transcripts in TALH and CCD are similar remains to be determined.

From a clinical perspective, the proposed role of the maxi-K channel as a K+ secretory channel recruited under conditions of high distal tubular flows may provide a route for urinary K+ excretion in the absence of functional SK (or ROMK) channels. In support of this proposed role are the observations that patients with Bartter's syndrome, associated, in some cases, with loss-of-function mutations in the ROMK gene, have modest hypokalemia (48). Although the hypokalemia may arise from K+ wasting associated with loss of Na-K-2Cl cotransport in the TALH, our data suggest that increased delivery of tubular fluid to the CCD may activate maxi-K channels, thereby augmenting urinary K+ losses.


    ACKNOWLEDGEMENTS

The authors thank Drs. Shaohu Sheng, Bill Miyawaki, and Lawrence Palmer for insightful discussions, Jim Wade for providing the anti-AQP2 antibody, and Beth Zavilowitz and James Bruns for technical support.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51391 (T. R. Kleyman) and DK-38470 (L. M. Satlin) and an American Heart Association Grant-in-Aid (L. M. Satlin). An abstract of this work was presented at the Annual Meeting of the American Society of Nephrology, October 2000, Toronto, Ontario, Canada.

Address for reprint requests and other correspondence: L. M. Satlin, Box 1664, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574 (E-mail: lisa.satlin{at}mssm.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11 October 2000; accepted in final form 10 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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