Methylation Increases the Open Probability of ENaC in A6 Epithelia. Andrea Becchetti1 Alexandra ...

46 Pages·2000·1.05 MB·English

JBC Papers in Press. Published on March 27, 2000 as Manuscript M000954200
M0:00954 1st Revision Becchetti et al. Page 1
Methylation Increases the Open Probability of
ENaC in A6 Epithelia.
Andrea Becchetti1
Alexandra E. Kemendy1
James D. Stockand1,2
Sarah Sariban-Sohraby3
and
Douglas C. Eaton1,2
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1Department of Physiology fro
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Emory University School of Medicine http
Atlanta, Georgia 30322 ://w
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2Center for Cell & Molecular Signaling .jbc
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Emory University School of Medicine rg
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3Université Libre de Bruxelles 27
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Laboratoire de Physiologie et Physiopatologie 0
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Bruxelles, Belgium.
Running title: Methylation and ENaC
Address reprint requests to : Douglas C. Eaton, Ph.D., Emory University School of Medicine,
Department of Physiology, 1648 Pierce Drive, N.E., Atlanta, GA 30322.
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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INTRODUCTION
The amiloride-blockable, highly selective, epithelial sodium channel (ENaC) present on the apical
surface of principal cells in mammalian renal cortical collecting tubules is the primary site for the
regulation of total body sodium balance and blood pressure. The cell line, A6, derived from distal
tubules of Xenopus laevis nephrons, is a good experimental model for the study of these sodium
channels. When grown on permeable supports in the presence of aldosterone, A6 cells express an
apical sodium channel with properties identical to those of channels in mammalian tissues 1.
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Despite many studies, the mechanism by which aldosterone stimulates apical sodium transport is still d
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poorly understood. It is known that the complex between aldosterone and its intracellular receptor h
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activates gene expression, and induces the synthesis of proteins 2-9; however, little is known about w
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the cellular functions of the induced proteins except that the final result is an increase in sodium rg
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transport 9-12. Originally, because of the observation that protein synthesis was required for ue
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aldosterone to increase sodium transport, it was postulated that aldosterone induced sodium channel a
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synthesis and insertion. However, earlier studies in A6 cells showed that aldosterone increases Na+ , 2
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entry at the apical membrane by changing the activity of channels that are already present in the
apical membrane and not by increasing the number of channels 13. Although interpretation of other
electrophysiological data remains controversial 14-16, biochemical methods support the original
observation that ENaC mRNA and ENaC protein in the apical membrane does not increase in the
presence of aldosterone (at least in the first two to four hours when the increase in sodium transport
is most dramatic) 10;17-19.
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Since the action of aldosterone appears to involve a mechanism which increases the P of sodium
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channels, an examination of post-translational mechanisms which alter P may offer some insight
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into the mechanism of aldosterone action, but identifying signal tranduction pathways which can
increase sodium channel P in A6 cells has been difficult. There have been many suggestions about
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potential aldosterone-induced post-translational modifications, but in the context of our previous
results 20-22, one is particularly interesting. Sariban-Sohraby et al. 23 demonstrated that the amount of
sodium transport which could be measured in apical membrane vesicles obtained from A6 cells, a
sodium-transporting, distal-nephron cell line, was markedly enhanced by prior application of agents
which methylate membrane proteins. There was no additional effect of SAM in the presence of D
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aldosterone and the effect was blocked by two methylation blockers: S-adenosyl homocysteine d
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(SAH) and 3-deazaadenosine (3-DZA). 3-DZA is a membrane permeable drug which blocks h
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transmethylation reactions by specifically inhibiting the S-adenosyl-homocysteine hydrolase, thereby w
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promoting the accumulation of S-adenosyl-homocysteine (SAH) and deaza-D-adenosylhomocysteine rg
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which produces end-product inhibition of SAM-dependent methyltransferases 24. Since they also ue
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demonstrated that application of aldosterone leads to the methylation of membrane protein and lipid, a
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their suggestion was that intracellular methyl transferases induced by aldosterone could be , 2
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responsible for the methylation and, therefore, modulate the sodium channel protein. As a post-
translational modification, methylation is analogous to phosphorylation (for reviews, see 25-28).
