Departments of 1 Medicine and 3 Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8856; 2 Department of Biomedical Science, University of Sheffield, Sheffield S102TN, United Kingdom; and 4 Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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ROMK channels are present in the cortical
collecting ducts of kidney and are responsible for K+
secretion in this nephron segment. Recent studies suggest that endocytosis of ROMK channels is important for regulation of
K+ secretion in cortical collecting ducts. We investigated
the molecular mechanisms for endocytosis of ROMK channels expressed in
Xenopus laevis oocytes and cultured Madin-Darby canine
kidney cells. When plasma membrane insertion of newly synthesized
channel proteins was blocked by incubation with brefeldin A, ROMK
currents decreased with a half-time of ~6 h. Coexpression with the
Lys44Ala dominant-negative mutant dynamin, but not wild-type
dynamin, reduced the rate of reduction of ROMK in the presence of
brefeldin A. Mutation of Asn371 to Ile in the putative NPXY
internalization motif of ROMK1 abolished the effect of the Lys44
Ala
dynamin mutant on endocytosis of the channel. Coimmunoprecipitation
study and confocal fluorescent imaging revealed that ROMK channels
associated with clathrin coat proteins in Madin-Darby canine kidney
cells. These results provide compelling evidence for endocytosis of
ROMK channels via clathrin-coated vesicles.
dominant-negative dynamin; Madin-Darby canine kidney cells; brefeldin A; Xenopus laevis oocytes; tyrosine-based consensus motif
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INTRODUCTION |
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THE PHYSIOLOGICAL ROLE OF the low-conductance K+ channels in apical membranes of principal cells of the cortical collecting ducts (CCDs) in the regulation of K+ secretion is well established (43). Secretion of K+ in this nephron segment is mediated by active transport of K+ into the cell through basolateral Na+-K+-ATPase and followed by passive movement of K+ into the tubular fluid through the apical K+ channels. cDNAs for ROMK1 and its isoforms ROMK2 and ROMK3 have been isolated (4, 15, 44). On the basis of the distribution of mRNA and proteins and biophysical characterization, it is known that the cDNAs for ROMK encode the low-conductance K+ channels in the apical membranes of the thick ascending limb of Henle's loop and CCD (11, 12).
As the final common pathway for K+ secretion in CCDs, ROMK channels are regulated by dietary K+ intake (43). To maintain K+ homeostasis, the ability of the kidney to secrete K+ increases when dietary intake increases. This response, called K+ adaptation, is associated with an increase in the number of active channels in rat CCDs. Studies using cell-attached patch-clamp recording have found that the number of active K+ channels in the apical membranes of CCDs increases by a factor of 3-4 when animals are fed a high-K+ diet for 1-2 wk (29). The increase in the density of active channels because of high K+ intake is not associated with an increase in the mRNA for ROMK in isolated rat CCDs (10). These results raise the possibility that ROMK channels in the apical membranes of CCD undergo active trafficking, and the increase in channel density in K+-adapted animals may be due to alterations in these processes.
Recently, Wang et al. (40) reported that Src kinase activity in rat kidney cortex is inversely correlated with dietary K+ intake in these animals. Low dietary K+ intake increases the activity of Src kinase, whereas high dietary K+ intake decreases the activity. Application of tyrosine kinase inhibitors and tyrosine phosphatase inhibitors increases and decreases, respectively, the number of channels in cell-attached recordings of CCDs isolated from these animals as well as in oocytes coexpressing ROMK1 and recombinant Src kinase (24, 41). The effects of these tyrosine kinase and phosphatase inhibitors on channel activity are prevented by pretreating the cells with inhibitors of endocytosis (24, 41). These results suggest that alteration of endocytosis of K+ channels is important for physiological regulation of the K+ channels in CCDs by dietary K+ intake.
