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Identification and localization of BK- subunits in the distal nephron of the mouse kidney

¹Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska; and ²Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas

Submitted 10 January 2007 ; accepted in final form 23 April 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Large-conductance, Ca²⁺-activated K⁺ channels (BK), comprised of pore-forming - and accessory -subunits, secrete K⁺ in the distal nephron under high-flow and high-K⁺ diet conditions. BK channels are detected by electrophysiology in many nephron segments; however, the accessory -subunit associated with these channels has not been determined. We performed RT-PCR, Western blotting, and immunohistochemical staining to determine whether BK-1 is localized to the connecting tubule's principal-like cells (CNT) or intercalated cells (ICs), and whether BK-2-4 are present in other distal nephron segments. RT-PCR and Western blots revealed that the mouse kidney expresses BK-1, BK-2, and BK-4. Available antibodies in conjunction with BK-1^–/– and BK-4^–/– mice allowed the specific localization of BK-1 and BK-4 in distal nephron segments. Immunohistochemical staining showed that BK-1 is localized in the CNT but not ICs of the connecting tubule. The localization of BK-4 was discerned using an anti-BK-4 antibody on wild-type tissue and anti-GFP on GFP-replaced BK-4 mouse (BK-4^–/–) tissue. Both antibodies (anti-BK-4 and anti-GFP) localized BK-4 to the thick ascending limb (TAL), distal convoluted tubule (DCT), and ICs of the distal nephron. It is concluded that BK-1 is narrowly confined to the apical membrane of CNTs in the mouse, whereas BK-4 is expressed in the TAL, DCT, and ICs.

connecting tubule; thick ascending limb; intercalated cells; mice; maxi K; BK_Ca

BK channels exhibit different pharmacological and biophysical properties depending on the tissue of expression. For example, BK have different Ca²⁺-sensitive properties in neuronal cells (4) than in mesangial cells (42, 44). It is now recognized that the diverse properties of BK are partially due to splice variants of the pore-forming -subunit (BK-) and the varied associations with the accessory -subunits (1–4) (19, 25). The BK-1 subunit, found in smooth muscle (7) and glomerular mesangial cells (20), bestows an increased Ca²⁺ sensitivity to BK- (34). In contrast, the BK-4 subunit, found in neuronal cells, decreases the Ca²⁺ sensitivity of BK- at Ca²⁺ concentrations less than 1.5 µM (5, 14).

A role for BK as a renal K⁺-secretory channel was first indicated in the isolated rabbit connecting tubule where it was found that high flow increased a K⁺ conductance that was inhibited by charybdotoxin (45), a pharmacological blocker of BK channels (27). Flow-induced K⁺ secretion (FIKS), first reported in early micropuncture studies of distal tubule function (24, 46), is also mediated by BK in isolated rabbit cortical collecting ducts (CCD) (52). While BK are relatively quiescent at resting membrane potentials and intracellular Ca²⁺ concentrations, increased fluid delivery to the distal tubule results in increased cell-surface shear stress which can elevate the intracellular Ca²⁺ concentration to a level sufficient to activate BK (22).

Within murine kidney tubules, the BK-1 subunit is only expressed on the apical membrane of the connecting tubule (40). Additionally, FIKS is significantly attenuated in mice null for the BK-1 subunit (BK-1^–/–) (39, 40). It is not understood how BK-1 increases channel activity in response to high flow; however, BK-1 bestows increased Ca²⁺ sensitivity to BK (34) and confers sensitivity of BK to activation by protein kinase G (PKG) (20, 33).

Patch-clamp studies have revealed that BK channels are in both the ICs and CNTs/PCs of the K⁺ secreting segments of the nephron (connecting tubule and CCD); however, the density of BK channels has been shown to be substantially higher in the ICs (11, 35, 36). These observations are counterintuitive with our current understanding of the biochemical and electrophysiological properties of these cells.

PCs and CNTs express high levels of 11- hydroxysteroid dehydrogenase, the enzyme which imparts the aldosterone sensitivity to these cells and makes them well-suited to regulate K⁺ homeostasis. Furthermore, PCs/CNTs express high levels of basolateral Na⁺-K⁺-ATPase which establishes the electrochemical driving force for K⁺ secretion and Na⁺ reabsorption, whereas ICs lack sufficient pump activity to support K⁺ secretion (36, 41). For these reasons, it is unlikely that ICs play a substantial role to regulate K⁺ balance.

As a first step in elucidating the purpose of BK in ICs, we determined which BK- accessory subunits are expressed at the RNA and protein levels in the murine kidney, whether BK-1 reside in the CNTs and/or ICs of the connecting tubule and determine the localization of the other -subunits along the nephron with particular interest in their expression in the CNTs/PCs and ICs of the distal tubule and collecting duct. Understanding the pattern of -subunit expression on the ICs, PCs and CNTs will provide insight into the function of BK channels in these cells.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Animal care and tissue preparation. All animals were maintained under the conditions approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center and the University of Texas Health Science Center at San Antonio. Male C57BL/6 mice (wild-type, BK-1^–/–, and BK-4^–/–), 20 wk old, were used in these experiments. The BK-4^–/– mice were bred and housed at the University of Texas Health Science Center at San Antonio, while the wild-type and BK-1^–/– mice were bred and housed at the University of Nebraska Medical Center Animal Facility. The animals had full access to food and water and were housed in cages with four other mice.

The animals were euthanized by pentobarbital sodium overdose, the kidneys were harvested, and the capsule was removed. The kidneys were cut in half through the long axis, maintaining the normal ration of cortex to medulla. Several other tissues (liver, testes, and brain) were also harvested and processed to be used as controls.