Highly specific methyl transferases promote the methylation of proteins at specific consensus sites
and much less specific esterases promote demethylation of the proteins. The difference between
phosphorylation and methylation is that methylation is in general more stable and, therefore, can be
used to alter the activity of proteins for a much longer period of time than is typical for
phosphorylation.
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The previous work on methylation implies an involvement of transmethylation in controlling sodium
transport; however, the studies all involved measuring changes in total sodium transport; therefore,
do not distinguish between the relative contribution of channel density, single-channel open
probability and conductance. In principle, any of these factors could contribute to a modification of
transepithelial transport. These considerations led us to study the effects of carboxymethylation
reactions on the activity of single-sodium channels of A6 cells using patch-clamp methods. In this
way, we hoped to clarify what aspect of channel function is altered and to confirm whether the
methylation effect is indeed membrane-directed.
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We found that methylation inhibitors reduce ENaC activity by decreasing P . Also, addition of the d
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methyl donor, SAM, to the cytosolic surface of ENaC in excised inside-out patches significantly h
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increased channel activity with respect to controls. The presence of SAM seems to maintain the w
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single channel mean open time even after excision. Addition of partially purified methyl-transferase rg
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produced little additional effect, suggesting that the endogenous methyl-transferase is probably ue
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membrane associated and close to the channel. Addition of GTP along with SAM increased channel a
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activity more than SAM alone. The present work has been previously reported in several brief , 2
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communications 13;22.
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MATERIALS AND METHODS
A6 Cell Culture Preparation. For single channel experiments, we used A6 cells from American
Type Culture Collection (Rockville, MD) in the 68th passage. Experiments were carried out on
passages 70-80, with no discernible variation between cells from different passages. Cells were
maintained in plastic tissue culture flasks (Corning, NY) at 26o C in a humidified incubator with 4%
CO in air. The culture medium was a mixture of Coon's medium F-12 (3 parts) and Leibovitz's
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medium L-15 (7 parts) modified for amphibian cells with 104 mM NaCl-25 mM NaHCO3, pH 7.4,
with a final osmolarity of 240 mosmol/kg H O. Besides these components, 10% (vol/vol) fetal D
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bovine serum (Irvine Scientific, CA), 1% streptomycin, and 0.6% penicillin (Hazleton Biologics, d
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KA) and, in most experiments, 1 (cid:58)M aldosterone were added. Cells grown on plastic tissue culture h
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dishes were detached when confluent by exposing them to divalent-free (Ca2+ and Mg2+) medium w
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containing 0.05% trypsin and 0.6 mM EDTA (Irvine Scientific, CA). The cells were then rinsed, rg
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centrifuged, resuspended, and finally replated. When used for patch-clamp experiments, A6 cells ue
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were replated at confluent density on collagen-coated CM permeable filters (Millipore, CA) attached a
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to the bottom of small Lucite disks with the disks suspended in 35-mm Petri plates as previously , 2
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described 29. This sided preparation forms a polarized monolayer with the apical surface oriented
upward and net sodium transport moving from the apical to basolateral surface. The cells were fed
with fresh medium every two days and sampled when fully differentiated (7 to 14 days after
replating). For experiments in which the aldosterone levels were reduced, after the cells reached
confluent density, the cells were incubated in medium free of both aldosterone and serum for two
days. Then half of the cells were washed with A6 saline and re-fed with serum-free medium
containing 1.5 (cid:58)M aldosterone. The remaining cells (aldosterone-free controls) were washed with
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A6 saline followed by re-addition of aldosterone-free and serum-free medium. Cells were incubated
for 4h followed by current or patch clamp measurements.