There are multiple pathways for endocytosis in mammalian cells, including phagocytosis, internalization via caveolae, and clathrin-dependent endocytosis (17, 23, 25). Phagocytosis (or "cell eating") occurs only in specialized cells. Caveolae are small microdomains of plasma membranes that are enriched in cholesterol and glycosphingolipids. Some glycophosphatidylinositol-anchored proteins and receptors of the seven-transmembrane-domain family are endocytosed via caveolae. Endocytosis via clathrin-coated vesicles is a well-characterized form of pinocytosis and is a common mechanism for retrieval of plasma membrane proteins (17, 23, 25).
The clathrin coat contains two oligomeric proteins, clathrin, and
clathrin adaptor protein (AP) complexes (37). Clathrin is
composed of three light (~33 kDa) and three heavy (~180 kDa) chains
that form a three-legged structure called triskelion. The AP complexes
are heterotetrameric. Thus far, three adaptor complexes, AP-1, AP-2,
and AP-3, have been found (18). The localization and
function of the three AP complexes are different. AP-2 complexes are
localized to the coated pits and are involved in plasma membrane endocytosis. Assembly of clathrin is not sufficient to drive vesicle budding. Budding of CCVs from plasma membranes requires dynamins, members of the ~100-kDa GTPase protein family
(34). The role of dynamins in clathrin-dependent
endocytosis from the plasma membrane is well established. Mutations of
dynamin that interfere with GTP binding and hydrolysis [such as
Lys44Ala (K44A)] severely inhibit CCV-mediated endocytosis from
plasma membranes (8). In the present study, we sought to
investigate the role of CCVs in endocytosis of ROMK.
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MATERIALS AND METHODS |
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Materials.
All chemicals are from Sigma-Aldrich (St. Louis, MO) unless stated
otherwise. Restriction endonucleases are from New England Biolabs,
GIBCO-BRL, or Roche-Mannheim. Brefeldin A (BFA) stocks (in ethanol; 15 mM) were stored at 20°C before use.
Molecular biology.
Wild-type (WT) ROMK1 cDNA was in the pSPORT plasmid (15).
Site-directed mutagenesis of ROMK1 was performed using a commercial mutagenesis kit (Quickchange, Stratagene, La Jolla, CA) and confirmed by nucleotide sequencing as previously described (16, 19, 20). mCAP cRNAs of WT ROMK1 channels were transcribed in vitro using T7 RNA polymerase (mMESSAGE kit, Ambion, Austin, TX) (16, 19, 20) after linearization of plasmid DNA with NotI
restriction endonuclease. cDNAs for syntaxin 3 and syntaxin 5 were in
pGEM vector that contained Xenopus laevis -globin 5'- and
3'-untranslated regions (3). mCAP cRNAs for syntaxins were
transcribed in vitro using T7 RNA polymerase after linearization of
plasmid DNA with NheI restriction endonuclease
(3). cDNAs encoding c-myc-tagged WT and K44A mutant rat
dynamin II were originally constructed in pCMV-5 vector
(2). An EcoRI-XbaI fragment was
excised from the original pCMV-dynamin plasmid and subcloned into
pBluescript SK vector doubly digested with EcoRI and
XbaI. mCAP cRNAs for WT and K44A dynamins were transcribed
in vitro using T7 RNA polymerase after linearization of
pBluescript-dynamin plasmid DNAs with XbaI restriction
endonuclease. Construction of the NH2-terminal enhanced green fluorescent protein (EGFP)-tagged ROMK2 in pEGFP-C2 vector (Clontech), pEGFP-ROMK2, has been described (28).