Two kidney halves were preserved for histochemical studies. One of these was fixed in Bouins solution (LabChem, Pittsburg, PA) for 24 h, preserved in a 70% ethanol solution, and embedded in paraffin for sectioning. The other was placed in O.C.T. compound (Tissue-Tek, Fort Washington, PA), frozen in liquid nitrogen, and stored at –80°C for later sectioning. The remaining two sections were immediately processed to collect RNA from one and protein from the other.

RNA isolation/RT-PCR. TRI reagent (Molecular Research Center, Cincinnati, OH) was used following the manufacturer's instruction to isolate RNA from all of the tissues used in this study. Briefly, the kidneys and other tissues from the BK-1^–/–, BK-4^–/–, and wild-type mice were homogenized in TRI reagent. All samples were phase separated by adding bromochloropropane followed by centrifuging. The aqueous phase was transferred to a clean Eppendorf tube and treated with isopropanol to precipitate the RNA and centrifuged. The isopropanol was removed, and the RNA pellet was washed with 75% ethanol. The samples were centrifuged a final time and the RNA pellet was resuspended in RNase-free water (DEPC treated). All samples were stored at –80°C for later analysis.

The RNA was treated with DNase (Promega) and then reverse transcribed using Superscript III (Invitrogen, Carlsbad, CA) and oligo (dT) primers as per manufacturer's protocol. The resulting cDNA was PCR amplified using Taq Master Mix (Qiagen, Valencia, CA) and primers specific for each of the BK- subunits. A second group of products was produced by omitting the addition of Superscript III from reverse-transcription reactions. The products from these reactions were carried through and used for PCR as negative controls. To ensure the reverse-transcription process was successful, positive control reactions were performed using primers specific for glyceraldehydes-3-phosphate dehydrogenase (GAPDH). PCR products were run on a 1% agarose gel containing ethidium bromide for visualization with UV radiation.

Primers were designed such that any pairing with genomic DNA contaminating the reaction would produce a distinguishable PCR product. Multiple splice variants for human BK-3 have been identified. However, a murine sequence for BK-3 has not been published. Using the Jellyfish program (Infotrieve), published mRNA sequences of human BK-3 were aligned to identify conserved regions. Primers were designed from these conserved regions.

Additional PCR primers were designed to copy the entire coding region of those -subunits identified within the murine kidney. Sequencing results of these products were used to identify any kidney-specific coding variations in the BK- subunits. The sequences of all primers are listed in Table 1.

Protein isolation/Western blotting. Kidney protein was homogenized in a standard RIPA buffer containing protease inhibitors (Complete, Roche Applied Science, Indianapolis, IN). The homogenate was sonicated twice for 5 s at low power and then incubated on ice for 30 min. The contents were centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was collected and the protein concentration was determined with Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The protein was stored at –80°C until use for SDS-PAGE and Western blot analysis.

The protein samples used for BK-2 and BK-4 Western blots were taken from whole kidney homogenate. For the BK-1 Western blots, the kidneys from wild-type, BK-1^–/–, and BK-4^–/– mice were cut into several coronal sections. The cortex tissue was then isolated from the medulla, and protein was isolated from each as described above. Protein samples from both the cortex and medulla were used to detect the presence of BK-1.

A standard Western blot protocol was followed as described previously (47). Forty micrograms of protein from each sample was combined 1:1 (volume) with a 2x Laemmli sample buffer containing 5% -mercaptoethanol and boiled for 10 min. The samples and molecular weight standards (All Blue, Bio-Rad) were loaded into a 12.5% Tris·HCl Criterion Precast Gel (Bio-Rad) and separated by SDS-PAGE using 100 V for 1 h. The separated proteins were transferred to a section of Hybond-P PVDF (Amersham Bioscience, Piscataway, NJ) and blocked overnight at 4°C in TBS containing 0.05% Tween (TBST) supplemented with 3% BSA, and 3% powdered milk. The following day the membrane was washed with TBST and incubated overnight at 4°C in a buffer (TBST + 3% BSA) containing the primary antibody (BK-, Santa Cruz sc-14749; BK- and -4, Alomone; GFP, Abcam, ab13970-50) diluted 1:1,000. The next day the membrane was washed in TBST and incubated at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (donkey-anti goat for 1, Molecular Probes; goat-anti rabbit for 2 and 4, Pierce; goat-anti chicken for GFP, Molecular Probes) diluted 1:5,000 in TBST containing 1% BSA for 1 h. After this final incubation, the membrane was rinsed and the HRP-labeling was developed using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). Images of the blots were produced using UVP Imager and Labworks software.

Immunohistochemistry. Kidney sections from wild-type, BK-1^–/–, and BK-4^–/– mice were fixed and embedded in paraffin. Two slices from each paraffin block were mounted onto a histological slide. Just before immunohistochemical staining, slides were cleared in xylene followed by rehydration in a series of ethanol washes. When necessary, antigen retrieval was performed by boiling slides in 10 mM calcium-citrate (pH 6.0). Autofluorescence was blocked by incubating slides in osmium tetraoxide for 2 min followed by rinsing the slides overnight in deionized water. Sections were incubated in blocking buffer (PBS with 1% BSA and 1% powdered milk) for 30 min at room temperature, after which the primary antibodies were added. The sections were incubated overnight at 4°C with diluted primary antibody (Table 2) or an equal concentration of IgG from the host species of the primary-antibody (negative controls). As an example, the anti-BK-4 antibody was raised in rabbit, the host species. The negative control was an equal concentration of rabbit IgG instead of the anti-BK-4 antibody.

