cAMP Increases Density of ENaC Subunits in the Apical Membrane of MDCK Cells in Direct ...

15 Pages·2002·4.12 MB·English

cAMP Increases Density of ENaC Subunits in the Apical Membrane of
(cid:2)
MDCK Cells in Direct Proportion to Amiloride-sensitive Na Transport
Ryan G. Morris and James A. Schafer
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294
abstract Antidiuretic hormone and/or cAMP increase Na(cid:2) transport in the rat renal collecting duct and sim-
ilar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was un-
dertaken to determine if that increment in Na(cid:2) transport could be explained quantitatively by an increased den-
sity of ENaC Na(cid:2) channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retro-
virally transfected with rat (cid:3)-, (cid:4)-, and (cid:5)ENaC subunits, each of which were labeled with the FLAG epitope in their
extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and
B.C. Rossier. 1996. Proc. Natl. Acad. Sci. USA. 93:15370–15375). The density of ENaC subunits was quantified by
specific binding of 125I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a satura- Do
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ble function of M2 concentration with half-maximal binding at 4–8 nM. Transepithelial Na(cid:2) transport was mea- nlo
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sured as the amiloride-sensitive short-circuit current (AS-I ) across MDCK cells grown on permeable supports. d
sc ed
Specific M2 binding was positively correlated with AS-Isc measured in the same experiments. Stimulation with fro
cAMP (20 (cid:6)M 8-p-chlorothio-cAMP plus 200 (cid:6)M IBMX) significantly increased AS-Isc from 11.2 (cid:7) 1.3 to 18.1 (cid:7) m h
1.3 (cid:6)A/cm2. M2 binding (at 1.7 nM M2) increased in direct proportion to AS-I from 0.62 (cid:7) 0.13 to 1.16 (cid:7) 0.18 ttp
fmol/cm2. Based on the concentration dependence of M2 binding, the quantitys cof Na(cid:2) channels per unit of AS-I ://ru
wtivaes lcy.a lTchuelastee dva tlou ebse w tohuel dsa mbee c ionn tshiset epnrte sweinthc ea asnindg aleb scehnacnen oefl ccAoMndPu, c0t.a2n3c (cid:7)e o0f. 0(cid:2)4 5a npdS 0(t.y2p1i c(cid:7)a0ll.y0 5re fpmoortl/ed(cid:6) Afo, rr eEsNpeacCsc- press.org
channels) only if the open probability is (cid:8)0.02, i.e., less than one-tenth of the typical value. We interpret the pro- /jg
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mpoermtiobnraanl ein ccarne aascecso iunn tb cinodminpgle taenlyd fAorS -tIhsce t Io iinndcirceaatsee tphraotd tuhcee din bcyr ecaAsMedP .density of ENaC subunits in the apical /article
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key words: retroviral transfection • FLAG epitope • channel number • membrane trafficking • short-circuit f/12
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current /1/7
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/1
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INTRODUCTION (Tomita et al., 1985; Reif et al., 1986; Garty and Benos, 17
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1988; Schafer and Hawk, 1992; Schafer, 1994; Garty p
Antidiuretic hormone (ADH),* in addition to its epon- 12
ymous action, also stimulates Na(cid:2) reabsorption in the and Palmer, 1997). As demonstrated previously in anu- 0171
ran epithelia, the increase in intracellular cAMP gener- .p
sthkien r aatn cdo rutriicnaal rcyo lblelacdtidnegr deupcitt h(eClCiaD o)f, aanmdp ihni bmiaannsy, iinn ated by the binding of ADH to V2 receptors results in df by g
an increase in the amiloride-sensitive conductance of ue
vaicttreor iastnicds csuimltuilraerd teop itthhee lima apmosmseaslsiainng ctorallnescptionrgt cdhuacrt- tlwehhceti cianhpg iu cdlatuli mcmt,a eatemllloyb wrraiennsuge l itons fci rnteh aiens ecpdrre iNanscaei(cid:2)dp eaNnl atcr(cid:2)ey l rlisen atoobf sttohhreep ctcieoolnll,- st on 04 Janu
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Ryan G. Morris’s present address is National Institutes of Health, Na- (Schafer and Troutman, 1990). ry 2
tional Heart, Lung, and Blood Institute, Laboratory of Kidney and The Na(cid:2) channels involved in this response to ADH 023
Electrolyte Metabolism, Bethesda, MD 20892.