Protein Carboxvmethvltransferase Isolation. Protein carboxvmethyltransferase was partially
purified from the membrane fraction of A6 cells as one of us has previously described 30. Medium
was removed, and cells were washed twice in a Ringer’s solution (85 mm NaCl, 18 mm NaHCO,,
4 mm KCI, 1 mM KH PO4, pH 7.4), scraped, and Dounce homogenized. Crude membrane and
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cytosolic fractions were separated by centrifugation at 50,000 x g for 1 h. Stripped membranes were
prepared by a modification of the method of Yamane and Fung 31. The membrane pellet was Do
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resuspended in Buffer A (20 mm Tris-HCl, pH 8, 1 mm EDTA, 1 mm dithiothreitol) along with 1% de
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sodium cholate and allowed to sit on ice for 1 h. The membranes were collected by centrifugation h
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as above, washed twice, and resuspended in Buffer A without sodium cholate. w
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The enzyme activity assay was similar to that described previously 32;33. In brief, 50 (cid:58)g protein of es
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the enzyme preparation was incubated with the incubation mixture (50(cid:58)l) contained 50mM Tris-HCl rc
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buffer (pH 8), 720nM [3H]adenosyl-methionine (15Ci/mmol), 100(cid:58)M of the methyltransferase 01
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substrate, N-Acetyl-S-Farnesyl-L-Cysteine (AFC), and a blocking concentration of a
methyltransferase inhibitor when appropriate (S-trans,trans-farnesylthiosalicylic acid, 3-
deazadenosine, or S-adenosylhomocysteine). After incubation for 1h at 37oC, the reaction was
stopped by the addition of 50(cid:58)l of 20% trichloroacetic acid and the reaction mix vortexed for 10s.
The isoprenyl cysteine methylesters were separated by the addition of 400(cid:58)l heptane to the reaction
mix. A fraction (200(cid:58)l) of the organic top layer containing the methylesters was transferred to a
small top-free Eppendorf tube and dried under vacuum. 200(cid:58)l of 1 M NaOH was added and the
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tube was placed upright in a vial containing a small volume of scintillation fluid. The vials were
sealed and incubated at 37oC overnight. The strong base hydrolyzes the methyl esters releasing
methanol vapor which partitions into the hydrophobic scintillant. The methylesters in the vials were
counted by liquid scintillation counting. The specific activity of the enzyme is defined as pMoles of
methyl-3H group transferred/mg/min of enzyme protein. The preparation catalyzed the incorporation
of base-labile isotope into AFC, which was 70-90% inhibited by SAH or FTS. The specific activity
of methyl groups transformed by the enzyme was 0.12 pmol/mg protein/min.
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Transepithelial current recording. A6 cells were grown for 14 days on permeable supports with a o
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surface area of approximately 9 cm2, at which time, transepithelial potential differences (PD) and de
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resistances (R) were measured using dual flexible electrodes, containing Ag/AgCl pellets (Millicell- h
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ERS, Millipore,Bedford MA). The resistance of the insert was obtained by passing an alternating w
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current of ± 20(cid:58)A at 12.5Hz. Transepithelial current (I ) was calculated from the PD and R values, rg
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and, in this system, the predominant component of the current is carried by Na+ through amiloride es
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sensitive Na+ channels at the apical membrane 34. Because measurements were made under sterile arc
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conditions, we were able to make several measurements over time on the same cells. 0
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Single-Channel Recordings. We used either the cell-attached or the inside-out configuration of the
patch-clamp technique (Hamill et al.). Before sampling, the apical cell surface was carefully washed
several times with our standard extracellular solution, containing (in mM): 95 NaCl, 3.4 KCl, 0.8
CaCl , 0.8 MgCl , 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; Sigma
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Chemical, MO); pH was adjusted to 7.4 with NaOH. Patch pipettes contained the same solution..
For inside-out experiments, the apical solution was substituted immediately before patch excision
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with a cytosol-mimicking solution (in mM: 85 KCl, 3 NaCl, 4 CaCl , 1 MgCl , 5 ethylene glycol-
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bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, potassium salt; SIGMA), 1 mM
Adenosine trisphosphate (ATP, sodium salt, SIGMA) and 10 HEPES at pH 7.4 adjusted with KOH).