To quantify the amount of colocalization between BK-4 and the various marker antigens (THP, NCC, NCX, and AQP2), we counted the number of stained tubules observed in multiple random sweeps across a section. For these fluorescent images, colocalization was determined by taking pictures using each filter and then merging the images. This was repeated multiple times as the sweep progressed across a given section. For each colocalization study, this procedure was performed on three sections, each from a different animal. Only tubules stained for a given marker antigen were counted. It was then determined how many of those tubules stained for that antigen (NCX alone, as an example) and how many stained with that antigen plus 4 (NCX + 4). Histograms represent, as a percentage of the total counted marker antigen-stained tubules counted, the staining observed with marker antigen and BK-4 on the same tubule and the marker antigen alone.

The CNT cell marker antigen, NCX, was used to quantify the BK-1 and BK-4 stainings in the connecting tubule (CNT) and CCD with respect to CNTs/PCs and intercalated cells (ICs). Similarly, aquaporin-2 (AQP2) was used as a marker for PCs in the collecting ducts. V-ATPase was the marker of ICs in both the CNT and collecting ducts. The quantification of BK-1 in the connecting tubule CNTs and ICs was based on its expression with or without V-ATPase. BK-4 staining in the CNTs and ICs of the connecting tubule was determined by its expression with or without NCX, while its expression in the PCs and ICs of the collecting duct was based on its expression with or without AQP2. Identification, verification, and counting of cells were performed as described above. Histograms represent, as a percentage of the total number of cells counted, the staining observed with marker antigen alone, the -subunit alone, or both antigens together.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
RT-PCR. RT-PCR was performed on isolated renal RNA to identify which of the -subunits (1–4) are expressed in wild-type, BK-1^–/–, and BK-4^–/– mice and to identify any kidney-specific splice variants. Figure 1A shows the results of the initial PCR experiment to identify which BK- subunits are present in the kidney of wild-type mice. Messenger RNA for BK-1, BK-2, and BK-4 was found. Each product corresponded to the correct size of the BK- segment intended to be copied: BK-1 561 bp, BK-2 694 bp, and BK-4 620 bp. No other products were present suggesting an absence of splice variants. No products were observed in the negative controls (columns labeled with –) while GAPDH (a positive control for the reverse-transcription process) was observed in all samples in which reverse transcriptase was used (columns labeled with +). Message for BK-3 was not detected in any of the samples from the wild-type mice. The PCR results from the BK-1^–/– and BK-4^–/– mice revealed the same products minus their respective BK- subunit knockout (not shown).

View larger version (35K): [in this window] [in a new window]   Fig. 1. Results of RT-PCR using wild-type mice and primers for BK-1-4. Lanes marked with + underwent reverse transcription while those marked with – were absent reverse transcriptase (negative controls). Primers for GAPDH were used as a positive control. A: results of the PCR amplification identify products of the correct size for BK-1 (lane 1), BK-2 (lane 2), and BK-4 (lane 4). Only one band was observed for each set of primers, suggesting the absence of splice variants. No products were observed in the negative controls (top). Bottom: positive control reactions for GAPDH. B: product of correct size for BK-3 was detected in the testes and liver but not in the kidney.

To confirm the absence of renal splice variants in BK-1, BK-2, and BK-4, alternate primers were designed which would anneal to the 5' and 3' untranslated regions (UTRs). Sequencing confirmed that the 676 bp of the renal BK-1 cloned were identical to that found in smooth muscle cells (40). Similarly, 708 bp of BK-2 was cloned, sequenced, and found to contain the identical coding region as BK-2 in endocrine tissue (43, 50). BK-4 was identical to BK-4 in brain (2, 26).

Western blot. Antibodies for BK-1, -2, and -4 confirmed the presence of these proteins in the murine kidney. Western blotting determined the specificity of these antibodies before immunohistochemical studies. Because of our inability to detect BK-3 message, Western blots were not performed for BK-3.

Attempts to detect BK-1 by Western blot using protein isolated from whole kidney homogenate produced faint but inconclusive results. Because previous studies identified BK-1 only within tubule segments located within the cortex, we dissected the cortex from the rest of the kidney in an attempt to concentrate BK-1 protein. Proteins isolated from each of these sections (cortex and medulla) were then used for Western blotting. An antibody for BK-1 detected a protein of the correct molecular weight (28 kDa) in protein isolated from the cortex of wild-type and BK-4^–/– but not in BK-1^–/– (Fig. 2A, top). Anti-BK-1 was never observed in proteins isolated from the medulla of any of the mice (Fig. 2A, bottom).

View larger version (74K): [in this window] [in a new window]   Fig. 2. Western blot determinations of antibody specificity for BK-1 (A), BK-2 (B), BK-4 (C), and GFP (D). A: anti-BK-1 detected a protein of correct molecular weight (28 kDa) in tissue from the kidney cortex of wild-type and BK-4–/– mice, but not BK-1–/– (top). No such product was detected in protein isolated from the medulla of any of the mouse strains (bottom). B: anti-BK-2 detected several proteins including one of correct molecular weight for BK-2 (39 kDa) in renal sections from BK-4–/–, BK-1–/–, and wild-type mice. C: anti-BK-4 detected proteins of the correct molecular weight for BK-4 (26 kDa) and its glycosylated forms (30 and 34 kDa) from the renal sections of the BK-1–/– and wild-type mice. No bands of similar molecular weight were detected in the BK-4–/– mice. D: anti-GFP detected a 30-kDa product in renal proteins from the BK-4–/– mice. No bands were detected in the wild-type and BK-1–/– mice.

The BK-4 antibody identified proteins of identical molecular weights of 26, 30, and 34 kDa from the kidneys of wild-type and BK-1^–/– mice. These molecular weights are consistent with those reported for the core BK-4 protein and both of its glycosylated forms, respectively (17). Deglycosylation experiments have not been performed to confirm the identity of the 30- and 34-kDa products as glycosylated forms of BK-4; however, the absence of all three bands in the blot from BK-4^–/– mice (Fig. 2C) supports this conclusion.