and cAMP are now known to be oligomeric complexes
Address correspondence to James A. Schafer, Department of
of three protein subunits, (cid:3)-, (cid:4)-, and (cid:5)ENaC, encoded by
Physiology and Biophysics, 1918 University Boulevard, Room 958
MCLM, Birmingham, AL 35294-0005. Fax: [phone]; E-mail: discrete genes (Canessa et al., 1994). All three subunits
[email protected] share a similar topology, consisting of two transmem-
*Abbreviations used in this paper: ADH, antidiuretic hormone; AS-Isc, brane regions, a large cysteine-rich extracellular loop,
amiloride-sensitive short-circuit current; B , maximal specific bind-
max and short COOH- and NH -terminal regions in the cyto-
ing; CCD, cortical collecting duct; CPT, 8-p-chlorophenylthio; 2
plasm. When the three cloned subunits are expressed to-
DMEM, Dulbecco’s modified Eagle’s medium; ENaC, epithelial
(amiloride-sensitive) Na(cid:2) channel; FLAG, octapeptide epitope gether in Xenopus oocytes, they form channels with char-
DYKDDDDY; IBMX, isobutylmethylxanthine; I , short-circuit cur- acteristics comparable to those found in the apical mem-
sc
rent; MDCK, Madin-Darby canine kidney; PKA, protein kinase A; RT, brane of intact epithelia such as the CCD, including: a
transepithelial resistance; VT, transepithelial voltage. single channel conductance of (cid:2)5 pS, a high Na(cid:2) to K(cid:2)
Portions of this work were previously published in abstract form
selectivity ratio, and inhibition by submicromolar con-
(Morris, R.G., and J.A. Schafer. 2000. J. Am. Soc. Nephrol. 11:34A; and
Morris, R.G., and J.A. Schafer. 2001. J. Am. Soc. Nephrol. 12:37A). centrations of amiloride (Canessa et al., 1994).
71 J. Gen. Physiol. © The Rockefeller University Press • 0022-1295/2002/07/71/15 $5.00
Volume 120 July 2002 71–85
http://www.jgp.org/cgi/content/full/120/1/71
Interestingly, not all epithelia expressing ENaC re- 1993; Bradford et al., 1995; Ismailov and Benos, 1995;
spond to ADH or cAMP with increased Na(cid:2) transport. Senyk et al., 1995).
For example, ADH-dependent cAMP production in the Although variable in magnitude, the increases in P
o
rabbit CCD produces either no change or a decrease in that occur in these patch-clamp and bilayer experi-
Na(cid:2) transport (Schafer and Hawk, 1992; Schafer, 1994, ments have been taken to support the view that direct
2002). Even in the same species, cAMP or ADH may activation of channels by cAMP/PKA can contribute to
have a different effect in different tissues, for example, the increased amiloride-sensitive Na(cid:2) transport pro-
stimulating Na(cid:2) transport in the rat CCD (Tomita et al., duced by ADH. This conclusion must, however, be tem-
1985; Reif et al., 1986), but not in the rat colon pered by the limitations of the experimental systems
(Bridges et al., 1984). This variability in the response to used. As indicated by the studies in lipid bilayers and
cAMP or ADH has been attributed to differences in the detached membrane patches (Prat et al., 1993; Berdiev
structure of the (cid:3)ENaC subunit (Schnizler et al., 2001), et al., 1996), the activity of ENaC channels depends on
or to differences in the actions of autacoids such as the presence of components of the cytoskeleton. In a
prostaglandin E that modify the response to ADH detached patch or in a reconstituted system in which
2
(Schafer, 2002). normal interactions of ENaC subunits with the cytoskel-
The increase in Na(cid:2) reabsorption that occurs with eton are disrupted, activation by PKA may involve D
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ADH or cAMP in the rat CCD and other responsive ep- changes in the interaction between residual or exoge- n
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ithelia represents an increase in the activity of ENaC nous actin and ENaC that do not occur in the intact d
e
d
hetero-oligomers in the apical membrane. Two general cell. It is also impossible to quantify the density of chan- fro
m
mechanisms have been put forward to explain this in- nels in the membrane of the native cells from the num- h
ttp
creased channel activity. First, modification of ENaC bers measured in detached patches. When multiple ://ru
subunits, occurring as one of the ultimate events of channels are present in a patch, not only is it techni- pre
ADH action, might cause an increase in the open prob- cally very difficult to determine their precise number, ss.o
rg
ability (P ) or an increase in the unit conductance of but the channels may also not be uniformly distributed, /jg
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tthhee ampuiclatilm meermic bcrhaannen weilt hc onmo pclheaxnegs ea ilnre tahdeyi rp nreusmenbte risn. rscersiubletidn gb yin M tahreu n“hakoat sapnodt s”E aotfo nch (a1n9n9e1l) aicnti vviatyso atos cdine-- /article-p
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Alternatively, or in addition to its effects on individual treated A6 cells. f/1
2
0
channel kinetics, ADH might cause the insertion of as- Both patch-clamp and biochemical approaches have /1
/7
sembled ENaC multimers into the apical membrane in been used in support of the alternative hypothesis that 1/1
2
a manner analogous to the ADH-stimulated insertion ADH increases Na(cid:2) transport by the insertion of addi- 17
4
1
of aquaporin-2 water channels in the CCD (Wade, tional channels into the apical membrane. Marunaka 7/jg
p
1985). and Eaton (1991) used patch clamping in A6 cell 1
2
0
The former mechanism of ADH action, i.e., an in- monolayers to show that both vasotocin and cAMP in- 17
1
crease in P of individual Na(cid:2) channels, has been sup- creased the number of Na(cid:2) channels per patch with no .pd
o f b
ported by studies using patch clamping and reconsti- change in single channel kinetics. Kleyman et al. y g
u
pturtoepde rNtiae(cid:2)s mchigahntn beels a. cCohnasnegqeuse nince tohfe p rsiontgeilne kcihnaansen eAl (N1a9(cid:2)9 4c)h aandnderelss siend A th6 ea pefifceacl tm oef mADbrHa noens tuhsein tgo taanl p aonotli iodf- est on 0
4
(PKA) activation by cAMP, and Shimkets et al. (1998) iotypic antibody (anti–anti-amiloride) that has been Ja
have shown that the COOH-terminal regions of (cid:4)- and shown to recognize (cid:3)ENaC (Kieber-Emmons et al., nua
(cid:5)ENaC are phosphorylated by this signaling cascade. 1995). In these experiments, apical membrane pro- ry 20
2
Using inside-out patches of A6 cell apical membranes, teins in intact A6 cells, with and without ADH treat- 3
Prat et al. (1993) showed that application of PKA plus ment, were radio iodinated and the Na(cid:2) channels were
ATP activated Na(cid:2) channels at least in part by an in- subsequently immunoprecipitated from extracted pro-
crease in P . They also showed, however, that this ef- teins using the antiidiotypic antibody. Kleyman et al.