Immunostaining and laser scanning confocal imaging. Madin-Darby canine kidney (MDCK) cell lines stably transfected with pEGFP-C2 empty vector or pEGFP-ROMK2 were grown in DMEM/F-12 media (1:1) with 10% fetal calf serum and selected with geneticin at 0.3 mg/ml as previously described (28). Cells were subcultured at 1:10 dilution twice a week. For immunostaining by monoclonal antibody against clathrin heavy chain, cells were grown on cover glasses to ~50% confluence at 37°C and further incubated at 25°C for 24-48 h. Surface expression of ROMK in MDCK cells is low at 37°C (6, 28). Growing cells at 25°C increases the stability and surface expression of the channel (6, 28). MDCK cells (on cover glasses) were fixed in 4% formalin in PBS and permeabilized by 0.1% Triton X-100. After blocking for the nonspecific staining by 5% BSA, cells were incubated sequentially with mouse monoclonal anti-clathrin heavy chain antibody (1:200 dilution; ICN Biochemicals), rabbit anti-mouse IgG (1:200), and rhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Fluorescent images were visualized through a Zeiss ×100 objective lens with a Zeiss LSM410 microscope. To detect the fluorescence of the green fluorescent protein (GFP), samples were excited with a 643 Kr/Ar ion laser at 488 nm. Fluorescent emissions were passed through a 510/540 band-pass filter. To detect fluorescence raised by rhodamine, samples were excited at 568 nm and emissions were passed through a 590-nm-long pass filter.
Coimmunoprecipitation of ROMK2 with clathrin coat proteins.
MDCK cells stably transfected with ROMK2 (cultured as above) were lysed
in a radioimmunoprecipitation assay buffer containing (in mM) 150 NaCl,
10 HEPES (pH 7.5), 1 EGTA, 1 DTT, 1 pheylmethylsulfonyl fluoride, as
well as 1% (vol/vol) Nonidet P-40, 1% (wt/vol) sodium deoxycholate,
0.1% (wt/vol) SDS, 10 µM leupeptin, and 1 µM pepstatin at 4°C
for 1 h. Lysates were centrifuged in a microfuge at 15,000 rpm at
4°C for 30 min. Supernatants were incubated with either monoclonal
antibody against -adaptin (1:30 dilution; Transduction Lab) or
control mouse IgG for 2 h and then precipitated by protein G-Sepharose. The immunoprecipitates were separated by 10% SDS-PAGE and
detected in immunoblot analysis by using monoclonal antibodies against
-adaptin and against clathrin heavy chain and a polyclonal antibody
against ROMK1.
Two-electrode voltage-clamp recording.
X. laevis oocytes were prepared as previously described
(16, 19, 20). Oocytes were injected with cRNA for ROMK1,
cRNA for WT or dominant-negative dynamin, and/or cRNA for syntaxin, as
indicated. Current-voltage (I-V) relationships
(100 to +100 mV, in 25-mV steps) were measured in oocytes at
~23°C by a two-electrode voltage clamp (TEVC) using an OC-725C
oocyte clamp amplifier (Warner Instruments, Hamden, CT), pCLAMP6
software, and Digidata 1200A digitizer (Axon Instruments, Foster City,
CA). The resistance of current and voltage microelectrodes (filled with
3 M KCl solution) was 1-2 M
. The bath solution contained (in
mM) 96 KCl, 1 MgCl2, 1 CaCl2, and 5 HEPES (pH
7.5 by KOH).
Whole-cell patch-clamp and single-channel patch-clamp recording.
For patch-clamp recording, MDCK cells were grown to ~50% confluence
at 37°C and further incubated at 25°C for 24-48 h. Cells were
dissociated by limited trypsin treatment and transferred to a recording
chamber. For ruptured whole-cell recording of MDCK cells, patch-clamp
pipettes (pulled from borosilicate glass, Warner Instruments) were
filled with solutions containing (in mM) 140 KCl, 1 MgCl2,
2 Na2ATP, 1 EGTA, and 5 HEPES (pH 7.4 titrated with KOH).
Bath solutions contained (in mM) 140 KCl, 1 MgCl2, 1 CaCl2, 5 HEPES (pH 7.4 with KOH), and 5 glucose. For
cell-attached single-channel recording (in MDCK cells and in oocytes),
pipette solutions were (in mM) 100 KCl, 1 MgCl2, 2 CaCl2, and 5 HEPES (pH 7.4 titrated with KOH). Pipette tip
resistance ranged from 3 to 5 M. Currents (whole cell or single
channel) were recorded with an Axopatch 200B patch-clamp amplifier
(Axon Instruments). Single-channel currents were low-pass filtered at 1 kHz using an eight-pole Bessel filter, sampled every 0.1 ms (10 kHz)
with a Digidata-1200A interface, and stored directly onto a computer
hard disk (100 GB) using pCLAMP7 software (19). Data were
transferred to CD for long-term storage. For analysis, event list files
were generated using the Fetchan program and analyzed for open
probability (Po) and amplitude histogram using
pCLAMP7 pSTAT (version 6.0.5, Axon Instruments).