The BK-4^–/– mice were previously generated by replacing the section of transcribed BK-4 genome with GFP mRNA (4). This replacement strategy for a gene knockout increases the efficiency of the knockout process. Additionally, because most of the transcription regulatory site is still present and used to drive GFP transcription, within a cell or tissue GFP expression can be used as a marker of the BK-4 knockout gene. Therefore, we used an anti-GFP antibody to localize GFP expression within the kidney of BK-4^–/– and to confirm the expression pattern of BK-4 in wild-type and BK-1^–/–. However, it was first confirmed that GFP was expressed in the kidney of BK-4^–/– and that the anti-GFP antibody was specific for GFP. Figure 2D is a Western blot of GFP expression (30 kDa) in the kidney of wild-type, BK-1^–/–, and BK-4^–/–. A single clear band is observed in BK-4^–/– and is absent in the wild-type and BK-1^–/–.

Immunolocalization BK-1. Figure 3A shows anti-BK-1 staining (red) with anti-NCX (green). As previously reported (41), we only observed anti-BK-1 staining in lumens showing basolateral anti-NCX staining. However, we observe in this study that anti-BK-1 staining is predominantly on the apical membrane of cells having basolateral anti-NCX staining. The arrowheads in Fig. 3A point to cells with neither anti-BK-1 nor anti-NCX staining.

View larger version (70K): [in this window] [in a new window]   Fig. 3. Double-immunohistochemical staining localizes BK-1 to connecting tubule's principal-like cells (CNTs) but not intercalated cells (ICs) of the connecting tubule. A: cortical sections (x100) showing anti-BK-1 (red) and the CNT marker, anti-NCX (green), within the same tubule. Arrows point to cells that do not stain for basolateral NCX. These cells are also absent of anti-BK-1 staining. B: representative image showing an absence of anti-BK-1 and anti-V-ATPase staining within the same cells (note: the CNT cross-section on the left is from a separate kidney). C: quantification of cellular colocalization for anti-BK-1 and anti-V-ATPase staining within the same tubule. Bars represent the percentage of cells that stained for anti-BK-1 alone, anti-V-ATPase alone, and both antigens together.

V-ATPase is localized on the apical membrane of the type-A IC and on the basolateral membrane of the type-B IC. A third type of IC, non-A-non-B, has both apical and basolateral V-ATPase and may be the dominant IC type within the mouse connecting tubule (18). This method of quantification has the added benefit of labeling all ICs within the connecting tubule allowing for a more accurate count. Figure 3B shows anti-BK-1 in red and anti-VATPase in green. These images illustrate that BK-1 does not colocalize with V-ATPase. Figure 3C is a histogram plot illustrating the negligible overlap between anti-BK-1 and anti-V-ATPase staining (BK-1 alone, 62.8 ± 5.0%; V-ATPase alone, 37.8 ± 4.0%; BK-1 + V-ATPase, 1.9 ± 1.1%). It is concluded that the BK-1 subunit is specifically localized on the apical membrane of CNTs.

Immunolocalization BK-4. We employed two different strategies to determine the localization of BK-4 in the mouse kidney: anti-BK-4 in wild-type and anti-GFP in BK-4^–/– in which the deleted BK-4 gene was replaced with GFP. Figure 4, A (anti-GFP) and B (anti-BK-4), are images of staining patterns that are consistent with the expression of BK-4 in several renal epithelial cells of the outer medullary region. No cells were stained in the inner medullary region. The specificity of the BK-4 antibody is supported by the lack of staining in BK-4^–/– (Fig. 4C). A negative control using rabbit IgG also showed a lack of staining for BK-4 (Fig. 4D). In most cases, anti-BK-4 produced a better signal to background ratio than the anti-GFP. Therefore, anti-BK-4 was used for specific localization of BK-4 in subsequent experiments.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Verification of BK-4 antibody. A: anti-GFP staining from section of GFP-replaced BK-4–/–. B: anti-BK-4 staining from a section of wild-type. C: negative control demonstrating lack of BK-4 staining in BK-4–/– mice. D: negative control using rabbit IgG for BK-4 from a renal section from wild-type mouse.

Figure 5A shows the colocalization of anti-THP (red) with anti-BK-4 (green). These results indicate that BK-4 is localized in the TAL. However, unlike anti-BK-1 which is clearly in the apical membrane of the CNT, the BK-4 staining appears cytoplasmic. The colocalization between anti-NCC (red) and anti-BK-4 (green) in Fig. 5B indicates that BK-4 has a cytoplasmic localization in the DCT. Figure 5C shows the histograms representing the percentage of THP-positive and NCC-positive tubules that stained for their antigens alone and with anti-BK-4. In both cases, greater than 90% of the tubules costained for the marker antigen and anti-BK-4 (THP + BK-4, 94.7 ± 4.4%; NCC + BK-4, 92.6 ± 5.1%). Thus BK-4 is localized to the TAL and DCT of the mouse. In both segments, the staining appears cytoplasmic.

View larger version (74K): [in this window] [in a new window]   Fig. 5. Double immunohistochemical staining showing anti-BK-4 in the thick ascending limb (TAL) and distal convoluted tubule (DCT). A: colocalization of anti-BK-4 (green) staining with anti-THP (red), a marker of the TAL (x40). B: colocalization of anti-BK-4 (green) staining with anti-NCC (red), a marker of the DCT (x100). C: bar plots summarizing the quantification of tubular colocalization of anti-BK-4 with anti-THP and anti-NCC. Bars represent the percentage of tubules counted that stained for both antigens together or the marker antigens (THP and NCC) alone.