o
fect of PKA was dependent on the presence of “short” (1994) reported there was approximately a doubling of
actin filaments and hypothesized that the action of the immunoprecipitated Na(cid:2) channels after treatment
PKA might be mediated by phosphorylation of G-actin with the hormone, which they concluded would ac-
rather than the channel itself. A role for actin in acti- count for the increase in Na(cid:2) transport produced by
vating ENaC was also supported by Berdiev et al. ADH. In another approach to quantifying Na(cid:2) channel
(1996), who demonstrated that PKA activation of (cid:3)-, density in the apical membrane, Snyder (2000) used
(cid:4)-, and (cid:5)ENaC subunits incorporated into planar lipid transient transfection to introduce human ENaC sub-
bilayers required the presence of short actin fila- units, which had been modified to allow fluorescence
ments. PKA has also been shown to increase the P labeling, into cultured epithelial cells of thyroid origin.
o
of several biochemically purified Na(cid:2) channel com- He demonstrated that cAMP stimulation produced an
plexes incorporated into planar bilayers (Oh et al., acute increase in the fluorescent labeling of ENaC in
72 cAMP Increases ENaC Membrane Density
the apical membrane that paralleled the increase in develop a line of MDCK cells expressing “flagged” rat
Na(cid:2) transport. ENaC subunits, i.e., subunits labeled in the extracellu-
The measurement of the surface density of ENaC lar loop-domain with FLAG as described by Firsov et al.
subunits in the intact epithelium has definite advan- (1996). We measured the density of total ENaC sub-
tages. Not only are the subunits in their native environ- units on the apical membrane surface and compared it
ment, but the use of epithelial monolayers with (cid:9)106 with the amiloride-sensitive short-circuit current (I )
sc
cells inherently provides the statistical averaging that is measured in the same cells in the presence and ab-
lost when examining individual patches or reconsti- sence of cAMP stimulation. Our results indicate that
tuted transporters. However, although the two surface- the increase in I produced by cAMP can be accounted
sc
labeling studies discussed above undeniably show an in- for entirely by a proportional increase in the surface
crease in the density of ENaC subunits in the apical density of ENaC.
membrane of A6 cells with ADH or cAMP, both are lim-
ited in their ability to relate the change in the surface MATERIALS AND METHODS
density of the label quantitatively to the change in Na(cid:2)
MDCK Cell Cultures
transport. In the experiments of Kleyman et al. (1994),
the identity and the number of antigenic sites per MDCK cells were obtained as a gift from Dr. D. Rotin (University Do
w
ENaC subunit, as well as the affinity of the antiidiotypic of Toronto, Toronto, Canada) with the permission of Dr. B. Ross- n
lo
antibody for these sites, are unknown. Similarly, in Sny- ier (University of Lausanne, Lausanne, Switzerland), whose labo- ad
e
der (2000) fluorescence could not be related quantita- ratory originally developed the clone. This cell line, which exhib- d fro
ited most of the characteristics of the type-1 MDCK cells, includ- m
tciovenlfyo ctaol mchiacnronseclo pdye nwseitrye, naontd r ethpere sceenlltsa teivxea mofi nthede abvy- ibnegc aau stera int shepadit hbeeleianl srheosiwstna ntcoe h (aRvTe) a (cid:9) n1e,g0l0ig0i b(cid:10)le·c amm2,i lworaisd ec-hseonsesni- http://ru