Po was analyzed on segments of continuous
recording from patches that contained only one active channel during
the lifetime of the recording. Po was determined
using the criteria of threshold crossing of currents (50%)
(19).
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RESULTS |
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Endocytosis of ROMK1 in oocytes.
X. laevis oocytes possess the machinery for endocytosis via
CCVs and have been used for studying CCV-mediated endocytosis of many
ion channels, including the epithelial Na+ channels (ENaCs)
and the CFTR Cl channels (5, 38).
I-V relationships were recorded in oocytes (~5
oocytes/time point) every 24 h postinjection of cRNA to monitor plasma membrane expression of ROMK1 channels. After the maximal steady-state expression of ROMK1 was reached (~72-96 h), BFA was added. BFA is a fungal metabolite that inhibits transport of the newly
synthesized channel proteins to plasma membranes by blocking anterograde trafficking of vesicles from the endoplasmic reticulum to
the Golgi complex (38, 42). BFA has been shown to be
active in X. laevis oocytes and inhibits protein trafficking
in these cells (26). BFA does not affect CCV-mediated
endocytosis in plasma membranes (42). I-V
relationships were again recorded from oocytes at 3, 6, 12, 24, and 48 h after addition of BFA (+BFA, Fig.
1A). After 3- or 24-h
incubation with BFA, some of the oocytes were transferred to BFA-free
solutions for further incubation (washout, Fig. 1A). Another
group of oocytes received vehicle (ethanol) instead of BFA from the
beginning and served as time controls (
BFA, Fig. 1A).
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Inhibition of ROMK1 currents by syntaxin 5. As an independent confirmation of the results from oocytes incubated with BFA, we used syntaxin 5 overexpression as an additional tool to inhibit transport of the newly synthesized ROMK1 to plasma membranes. Syntaxins are a family of membrane proteins that are involved in the trafficking of membrane vesicles (39). Syntaxin 5 is expressed in the Golgi complex and plays an important role in regulating vesicle transport between endoplasmic reticulum (ER) and the Golgi (9). Overexpression of syntaxin 5 disrupts the stoichiometric interactions among the endogenous transport-associated regulatory proteins and thus inhibits ER to Golgi transport (33, 35).
ROMK1 currents in plasma membranes of oocytes were monitored using TEVC recording (Fig. 2). After reaching the maximal steady-state expression of ROMK1 currents, one group of oocytes were injected with cRNA for syntaxin 5 (3 ng), whereas the other group of oocytes were injected with water to serve as controls (Fig. 2A). As shown, ROMK1 currents decreased by 76 ± 12% over 24 h in oocytes injected with cRNA for syntaxin 5 but not in water-injected oocytes (
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Inhibition of endocytosis of ROMK in oocytes by coexpression with a dominant-negative dynamin. The K44A mutation of dynamin impairs GTP binding and hydrolysis. Overexpression of this K44A dominant-negative mutant dynamin inhibits budding of CCVs and endocytosis from plasma membranes but does not inhibit budding of vesicles from the trans-Golgi network (TGN) (1, 8).