View larger version (71K): [in this window] [in a new window]   Fig. 6. Immunohistochemical staining showing anti-BK-4 in the ICs of the connecting tubule. A: colocalization of anti-BK-4 (green) in tubules stained for anti-NCX (red), indicating BK-4 expression in the connecting tubule. Colocalization was not observed within a given cell, suggesting BK-4 is expressed in the ICs of the connecting tubule (x100). B: triple immunohistochemical staining with anti-BK-4 (green), anti-NCX (red), and anti-V-ATPase (blue) of renal section from wild-type mouse. Green and blue overlap confirms BK-4 expression in the ICs of connecting tubule (x100). C: summary bar plots showing the quantification of cellular colocalization of anti-BK-4 and anti-NCX within the same tubule. Bars represent the percentage of cells counted that stained for anti-BK-1 alone, anti-V-ATPase alone, and both antigens together.

View larger version (69K): [in this window] [in a new window]   Fig. 7. Double immunohistochemical staining localizes BK-4 on ICs of the collecting ducts. A: anti-BK-4 (green) is observed in cortical collecting duct cells that are absent of anti-AQP2 (red), indicating anti-BK-4 staining on ICs but not PCs. B: anti-BK-4 staining is absent in anti-AQP2-positive tubules of the inner medulla. Arrows indicate cells not having anti-AQP2 staining.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

BK-1 in CNT cells. As shown previously (40), the BK-1 subunit is confined to only the connecting tubule segment where it has a role in volume expansion-evoked K⁺ secretion. We previously showed that the full-length BK-1 in the rabbit connecting tubule was identical to that found in smooth muscle (40). That BK-1 is present only on the apical membrane of CNTs, and not on ICs, is consistent with the finding that CNTs, but not ICs, have substantial apical ENaC and basolateral Na⁺-K⁺-ATPase to supply the forces for K⁺ transport (3, 17, 36, 41). Previously, microperfusion of the isolated rabbit connecting tubule revealed a charybdotoxin-sensitive, flow-induced K⁺ conductance with microelectrodes (45). More recently, micropuncture of the mouse kidney revealed iberiotoxin-sensitive K⁺ secretion in the late distal tubule (1). Thus the microperfusion and micropuncture studies are consistent with a channel comprising BK-/1, which is charybdotoxin and iberiotoxin sensitive.

Interestingly, patch-clamp experiments report very few BK channels in CNTs and PCs of rat kidneys (36). However, a recent study reported that BK channels are observed with equal frequency in PCs and ICs of kidneys when MAPK is inhibited or when rats are placed on a high-K⁺ diet (21). It was proposed that the high plasma [K⁺] would activate BK by inhibiting MAPK.

The isolated rabbit CCDs exhibit flow-induced K⁺ secretion (52). It was therefore surprising that BK-1 was not present in the CCD of the mouse. However, it was shown previously that BK-1 was localized outside of the connecting tubule of rabbit renal sections, in an area consistent with the initial CCD (40). Thus the BK-1 subunit may be isolated to the connecting tubule in the mouse but also reside in the initial CCD of the rabbit, accounting for the observed inhibition of FIKS by iberiotoxin in the isolated rabbit CCD. It is also possible that BK-2 or BK-3 contributes to FIKS in the rabbit CCD.

Expression of BK-2-3 subunits in mouse kidney. Although BK-2 was not localized to specific segments in this study, it was found that BK-2 was expressed abundantly at the RNA and protein levels in mouse kidneys. These results are in agreement with previous real-time PCR experiments revealing the expression of BK-2 in the human kidney (49) and the isolated rabbit CCD (32). That BK-2 expression was increased in the kidneys of rabbits on a high-K⁺ diet suggests the involvement of this subunit in K⁺ secretion in the isolated CCD (32). The development of a BK-2 knockout will help address this issue.

The BK-3 subunit, abundantly expressed in the positive controls (testis and liver), was not detectable in mouse kidney. This result agrees with the findings by Xia et al. (53) who did not detect BK-3 RNA in rat and human kidneys by Northern blot. However, other studies found BK-3 RNA in human kidney (48) and isolated rabbit CCDs (32). Either the BK-3 is not expressed in the kidneys of the mouse as in the other species or our method was not sensitive enough to detect BK-3 transcript which may only reside in the isolated CCD.

Expression of BK-4. Anti-BK-4 and anti-GFP in GFP-replaced BK-4^–/– mice detected BK-4 in TAL, DCT, and ICs of the connecting tubule, CCD, and a portion of the outer medullary collecting duct. BK-4 was not detected in ICs of the inner medullary collecting duct. The abundant expression of BK-4 at the RNA and protein levels is consistent with a previous study (32) that found more than twice the RNA expression of BK-4 compared with the other three -subunits. The BK-4 subunit, found primarily in neuronal cells (26), has complicating effects on the Ca²⁺ and voltage sensitivity of BK (14, 51). At lower physiological intracellular Ca²⁺ concentrations, the BK-4 renders the BK- less sensitive to Ca²⁺ (51). Therefore, BK-/4 would be quiescent at resting membrane potentials. However, at Ca²⁺ concentrations greater than 1.5 µM, BK-/4 is more sensitive to activation by depolarization (14, 51). Since the TAL and DCT are not segments associated with K⁺ secretion, it is possible that BK-4 confers to the BK- pore other unknown properties that are required for the specialized functions of these cells.

The finding that ICs contain BK-/4 is consistent with the negligible apical membrane conductance in ICs found with microelectrodes (31). BK channel activity exhibited by cell-attached patches is consistent with negligible open probability of expressed BK-/4 at 100 nM Ca²⁺ (8, 36). Moreover, the insensitivity of BK-/4 to IBTX explains the findings by Grunnet et al. (12) who found IBTX binding to PCs but not ICs of the rat and rabbit distal nephron.