erage labeling of the whole epithelium, because not all tive conductance (Ishikawa et al., 1998). The unmodified wild- pre
cells expressed ENaC subunits in this transient transfec- type cells are referred to below as parental MDCK cells. We also ss.o
tion system. obtained a different clone of type-1 MDCK cells (a gift of Dr. D. rg/jg
ingT heexsep elirmimiteantitosn sd ewsecrreib oevde rcboym Fei risno tvh ee ts uarlf.a c(e1-l9a9b6e)l.- Bosofa mlAkeloa vobefat tmzh, aeN iaemtp mBhriuromnlooignbygl ohRtaesm sdee)a,sr cctrohi b saeendrdv be Te arloas wian. isTnoghu eCrsceeen aotrefer ,pr Uerofnetreivrienerd sf iottoyr p/article-p
These investigators modified rat (cid:3)-, (cid:4)-, and (cid:5)ENaC by as type-1 MDCK cells. df/1
2
introducing the octapeptide FLAG epitope (DYKD- Cultures of MDCK cells were expanded in T-75 flasks in a hu- 0/1
DtraDcDelYlu) lianrt olo tohpe oefa relayc (hN. HW2h-teenrm thineasel) sruebguionnit so wf tehree eexx-- dmiuidmifi ewda si nDcuulbbaetcocro a’st 3m7o(cid:11)Cd iifine dth Ee apgrlees’se nmcee doifu m4% ( DCOM2E. MT)h e( Lmifee- /71/1217
Technologies) supplemented with 50 mM HEPES (pH 7.4), 1% 41
pressed in Xenopus oocytes, the binding of 125I-labeled Pen/Strep-fungizone, and 10% FBS. The medium was ex- 7/jg
p
monoclonal antibody to FLAG allowed these investiga- changed as needed, usually daily, and the cells were split upon 12
0
tors to determine the surface density of the ENaC sub- confluence. For the both electrophysiological and surface label- 17
1
units on a femtomole per oocyte basis. The macro- ing experiments, the cells were seeded on inserts with permeable .pd
scopic amiloride-sensitive Na(cid:2) current and the surface mcoenm) borra 2n4e-sm: emit htrearn 2sw4e-mllsm ( Ccyactlaolopgo rNeos .( 3C4a1ta2l;o Cgo Nstoa.r )3 5a-t3 a0 9d0e;n Fsiatly- f by gu
density of ENaC subunits measured in the same oocyte of (cid:2)400,000 cells/insert, after which they were grown in the est o
were linearly related over a wide range with a slope of modified DMEM without selection antibiotics (see below). n 0
1.1 (cid:6)A/fmol. Although Firsov et al. (1996) did not ex- Other Tissues 4 Ja
n
amine the effect of cAMP,1 they found that a mutation ua
in the (cid:4) subunit associated with Liddle’s syndrome re- In some of the immunoblotting and RT-PCR studies described ry 20
sulted in an increase in the surface density of ENaC below, we also used protein and total RNA extracted from fresh 23
samples of dog and rat renal cortex and inner medulla. These tis-
subunits and an increase in amiloride-sensitive current.
sues were obtained from cadarveric animals that had been used
Our objective in the present studies was to use FLAG in other studies in our own laboratory or in other laboratories at
antibody surface labeling to quantify any change in the this university. All such studies were approved by the Institutional
surface density of ENaC in the apical membrane of Animal Care and Use Committee at the University of Alabama at
Birmingham.
mammalian epithelial cells with cAMP treatment and
to relate it to transepithelial Na(cid:2) transport measured in Retroviral Transfection of Parental MDCK Cells
the same epithelia. We used retroviral transfection to Three plasmids containing rat (cid:3)-, (cid:4)-, and (cid:5)ENaC subunits, into
each of which the FLAG epitope had been introduced in the
1In the course of the present experiments, we also measured whole early portion of the extracellular loop as described by Firsov et al.