The role of CCVs in endocytosis of ROMK1 was examined using K44A mutant rat dynamin II (14). Oocytes were injected with cRNA for ROMK1 combined with cRNA for either WT or K44A mutant dynamin. Seventy-two hours after injection of cRNAs, I-V relationships were recorded in oocytes by TEVC. Afterward, oocytes received either BFA (5 µM) or vehicle (ethanol), and I-V relationships were recorded every 6 h for an additional 24 h. As shown in Fig. 3A, oocytes coexpressing ROMK1 and K44A mutant dynamin (Fig. 3A) for 72 h after injection of cRNA had higher currents (Ik 54 ± 8 µA at
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Internalization signal for endocytosis via CCVs.
The COOH-terminal tail of ROMK1 (amino acids 373-378) contains the
sequence YDNPNF, which is highly homologous to the (Y/F)(D/E)NPXY internalization motif of many membrane proteins that are internalized via CCVs (23), such as the LDL receptor, -amyloid
precursor protein, and mannose receptor (Fig.
4A). To test the importance of
this region for endocytosis of ROMK1, Asn375 was mutated to Ile, and
the effect on ROMK1 currents in oocytes was examined. We found that
mutation of Asn375, but not of Asn377, abolished the ability of the
K44A dynamin mutant to enhance ROMK currents (Fig. 4B; also
compare with Fig. 3A). Moreover, mutation of Asn375, but not
Asn377, abolished the decay of ROMK1 in the presence of BFA for 6 h (Fig. 4C, also compare with Fig. 1A).
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Endocytosis of ROMK2 in MDCK cells.
To examine endocytosis of ROMK in a mammalian cell system, we used an
MDCK cell line stably transfected with a GFP-tagged ROMK2. Fusion of
the GFP NH2 terminus to the ROMK peptide does not affect
trafficking and function of the channel (28). Stable transfection avoids potential artifacts of overexpression of proteins commonly observed with transient transfection. As reported previously (28), GFP was distributed throughout the cytoplasm and
nuclei and GFP-tagged ROMK2 was targeted to the plasma membrane of MDCK cells (top left and bottom left, respectively,
Fig. 5A). Functional channel
activity on the cell surface was confirmed by patch-clamp recording.
Whole-cell inwardly rectifying K+ currents were recorded
from cells expressing GFP-ROMK2 (Fig. 5, A and
B). Currents recorded from cells expressing GFP alone (Fig.
5, A and B) were not significantly different from
background currents in the control untransfected cells (not shown). The
single-channel kinetics and Po of GFP-ROMK2 in
MDCK cells (Fig. 5C and data not shown) were not
significantly different from channels expressed in oocytes
(19). When exocytotic insertion of ROMK was blocked by
BFA, whole-cell inwardly rectifying K+ currents in MDCK
cells decreased by 56% over 6 h (open bars, Fig. 5D).
Currents did not change significantly if cells were incubated with
vehicle (ethanol; shaded bars, Fig. 5D). Incubation with BFA
did not affect single-channel conductance or Po
of GFP-ROMK2 in MDCK cells (not shown).
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Localization of ROMK in CCVs.
To provide direct evidence for endocytosis of ROMK via CCVs, we
examined the subcellular distribution of GFP-ROMK2 in MDCK cells. The
distribution of CCVs was examined by labeling with a monoclonal
antibody against clathrin heavy chain. Because of nonspecific
interactions of antibodies with the filter membrane, it was not
feasible in these studies to grow cells in Transwells for obtaining
optimal confocal images along the z-axis, as shown in Fig.
5A. Nevertheless, as shown in the image sectioned at the xy-plane (left, Fig.
6A), GFP-ROMK2 in MDCK cells
was evidently distributed to the plasma membrane. Additionally,
GFP-ROMK2 was present intracellularly in a punctate pattern, suggesting
localization to the intracellular organelles. The staining by
anti-clathrin heavy chain antibody was membranous as well as punctate
intracellularly (middle, Fig. 6A). This
distribution is consistent with the pattern of distribution of CCVs
(1). Partial colocalization of GFP-ROMK2 with CCVs was
evident from the merged image of distribution of GFP-ROMK2 and clathrin
heavy chain (yellow, right, Fig. 6A). The localization of ROMK to the plasma membrane-associated CCVs suggests endocytosis of the channel at the plasma membrane. Besides endocytosis of plasma membrane proteins, CCVs are also involved in transport of
proteins from TGN to lysosomes (37). ROMK channels
expressed in MDCK cells are labile (6, 28). A significant
fraction of channel protein is degraded before being targeted to the
surface membrane (6, 28). The colocalization of ROMK with
CCVs in the perinuclear region likely represents proteins in the
synthetic pathway or the pathway for degradation. As reported
previously (28) and also shown in Fig. 5A, GFP
was distributed in cytoplasm and nuclei (left, Fig.