There are several arguments against a role for BK-/4 in flow-mediated K⁺ secretion in ICs. The BK channel should be very Ca²⁺ sensitive if Ca²⁺ is a mediator of FIKS. Flow or stretch increases the Ca²⁺ concentration in ICs to 300 nM (22). However, BK-4 renders BK- relatively unresponsive to intracellular Ca²⁺ concentrations less than 1.5 µM (51). Therefore, a Ca²⁺ concentration of 300 nM would be insufficient to activate BK-/4 significantly, even at an intracellular potential of 30 mV, which is the value calculated for the ICs of the connecting tubule of animals on a high-K⁺ diet (38). Second, BK-4 confers IBTX and ChTX resistance to BK. However, flow-induced K⁺ secretion is IBTX (1) and ChTX (45) sensitive.

On the other hand, a previous finding that a high-K⁺ diet induces an increase in BK-4 transcript in the isolated rabbit CCD suggests a secretory role for BK-4 in this segment of the rabbit kidney (32). Moreover, it is possible that the local Ca²⁺ concentration near BK-/4 becomes much higher than the 300 nM measured with high-flow conditions. However, the quantity of Na⁺-K⁺-ATPase is probably too low in ICs to serve as a source for K⁺ secretion (36). It is doubtful that lumenal H⁺-K⁺-ATPase is the source of cell K⁺ entry because a low-K⁺ diet increases H⁺-K⁺-ATPase yet decreases BK- and BK-4 (32).

A study by Hirsch et al. (15) is consistent with the notion that BK-/4 responds to cell swelling in ICs. In the rat CCD, hypo-osmotic swelling increased intracellular Ca²⁺ concentration and activated BK channels. That the steepest part of the Po vs. Ca²⁺ activation curve was between 1 and 10 µM is consistent with the properties of BK-/4. However, this study did not distinguish between PCs and ICs and the absolute increase in cell [Ca²⁺] was not determined. It therefore remains to be established whether BK-/4 in ICs is activated by physiological increases in Ca²⁺ concentrations induced by cell volume changes.

In summary, the murine kidney contains BK-1, BK-2, and BK-4 subunits. BK-1, previously found to have a role in volume expansion-induced K⁺ secretion, is narrowly confined to CNTs. The BK-4 subunit is more ubiquitously distributed with a presence in the TAL, DCT, and ICs. While it is likely that the BK-/1 has a secretory function in CNT cells, the function of BK-/4 in the TAL, DCT, and IC is still uncertain.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-49461, RO1-DK-73070, and RO1-DK-71014 (to S. C. Sansom), a fellowship (no. 0610059Z) from the American Heart Association-Heartland Affiliate (to P. R. Grimm), and American Heart Association Grant 0335007N (to R. Brenner).