cell amiloride-sensitive currents in Xenopus oocytes expressing wild- (1996), were provided by Dr. B. Rossier (University of Lausanne,
type and FLAG-labeled rat ENaC subunits. Using a variety of cAMP Lausanne, Switzerland). These flagged subunits are designated
analogs, we were unable to show any stimulatory effect, even at high hereafter by a subscript F, e.g., (cid:3)ENaC. These subunit cDNAs
F
concentrations in the presence of IBMX. Most likely the lack of effect were provided in mammalian plasmids (a modification of
is due to the high rate of endogenous cAMP production that we con- pSD65), and new restriction sites appropriate for subcloning into
firmed in these oocytes (unpublished data). the retroviral vectors (5(cid:12) XhoI and 3(cid:12) NotI) were added to each
73 Morris and Schafer
by PCR. The resulting cDNA constructs were bidirectionally se- served as a water bath whose temperature was maintained at 37(cid:11)C
quenced in order to verify that the ENaC subunits had not been by an immersion heater and temperature regulator. The baso-
modified, and they were then subcloned into bicistronic retrovi- lateral current–receiving electrode was a stainless-steel sheet,
ral vectors, which were generously provided by Dr. R. Boucher whereas the basolateral voltage–sensing electrode, an Ag-AgCl
2
(University of North Carolina, Chapel Hill, NC). The three vec- electrode, was connected to the basolateral medium by an agar
tors, LXPIN, LXPIH, and LXPIP, contained antibiotic resistance bridge. These basolateral electrodes served as a common elec-
genes for, respectively, neomycin (G418), hygromycin, and puro- trode pair for all 24 inserts mounted in the Lucite manifold. The
mycin, in one cloning position, with the (cid:3)-, (cid:4)-, or (cid:5)ENaC sub- apical electrodes consisted of a small stainless-steel disc for cur-
F F F
unit in the other. Sequencing from the 5(cid:12) and 3(cid:12) termini verified rent passing and a central agar bridge connected to an Ag-AgCl
2
the orientation and nucleotide fidelity of the subcloned ENaC electrode. The apical electrode pair was encased in a housing
subunits. Using the protocol of Comstock et al. (1997), PA317 made from a rubber stopper, which rested on the top rim of the
packaging cells (American Type Culture Collection) were trans- cell culture inserts with the current-passing electrode and the
fected with the retroviral vectors to produce three retrovirions: agar bridge extending into the apical solution. Using this hous-
L(cid:3) PIN, L(cid:4) PIH, and L(cid:5) PIP, each containing the indicated ing, the apical electrode pair could be moved rapidly from insert
F F F
flagged ENaC subunit and the respective antibiotic resistance to insert for sequential electrophysiological measurements with
gene. the VCC600 voltage-clamp. A MacLab model 8e (ADInstru-
Parental MDCK cells were first infected with one of the three ments) interface and a Macintosh computer were used to control
retrovirions, and then selected with the corresponding antibi- the clamp and perform a rapid sequence of short-circuit, open-
otic, followed by transfection and selection cycles with the other circuit, and current-passing recordings, and the data were stored D
o
two virions. Selection media included (in combinations appro- on the computer. wn
priate to the history of infection): G418 (800 (cid:6)g/ml), hygromy- Two solutions were used in the Ussing chambers, as well as in loa
d
cin (300 (cid:6)g/ml), and puromycin (5 (cid:6)g/ml) DMEM. At the end the multiinsert apparatus: unmodified DMEM or a “chloride- ed
of three rounds of infection and selection, colonies of cells that free” solution containing (in mM): 140 Na(cid:2) aminobenzene- from
hreasdp obnedenin ign afencttibedio twicit hre asilsl ttahnrceees swuebruen isitosl aatnedd eaxnhdi bexitpeadn tdheed c foorr- nsualtfeo,n 5a tKe(cid:2), 2g4lu Ncoan(cid:2)a tbei,c aanrbdo 1n0a tDe -g6l uCcao2s(cid:2)e .gluconate, 3 Mg2(cid:2) gluco- http://ru
screening while maintaining selection pressure with all three an- pre
tibiotics. Surface Labeling of Flagged ENaC Subunits ss.o
Clones of the triply transfected MDCK cells (referred to as rg
(cid:3) (cid:4)(cid:5) MDCK cells) were subsequently grown on permeable sup- Anti-FLAG antibody (Sigma-Aldrich; referred to below as M2) /jgp
apsumoFbreutFtsnh. FiAatssfo twenrae s4 p–inl5uc dsr e 2ian sm ecdMu lbt uNy raoe(cid:2)v, e ebrxnupitgryerhastst ie“oi nnind o utfhc ttehi oecn ut”rl tawunristefh e mc1t ee(cid:6)ddMi uE mNde aaxCs- i(LwtPyaai sbe aerrtacl edetd ihoC elMh abCe2em olameincdpat ilrwb eiCohthdoe y.n1) 2s w5ipIva eeis nr Ci nttahhinteeic aUemlrlA yaB Cnp uerunfaradticefiitroue lradueb sroei’nlsvig eni rgnI soactdnoruo raec-Bn tfieioaoacndnisls-.- /article-pdf/12