Only one inside-out experiment was performed per dish to avoid any possibility of examining cells
whose properties might have been altered by extended exposure to high potassium solution. In some
of the inside-out experiments, 300 (cid:58)M S-adenosyl-L-Methionine (SAM, SIGMA) or GTP was added
to the "intracellular" solution. Solutions containing SAM, GTP, or ATP were prepared fresh every
day to prevent possible degradation. All experiments were performed at room temperature (22-24o
C, very close to the physiological temperature for amphibian cells) within 45-60 minutes of D
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removing the A6 cells from the incubator. Patch pipettes with a tip diameter (cid:35) 1 (cid:58)m were fabricated ad
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from WPI TW 150 glass (New Haven, CT) and fire-polished. Since the voltage-dependence of h
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apical sodium channels in A6 cells is relatively small 35, we have not corrected all our data for the w
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small (a few mV) junction potential present after patch excision in a high potassium solution. rg
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Data Acquisition and Analysis. Single channel currents from cell-attached patches were measured a
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with an Axopatch 1-B current-voltage clamp amplifier (Axon Instruments, Inc. Burlingame, CA), , 2
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low-pass filtered at 5 KHz, recorded on a digital video recorder (Sony, Japan), and then the recorded
signal was refiltered and digitized at twice the corner frequency (usually 1 KHz) using a Scientific
Solutions A/D converter and a IBM PC computer equipped with Axotape software (Axon
Instruments, CA). The data were subsequently transferred to a Micro Vax II computer (Digital
Equipment, MA) for single-channel analysis. Data records from cells grown in the presence of
aldosterone were low-pass filtered at 100 Hz while those from aldosterone-depleted cells were
filtered at 300 or 500 Hz using a software Gaussian filter. Events were detected by setting the
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threshold level at 50 % of the estimated single-channel current amplitude. Because of the necessity
for analyzing long continuous records, programs that closely follow the strategy of Colquhoun and
Sigworth 36 were written for use on the VAX family of computers. These programs produce tables
of event durations and amplitudes based on a 50% threshold crossing algorithm, and allowed the
analysis of long continuous records, necessary for the interpretation of single-channel experiments
on renal epithelial tissue.
One method for calculating NP from single-channel records without making any assumptions about
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the total number of channels in a patch or the P of a single channel is given by : o
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(2) from
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where T is the total recording time, N is the observable number of current levels (corresponding to ww
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the apparent number of channels) within the patch determined as the highest observable current rg
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level, i is the number of channels open, and t is the time during which i channels are open. If es
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channels open independently of one another and the exact number of channels in a patch is known, arc
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then the P of a single channel can be calculated by dividing NP by the number of channels in a , 20
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patch. The total number of functional channels (N) in the patch was determined by observing the
number of peaks detected in all points amplitude histograms constructed, when possible, from event
records of long enough duration to provide 95% confidence of determining the correct N according
to methods we have previously described 37;38. However, especially in some of the untreated excised
patches, we could not record long enough to reach a 95% confidence level and the values for N in
these patches may be an underestimate. The mean open time (t ) of N channels can be calculated
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as follows:
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(3)
where n is the total number of transitions between states during the total recording period, T, and the
other parameters are the same as in Equation 2. This value represents the average time the channel
spends open (in any open state) and should not be confused with the mean residency time of the
channel in a specific state (sometimes called the mean open time for the state). Nonetheless, this D
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measure provides an easy way to distinguish whether experimental manipulations (e.g., SAM or 3- d
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DZA) modify P by affecting the channel's open states or closed states. h
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Statistics. Unless otherwise stated, data are presented as the means ± standard errors of the means. rg
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Unpaired or paired Student's t test as appropriate were used to test for significance between two e
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treatments. ANOVA with Student-Neumann-Keuls post-test was used for multiple comparisons. a
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Probabilty < 0.05 was considered significant. , 2
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