6B). For comparison, GFP was not localized to the plasma
membrane-associated CCVs (right, Fig. 6B). A
small amount of GFP was found in the perinuclear CCVs, probably
representing proteins in the synthetic and/or degradation pathway.
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Coimmunoprecipitation of ROMK with clathrin coat proteins.
We further investigated endocytosis of ROMK via CCVs using
coimmunoprecipitation. Besides endocytosis of plasma membrane proteins, CCVs are also involved in transport of proteins from TGN to lysosomes (37). CCV-mediated endocytosis at the plasma membrane
involves AP2 (containing -adaptin subunit), whereas transport of
proteins between TGN and lysosomes involves AP1 (containing
-adaptin). To examine association of ROMK with plasma
membrane-associated CCVs, we used antibody against
-adaptin for
immunoprecipitation. As shown in Fig. 7,
antibody against
-adaptin, but not control IgG, coimmunoprecipitated
clathrin heavy chain and ROMK from MDCK cells expressing ROMK2.
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DISCUSSION |
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Several studies have suggested that ROMK channels undergo endocytosis in the plasma membrane of X. laevis oocytes and rat CCDs (24, 40, 41). The purpose of this study was to examine the kinetics and the role of CCVs in endocytosis of ROMK channels. The abundance of ROMK proteins in plasma membranes in the steady state represents the balance between exocytotic insertion of the newly synthesized proteins and retrieval of the proteins. Using BFA (or overexpression of syntaxin 5) to block the anterograde transport of the newly synthesized proteins to plasma membranes, we found that ROMK1 channels undergo endocytosis in plasma membranes of oocytes with a half-time of ~6 h by electrophysiological recording. Electrophysiological recording was chosen for these studies because it is highly quantitative and selective for active channels that are present in the plasma membranes only. Additionally, it allows us to compare our results with the published literature on regulation of the K+ channel by changes in dietary K+ intake [which mainly examines density of active channels using electrophysiological recordings (10, 24, 29, 30, 40, 41); see below]. ROMK channels are expressed in renal tubular epithelial cells (12). To know whether the endocytosis we observed also occurs in renal epithelial cells, we further examined endocytosis of the channel using a MDCK cell line stably expressing ROMK2. We found that endocytosis of ROMK2 also occurs in these cells with a time course similar to the endocytosis of ROMK1 in oocytes. ROMK2 lacks the first 19 amino acids of ROMK1 and is otherwise identical to ROMK1 (12). The similar rate of endocytosis of ROMK1 and ROMK2 is also consistent with the finding of an NPXY internalization motif in the COOH terminus of both ROMK1 and ROMK2.
There are multiple pathways for endocytosis (17). Endocytosis of ENaC and CFTR both involve clathrin-coated pits (5, 38). We therefore examined whether CCVs are involved in endocytosis of the ROMK channels. Dynamins are a ~100-kDa GTPase protein family that play an important role in endocytosis mediated via CCVs (1, 8). The role of dynamin-dependent CCVs in endocytosis of ROMK in oocytes is demonstrated by inhibition of endocytosis because of coexpression of the channels with the dominant-negative dynamin mutants. Dynamin has also been reported to be involved in internalization of caveolae (13). Direct demonstration of ROMK in CCVs will provide further confirmation of endocytosis of ROMK via CCVs. We attempted to examine the localization of ROMK in CCVs in oocytes using immunofluorescent staining. However, specific staining of CCVs in X. laevis oocytes was not feasible using available commercial antibodies. Localization of ROMK with plasma membrane-associated CCVs, nevertheless, is evident in MDCK cells by using immunofluorescent colocalization and coimmunoprecipitation.