described by Stutts et al. (1995). Functional expression of the 0
transfected ENaC subunits was monitored by the amiloride-sensi- exchange column, eluted, and subsequently placed in a 10,000 /1/7
tIinvset rturamnesenptsi)th. eTlihale vocllotangee (wVitTh) utshine gm ano sEt VrOobMu s(tW torarlnds ePprietchiesiloianl MdiaWlyCz eSdli dfoer-A a-tL ylezaesrt d2i4al yhs ias gcaaisnssett tPeB (SP.i eTrhcee Camheomunicta ol fC loa.b) ealnedd 1/12174
aifmy tilhoer ipdree–sseenncseit iovfe flvoagltgaegde (wbaus ts enloetc tweidld. -RtyTp-Pe)C REN waaCs ususebdu ntoit sv einr- aPnietribceo dCyh wemasi cdael teCrom.)i naendd bwya sa dmiluictreodp rtoo tethine adsessaiyr e(d# sp23e2ci3fi5c; 17/jgp1
tEhNea Ctr asnusbfeucntietd c DceNllAs, iann tdh et op acroennfitarlm c etlhl el inaeb.s eTnhcee soufi tawbilidli-ttyy poef aaccttiivviittyy wbya st htyep aicdadlliyti o(cid:2)n1 .o8f (cid:6)uCnil/ab(cid:6)egl eadn tMib2o dayn,t bibuotd ity .w Tahs em sopdeicfiiefidc 20171.p
as needed to give sample count rates that ranged from at least d
tehxetr apcrtiemde frrso umse fdre fsohr dtohgis kpidunrpeoy saes wa apso csoitnivfier cmoendtr obly. Euxsipnrge sRsiNoAn 20 times background to (cid:9)10,000 cpm, depending on the anti- f by g
boyf aimll mthureneo sbulobtutinnigts ains dthesec (cid:3)riFb(cid:4)eFd(cid:5) Fb MelDowC.K cells was also confirmed boEdqy uciolinbcreiunmtr abtiinodni nugse ads.says were performed on monolayers of uest on
Electrophysiological Measurements (cid:3)mFm(cid:4)F (cid:5)cFe lMl cDuCltKur cee ilnlss egrrtos.w Anf toenr eexitpheerri mcyecnlotaplo mree aosru rtreamneswnetsl la 2n4d- 04 Jan
treatments, the inserts were placed in an ice bath and briefly ua
Short-circuit currents (Isc) were measured by mounting 12-mm rinsed with ice-cold PBS, and then incubated for 30 min with ry 20
diameter cell culture inserts (Millicell PCF; Millipore) in Ussing- ice-cold blocking solution (PBS (cid:2) 5% FBS). The apical blocking 23
type chambers (Jims Instruments) enclosed in a circulating wa- solution was aspirated and replaced with 500 (cid:6)l of ice-cold
ter-jacket maintained at 37(cid:11)C and equipped with bubble-lifts blocking solution containing the desired concentration of 125I-
driven by the 95% O /5% CO gassing. The half-chambers were labeled M2 antibody and incubated for 1 h. To determine the
2 2
connected via agar bridges to potential-sensing and current-pass- nonspecific binding, M2 antibody was added to paired inserts
ing Ag-AgCl electrodes. All electrodes were connected to a volt- together with a 100-fold excess (by weight) of FLAG peptide
age clamp (model VCC600; Physiological Instruments). I was (Research Genetics). After a 1-h incubation, excess label was as-
sc
continuously recorded on a strip-chart recorder. pirated and monolayers were washed four times with 1 ml of ice-
In addition to the traditional Ussing-type chambers, I was also cold blocking solution. Label remaining bound to the apical
sc
measured in the 24-mm diameter cyclopore and transwell cul- surface of the monolayers was then removed by “acid-stripping”
ture inserts that were used to conduct binding experiments. For as per Wiley and Cunningham (1982). Briefly, 750 (cid:6)l of ice-cold
these experiments, we built a special “multiinsert apparatus” that acid wash solution (0.5 M NaCl, 0.2 M Na(cid:2) acetate, pH 2.4) was
allowed rapid sequential recording of I and R, as well as the aliquoted onto the apical membrane of monolayers, incubated
sc T
open-circuit V. The inserts were held in a manifold constructed on ice for 5 min, and then harvested for gamma counting. Two
T
from a Lucite sheet with 24 equally spaced openings. The lower acid strips were performed per monolayer to confirm that
portions of the inserts protruded below the manifold into the ba- (cid:13)80% of the bound label was removed. The counts from both
solateral medium, which was contained in an inexpensive plastic acid strips were combined for data analysis. For each experi-
container and was gassed with a mixture of 95% O /5% CO. mental point, triplicate inserts were labeled with 125I-labeled M2
2 2
This container was placed in a larger plastic container that antibody in the absence of competing peptide and a paired trip-
74 cAMP Increases ENaC Membrane Density
licate in its presence. Specifically, bound counts were taken to RESULTS
be the difference in average counts obtained in the presence