	ACKNOWLEDGMENTS

 
We thank Dr. D. H. Ellison (Oregon Health and Science University) for the kind gift of the NCC antibody, Dr. J. B. Wade (University of Maryland Medical School) for the kind gift of the AQP2 antibody, and Dr. J. L. Pluznick (Yale University) for assistance and suggestions in the writing of this manuscript. We thank the University of Nebraska-Lincoln DNA-sequencing facility.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. C. Sansom, Dept. of Cellular and Integrative Physiology, Univ. of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850 (e-mail: ssansom{at}unmc.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.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Bailey MA, Cantone A, Yan Q, MacGregor GG, Leng Q, Amorim JB, Wang T, Hebert SC, Giebisch G, Malnic G. Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of Type II Bartter's syndrome and in adaptation to a high-K diet. Kidney Int 70: 51–59, 2006.[CrossRef][Web of Science][Medline]
2. Behrens R, Nolting A, Reimann F, Schwarz M, Waldschütz R, Pongs O. HKCNMB3 and hKCNMB4, cloning and characterization of two members of the large-conductance calcium-activated potassium channel subunit family. FEBS Lett 474: 99–106, 2000.[CrossRef][Web of Science][Medline]
3. Biner HL, Arpin-Bott MP, Loffing J, Wang X, Knepper M, Hebert SC, Kaissling B. Human cortical distal nephron: distribution of electrolyte and water transport pathways. J Am Soc Nephrol 13: 836–847, 2002.[Abstract/Free Full Text]
4. Brenner R, Chen QH, Vilaythong A, Toney GM, Noebels JL, Aldrich RW. BK channel beta4 subunit reduces dentate gyrus excitability and protects against temporal lobe seizures. Nat Neurosci 8: 1752–1759, 2005.[CrossRef][Web of Science][Medline]
5. Brenner R, Jegla TJ, Wickenden A, Liu Y, Aldrich RW. Cloning and functional characterization of novel large conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. J Biol Chem 275: 6453–6461, 2000.[Abstract/Free Full Text]
6. Campean V, Kricke J, Ellison D, Luft FC, Bachmann S. Localization of thiazide-sensitive Na⁺-Cl^– cotransport and associated gene products in mouse DCT. Am J Physiol Renal Physiol 281: F1028–F1035, 2001.[Abstract/Free Full Text]
7. Dick GM, Rossow CF, Smirnov S, Horowitz B, Sanders KM. Tamoxifen activates smooth muscle BK channels through the regulatory beta 1 subunit. J Biol Chem 276: 34594–34599, 2001.[Abstract/Free Full Text]
8. Dworetzky SI, Boissard CG, Lum-Ragan JT, McKay MC, Post-Munson DJ, Trojnacki JT, Chang CP, Gribkoff VK. Phenotypic alteration of a human BK (hSlo) channel by hSlo subunit coexpression: changes in blocker sensitivity, activation/relaxation and inactivation kinetics, and protein kinase A modulation. J Neurosci 16: 4543–4550, 1996.[Abstract/Free Full Text]
9. Fallet RW, Bast JP, Fujiwara K, Ishii N, Sansom SC, Carmines PK. Influence of Ca²⁺-activated K⁺ channels on rat renal arteriolar responses to depolarizing agonists. Am J Physiol Renal Physiol 280: F583–F591, 2001.[Abstract/Free Full Text]
10. Frindt G, Palmer LG. Ca-activated K channels in apical membrane of mammalian CCT, and their role in K secretion. Am J Physiol Renal Fluid Electrolyte Physiol 252: F458–F467, 1987.[Abstract/Free Full Text]
11. Frindt G, Palmer LG. Apical potassium channels in the rat connecting tubule. Am J Physiol Renal Physiol 287: F1030–F1037, 2004.[Abstract/Free Full Text]
12. Grunnet M, Hay-Schmidt A, Klaerke DA. Quantification and distribution of big conductance Ca²⁺-activated K⁺ channels in kidney epithelia. Biochim Biophys Acta 1714: 114–124, 2005.[Medline]
13. Guggino SE, Guggino WB, Green N, Sacktor B. Ca²⁺-activated K⁺ channels in cultured medullary thick ascending limb cells. Am J Physiol Cell Physiol 252: C121–C127, 1987.[Abstract/Free Full Text]
14. Ha TS, Heo MS, Park CS. Functional effects of auxiliary beta4-subunit on rat large-conductance Ca²⁺-activated K⁺ channel. Biophys J 86: 2871–2882, 2004.[Web of Science][Medline]
15. Hirsch J, Leipziger J, Frobe U, Schlatter E. Regulation and possible physiological role of the Ca²⁺-dependent K⁺ channel of cortical collecting ducts of the rat. Pflügers Arch 422: 492–498, 1993.[CrossRef][Web of Science][Medline]
16. Hunter M, Lopes AG, Boulpaep EL, Cohen B, Giebisch G. Single channel recordings of calcium-activated potassium channels in the apical membrane of rabbit cortical collecting tubules. Proc Natl Acad Sci USA 81: 4237–4239, 1984.[Abstract/Free Full Text]
17. Kashgarian M, Biemesderfer D, Caplan M, Forbush B III. Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments. Kidney Int 28: 899–913, 1985.[Web of Science][Medline]
18. Kim J, Kim YH, Cha JH, Tisher CC, Madsen KM. Intercalated cell subtypes in connecting tubule and cortical collecting duct of rat and mouse. J Am Soc Nephrol 10: 1–12, 1999.[Abstract/Free Full Text]
19. Korovkina VP, England SK. Molecular diversity of vascular potassium channel isoforms. Clin Exp Pharmacol Physiol 29: 317–323, 2002.[CrossRef][Web of Science][Medline]
20. Kudlacek PE, Pluznick JL, Ma R, Padanilam B, Sansom SC. Role of hbeta1 in activation of human mesangial BK channels by cGMP kinase. Am J Physiol Renal Physiol 285: F289–F294, 2003.[Abstract/Free Full Text]
21. Li D, Wang Z, Sun P, Jin Y, Lin DH, Hebert SC, Giebisch G, Wang WH. Inhibition of MAPK stimulates the Ca²⁺-dependent big-conductance K channels in cortical collecting duct. Proc Natl Acad Sci USA 103: 19569–19574, 2006.[Abstract/Free Full Text]
22. Liu W, Xu S, Woda C, Kim P, Weinbaum S, Satlin LM. Effect of flow and stretch on the [Ca²⁺]_i response of principal and intercalated cells in cortical collecting duct. Am J Physiol Renal Physiol 285: F998–F1012, 2003.[Abstract/Free Full Text]
23. Loffing J, Loffing-Cueni D, Valderrabano V, Klausli L, Hebert SC, Rossier BC, Hoenderop JG, Bindels RJ, Kaissling B. Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron. Am J Physiol Renal Physiol 281: F1021–F1027, 2001.[Abstract/Free Full Text]
24. Malnic G, Berliner RW, Giebisch G. Flow dependence of K⁺ secretion in cortical distal tubules of the rat. Am J Physiol Renal Fluid Electrolyte Physiol 256: F932–F941, 1989.[Abstract/Free Full Text]