The cytoplasmic domains of many membrane proteins that are internalized via CCV-mediated endocytosis contain specific sequence information that facilitates localization to coated pits (23). Several types of internalization signals have been found (23). One of these is the NPXY motif, which contains consensus amino acid sequence asparagine, proline, any amino acids, and tyrosine, respectively (7, 23). In this study, we also reported that the COOH terminus of ROMK1 contains an NPXY internalization motif. These results, together with the results of dominant-negative dynamin and the results of association of ROMK with CCVs, provide strong evidence that ROMK channels are regulated by CCV-mediated endocytosis. The possibility that ROMK may also be regulated by other internalization pathways, however, cannot be excluded.
Endocytosis via CCVs in plasma membranes has multiple roles. These roles include maintenance of cellular homeostasis by recovering protein components that are inserted into the plasma membrane by ongoing secretory activity, uptake of molecules from the extracellular space, and regulation of the number of membrane proteins in physiological and pathophysiological conditions (17). Several lines of evidence suggest that regulation of the apical membrane channel density by dietary K+ intake may occur by means of alterations of endocytosis of the channels (24, 40, 41). High K+ intake may decrease CCV-mediated endocytosis of the channels in the apical membrane and thus lead to the increase in the density of the channels. A recent study reported that the increase in the density of active K+ channels in rat CCDs occurs within 6 h (essentially 1 meal) of a high-K+ diet (30). We found that the half-time for reduction in ROMK currents in oocytes in the presence of BFA is ~6 h. This value may represent an underestimation of the rate of endocytosis of ROMK in plasma membranes if significant amounts of the endocytosed channels are recycled back to the plasma membranes. On the other hand, it may be an overestimation if significant pools of inactive channels not readily available for endocytosis are present in the plasma membranes. Nevertheless, the time course of endocytosis of ROMK measured by electrophysiological recording in our studies is in agreement with the time course of changes in the density of active K+ channels in CCDs associated with alterations of dietary K+ intake. The similar time course supports the hypothesis that variations of dietary K+ intake regulate K+ channel density by altering CCV-mediated endocytosis of the channels.
What might the signaling mechanism(s) be that links the changes in dietary K+ intake to the control of channel density by means of alterations of endocytosis of the channels? Studies by Wang and colleagues (24, 40, 41) suggest that changes in dietary K+ intake affect the activity of the Src tyrosine kinase and the related tyrosine phosphatases, which in turn alters the rate of endocytosis and/or exocytosis of the channels. Having found that the mechanism of the endocytosis of ROMK is via CCVs and determined the kinetics of endocytosis, one can examine in future studies how tyrosine kinases and/or phosphatases regulate the CCV-mediated endocytosis of the channels.
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ACKNOWLEDGEMENTS |
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We thank Drs. Barbara Barylko and Joe Albanesi for wild-type and mutant dynamin cDNA, Dr. I. Bezprozvanny for syntaxin 3 and syntaxin 5 cDNA, Dr. Y. M. Leung for participation in the work in the early phase, Dr. Xin-Ji Li and Wei Ding for technical assistance, and Dr. Charles Pak for support and encouragement.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54368 (C.-L. Huang) and DK-54231 (P. A. Welling), American Heart Association National Center Grant-in-Aid 0150179N (C.-L. Huang), and a seed fund from the Center for Mineral Metabolism.
Address for reprint requests and other correspondence: C.-L. Huang, Dept. of Medicine, UT Southwestern Medical Ctr., 5323 Harry Hines Blvd., Dallas, TX 75390-8856 (E-mail: Chou-Long.Huang{at}UTSouthwestern.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.
April 16, 2002;10.1152/ajprenal.00378.2001
Received 28 December 2001; accepted in final form 9 April 2002.
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