and absence of peptide. Expression of ENaC Subunit Proteins in MDCK Cells
Western Blotting and Immunoprecipitation of ENaC Subunits In preliminary experiments, we first tested the antibod-
ies to the rat (cid:3)-, (cid:4)-, and (cid:5)ENaC subunits in immuno-
For each Western blot, protein was harvested from two confluent
blots with protein extracted from rat kidney cortex, and
24-mm monolayers of (cid:3)(cid:4)(cid:5) or parental MDCK cells. Monolay-
F F F we obtained the same bands reported by Masilamani et
ers were washed extensively with ice-cold PBS, removed from the
filter inserts with cell scrapers, and briefly centrifuged. After aspi- al. (1999) in their original description of the antibod-
rating the PBS, 600 (cid:6)l of ice-cold lysis buffer (10 mM triethanola- ies. Because MDCK cells were derived from dog kidney,
mine, 250 mM sucrose, pH 7.6, plus PMSF [1 mg/10 ml] and leu- we also tested the same rat antibodies against protein
peptin [1 (cid:6)g/ml]) was added to the pellet, and the cells were ho-
isolated from dog kidney and from type-1 MDCK cells
mogenized with a glass mortar and pestle. SDS was then added
from which our parental strain was derived. Unfortu-
(1% final concentration) to the homogenate, which was mixed
with a 21-ga needle and syringe. The sample was transferred to a nately, only the antibody for the rat (cid:4)ENaC was found
1.5-ml tube and sheered with a 30-ga needle 3–4 times before to also recognize the dog subunit as shown in Fig. 1. Us-
centrifuging 15 min at 14,000 g. Approximately 90% of the su- ing the (cid:4)ENaC antibody, a single band at (cid:2)101 kD was
pernatant was then recovered and concentrated using a Micro- obtained in proteins isolated from dog kidney cortex Do
con 30 (Millipore) as per the manufacturer’s instructions. Pro- w
tein determinations were made with a MicroBCA Kit (Pierce and inner medulla, type-1 MDCK cells, and triply-trans- nloa
Cadhdeemdi ctoa l1 C0o-(cid:6).)g. sLaamemplmesl io bf upfrfoetre cino,n wtahiincihn gw emree rbcraipetfloye bthoailneodl awnads fbeacntedd i n( (cid:3)thF(cid:4)e Fp(cid:5)aF)r eMntDalC MKD cCeKll sc. eHllso uwseevde rf, otrh terraen swfeacst inono ded fro
m
rtarunandn sopfneror 1rce0ed%ss te opd o af lonyrai tcirmroycmlaemullnuidoloebs lego etmtlsien. mgP.rbortaenine (wNaist reolebcintrdo;p Ohosmreotinciaclsl)y iuns etdh efsoer stpurdoiteesi n( fierxstt rlaacntieo nin hFaigd. b1)e.e n(A ilnl dMuDceCdK ocveellrs- http://rup
night with butyrate and dexamethasone, and equal pro- re
et Aaln.,t i1b9o9d9ie) sw sepreec ipfirco vtiod ethdr beey Drart. EMN. aKCn espupbeurn i(tNs a(tMioansailla mInasntii- tein loading on each blot was independently verified ss.org
tutes of Health, Bethesda, MD). The membrane blots were among all MDCK cell lanes.) We also performed RT- /jgp
bolvoecrkneigdh wt iatht B4(cid:11)lCot twoi tfho rp 1r ihm aatr ryo EoNma tCe manpetirbaotudriees a antd t ihnec uabpaptreod- PCR using degenerate, ENaC subunit–specific primer /article
1p:r1i,a5t0e0 ,d ailnuttii-o(cid:5)EnNs a(Can Lti5-(cid:3)50E N1:a2C,0 0L07)6. 6T h1e:2y ,w50er0e, tahnetni- (cid:4)wEaNshaeCd tLh5r5e8e preaviersr steh-tarta nwsecrrei bsehdo wtont atlo m aRmNpAli fysa EmNpaleCs sfurobmun ditos gf rkoimd- -pdf/120
/1
times with T-TBS (TBS/0.05% Tween-20), incubated for 1 h at /7
1
room temperature with secondary antibody diluted 1:5000 in /1
2
1
Blotto, and washed again with T-TBS. Blots were visualized with 7
4
1
the ECL detection system (Amersham Pharmacia Biotech) per 7
/jg
the manufacturer’s instructions. Autoradiograms of immuno- p
1
2
blots were digitized on an Epson transparency scanner (Expres- 0
1
7
sion Model 1600). Densitometric comparisons of bands on the 1
.p
same blot were made using the NIH Image program and cali- df b
brated gel analysis subroutine (version 1.62; National Institutes y g
u
of THoe avlethri)f.y that the expressed ENaC subunits were indeed est on
pflraogtgeeind, h(cid:3)aFr, v(cid:4)esF,t eadn dfr o(cid:5)mF stuwbou cnoitnsfl wueernet i2m4-mmumn odpiarmeceipteitra mteodn forolamy- 04 Jan
ea rps rooft einind-uGc eimd m(cid:3)Fu(cid:4)nF(cid:5)oFp MreDciCpiKta ctieolnls ukisti n(g1 7M129 3a8n6t;i bRoodcyh (e5) (cid:6)acl)c oarndd- uary 20
2
ing to the manufacturer’s instructions. After immunoprecip- 3
itation, samples were boiled briefly in Laemmli buffer and
processed for Western blotting as described above. In other ex-
periments, the subunit-specific antibodies were used to immuno-
precipitate the proteins, and the immunoblots were probed with
the M2 antibody.