25. McManus OB, Helms LMH, Pallanck L, Ganetzky B, Swanson R, Leonard RJ. Functional role of the subunit of high conductance calcium- activated potassium channels. Neuron 14: 645–650, 1995.[CrossRef][Web of Science][Medline]
26. Meera P, Wallner M, Toro L. A neuronal beta subunit (KCNMB4) makes the large conductance, voltage- and Ca²⁺-activated K⁺ channel resistant to charybdotoxin and iberiotoxin. Proc Natl Acad Sci USA 97: 5562–5567, 2000.[Abstract/Free Full Text]
27. Miller C, Moczydlowski E, Latorre R, Phillips M. Charybdotoxin, a protein inhibitor of single Ca²⁺-activated K⁺ channels from mammalian skeletal muscle. Nature 313: 316–318, 1985.[CrossRef][Medline]
28. Montrose-Rafizadeh C, Guggino WB. Role of intracellular calcium in volume regulation by rabbit medullary thick ascending limb cells. Am J Physiol Renal Fluid Electrolyte Physiol 260: F402–F409, 1991.[Abstract/Free Full Text]
29. Morita T, Hanaoka K, Morales MM, Montrose-Rafizadeh C, Guggino WB. Cloning and characterization of maxi K⁺ channel -subunit in rabbit kidney. Am J Physiol Renal Physiol 273: F615–F624, 1997.[Abstract/Free Full Text]
30. Morton MJ, Hutchinson K, Mathieson PW, Witherden IR, Saleem MA, Hunter M. Human podocytes possess a stretch-sensitive, Ca²⁺-activated K⁺ channel: potential implications for the control of glomerular filtration. J Am Soc Nephrol 15: 2981–2987, 2004.[Abstract/Free Full Text]
31. Muto S, Yasoshima K, Yoshitomi K, Imai M, Asano Y. Electrophysiological identification of alpha- and beta-intercalated cells and their distribution along the rabbit distal nephron segments. J Clin Invest 86: 1829–1839, 1990.[Web of Science][Medline]
32. Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Dietary K⁺ regulates apical membrane expression of maxi-K channels in rabbit cortical collecting duct. Am J Physiol Renal Physiol 289: F922–F932, 2005.[Abstract/Free Full Text]
33. Nara M, Dhulipala PDK, Ji GJ, Kamasani UR, Wang YX, Matalon S, Kotlikoff MI. Guanylyl cyclase stimulatory coupling to K_Ca channels. Am J Physiol Cell Physiol 279: C1938–C1945, 2000.[Abstract/Free Full Text]
34. Nimigean CM, Magleby KL. The beta subunit increases the Ca²⁺ sensitivity of large conductance Ca²⁺-activated potassium channels by retaining the gating in the bursting states. J Gen Physiol 113: 425–440, 1999.[Abstract/Free Full Text]
35. Pacha J, Frindt G, Sackin H, Palmer LG. Apical maxi K channels in intercalated cells of CCT. Am J Physiol Renal Fluid Electrolyte Physiol 261: F696–F705, 1991.[Abstract/Free Full Text]
36. Palmer LG, Frindt G. High-conductance K channels in intercalated cells of the rat distal nephron. Am J Physiol Renal Physiol 292: F966–F973, 2007.[Abstract/Free Full Text]
37. Park SY, Lee JH, Kim CD, Lee WS, Park WS, Han J, Kwak YG, Kim KY, Hong KW. Cilostazol suppresses superoxide production and expression of adhesion molecules in human endothelial cells via mediation of cAMP-dependent protein kinase-mediated maxi-K channel activation. J Pharmacol Exp Ther 317: 1238–1245, 2006.[Abstract/Free Full Text]
38. Pluznick JL, Sansom SC. BK channels in the kidney: role in K⁺ secretion and localization of molecular components. Am J Physiol Renal Physiol 291: F517–F529, 2006.[Abstract/Free Full Text]
39. Pluznick JL, Wei P, Carmines PK, Sansom SC. Renal fluid and electrolyte handling in BKCa-beta1–/– mice. Am J Physiol Renal Physiol 284: F1274–F1279, 2003.[Abstract/Free Full Text]
40. Pluznick JL, Wei P, Grimm PR, Sansom SC. BK-1 subunit: immunolocalization in the mammalian connecting tubule and its role in the kaliuretic response to volume expansion. Am J Physiol Renal Physiol 288: F846–F854, 2005.[Abstract/Free Full Text]
41. Sabolic I, Herak-Kramberger CM, Breton S, Brown D. Na/K-ATPase in intercalated cells along the rat nephron revealed by antigen retrieval. J Am Soc Nephrol 10: 913–922, 1999.[Abstract/Free Full Text]
42. Sansom SC, Stockand JD. Physiological role of large, Ca-activated K channels in human glomerular mesangial cells. Clin Exp Pharmacol Physiol 23: 76–82, 1996.[Web of Science][Medline]
43. Solaro CR, Lingle CJ. Trypsin-sensitive, rapid inactivation of a calcium-activated potassium channel. Science 257: 1694–1698, 1992.[Abstract/Free Full Text]
44. Stockand JD, Sansom SC. Glomerular mesangial cells: electrophysiology and regulation of contraction. Physiol Rev 78: 723–744, 1998.[Abstract/Free Full Text]
45. Taniguchi J, Imai M. Flow-dependent activation of maxi K⁺ channels in apical membrane of rabbit connecting tubule. J Membr Biol 164: 35–45, 1998.[CrossRef][Web of Science][Medline]
46. Terauchi R, Kitamura N. Requirement of regulated activation of Ras for response of MDCK cells to hepatocyte growth factor/scatter factor. Exp Cell Res 256: 411–422, 2000.[CrossRef][Web of Science][Medline]
47. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979.[Abstract/Free Full Text]
48. Uebele VN, Lagrutta A, Wade T, Figueroa DJ, Liu Y, McKenna E, Austin CP, Bennett PB, Swanson R. Cloning and functional expression of two families of -subunits of the large conductance calcium-activated K⁺ channel. J Biol Chem 275: 23211–23218, 2000.[Abstract/Free Full Text]
49. Wagner S, Harteneck C, Hucho F, Buchner K. Analysis of the subcellular distribution of protein kinase C using PKC-GFP fusion proteins. Exp Cell Res 258: 204–214, 2000.[CrossRef][Web of Science][Medline]
50. Wallner M, Meera P, Toro L. Molecular basis of fast inactivation in voltage and Ca²⁺-activated K⁺ channels: a transmembrane beta-subunit homolog. Proc Natl Acad Sci USA 96: 4137–4142, 1999.[Abstract/Free Full Text]
51. Wang B, Rothberg BS, Brenner R. Mechanism of beta4 subunit modulation of BK channels. J Gen Physiol 127: 449–465, 2006.[Abstract/Free Full Text]
52. Woda CB, Bragin A, Kleyman TR, Satlin LM. Flow-dependent K⁺ secretion in the cortical collecting duct is mediated by a maxi-K channel. Am J Physiol Renal Physiol 280: F786–F793, 2001.[Abstract/Free Full Text]
53. Xia XM, Ding JP, Lingle CJ. Molecular basis for the inactivation of Ca²⁺- and voltage-dependent BK channels in adrenal chromaffin cells and rat insulinoma tumor cells. J Neurosci 19: 5255–5264, 1999.[Abstract/Free Full Text]

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