Statistics and Nonlinear Regression of Binding Saturation Plots Figure 1. Western blot demonstrating the expression of the
(cid:4)ENaC subunit in MDCK cells and dog kidney tissues. The lanes
StatView for Macintosh (version 4.5.1, SAS Institute Inc.) was contained protein extracts from: parental MDCK cells (the line
used for standard statistical calculations. Numerical results are subsequently used for transfection of flagged ENaC subunits), an-
presented as averages (cid:7)SEM. ANOVA with Bonferroni-Dunn other clone of Type-I MDCK cells, (cid:3)(cid:4)(cid:5) MDCK cells (the triply
F F F
post-hoc testing was used for comparisons of paired and non- transfected cell line developed in this study), dog kidney cortex,
paired averages as appropriate, with significance ascribed at and inner medulla. All three lines of MDCK cells were induced
P (cid:8) 0.05. overnight with 2 mM butyrate plus 1 (cid:6)M dexamethasone, and 20
Kaleidagraph for Macintosh (version 3.5.1; Synergy Software) (cid:6)g of extracted protein was present in each lane. In the case of the
was used for nonlinear least squares fitting of specific binding dog kidney tissues, 50 (cid:6)g of protein was present for each. The
data to the Michaelis-Menten relationship. Quality of fit is indi- numbers on the left side indicate the molecular weights (kD) of
cated by the correlation coefficient (r) and associated P value. proteins in the molecular weight calibration ladder.
75 Morris and Schafer
ikawa et al., 1998), and confirmed in our electrophysio-
logical studies below.
As shown in Fig. 2 A, the subunit-specific antibodies
to rat ENaCs showed the presence of all three ENaC
subunits in the retrovirally transfected (cid:3) (cid:4) (cid:5) MDCK
F F F
cells with and without induction, and the approximate
molecular weights were: (cid:3), 94 kD; (cid:4), 98 kD; (cid:5), 89 kD. It
should be noted that, whereas only a single band was
obtained in all immunoblots for (cid:3)ENaC and (cid:5)ENaC, in
some blots for (cid:4)ENaC we obtained two bands (ranges:
93–102 kD and 105–116 kD) as shown in Fig. 2 B. For
nine separate protein samples from induced (cid:3) (cid:4) (cid:5)
F F F
MDCK cells, the (cid:4)ENaC doublet was present in six. In
six of these blots in which protein samples from unin-
duced and induced (cid:3) (cid:4) (cid:5) MDCK cells were paired,
F F F
three showed the doublet and it was present in both in- D
o
w
duced and uninduced samples. The source of the n
lo
a
higher molecular weight member of the doublet is un- d
e
d
known, and it was not observed in protein isolates from fro
m
rat kidney by Masilamani et al. (1999). The 110-kD h
ttp
band was variable among different protein isolates, ://ru
ranging from barely detectable in comparison with the pre
98-kD band to almost as dense (as in Fig. 2 B). The mo- ss.o
rg
lecular weight difference between the bands of the /jg
p
depoiutobpleet (w(cid:2)as1 .t1e nk Dti)m. eTsh liasr gceorm tphaarnis owne igahntd otfh eth aeb FseLnAcGe /article-p
of any (cid:4) band in the parental cells (Fig. 1) rules out the df/1
2
0
possibility that the doublet was due to the presence of /1
both wild-type and flagged (cid:4)ENaC in the transfected /71/1
2
cells. 17
4
fFeicgtuedre M 2D.CKE xcperlless swioitnh aonf dE wNitahCo ustu binudnuitcst ioinn . r(eAtr)o Wvireasltleyr ntr banlost- In the representative blot shown in Fig. 2 A, the levels 17/jgp
ogrf opurpoetedi na cfrcoomrd itnrigp lteo-fl tahgeg esudb (u(cid:3)nFi(cid:4)t-Fs(cid:5)pFe McifiDcC aKn)t cibeollds.y ,T ahned l ainne es aacrhe oofv earlnl tighhret ein EdNuacCtio snu bwuitnhit bs uaptypraetaer eadn dto d beex ainmcertehaasesodn bey, 120171
subunit group extracts are from (cid:3)(cid:4)(cid:5) MDCK cells without (first whereas only (cid:3)ENaC appears to be significantly in- .pd
F F F f b
lane of pair) and with (second lane) overnight induction with 2 creased in Fig. 2 B. Densitometric ratios of paired in- y g
smidMe ibnudtiycraattee tphleu sm 1o (cid:6)leMcu dlaerx wameigehthtsa s(oknDe). oTfh per nouteminbse irns tohne tmheo lleecft- duced and uninduced bands for each of the three uest o
uplaarre dw eiing htth cea lsiabmraet iowna yl aadsd pera.n (eBl )A A, nboutht eshr ocowminpgo tshitee odfo bulboltest p froer- EthNeareC wsuasb usingintsifi acraen pt rinesdeunctteido nin o nFilgy .o 2f C(cid:3)E aNnadC s h(orawt ioth oaft n 04 Ja
(cid:4)ENaC (see text). (C) Ratios of the densitometry measurements induced to uninduced 4.4 (cid:7) 1.1, P (cid:8) 0.02) and (cid:4)ENaC nua
for the (cid:3)-, (cid:4)-, and (cid:5)ENaC subunit bands in the presence of induc- (2.4 (cid:7) 0.5, P (cid:8) 0.01), but not (cid:5)ENaC (3.2 (cid:7) 1.6, not ry 20
tion to that in the absence of induction. The number of separate significantly different from 1.0). However, as shown be- 23
protein extractions and immunoblots is indicated in parentheses low, induced (cid:3) (cid:4) (cid:5) MDCK cells exhibited a much
in each bar. Asterisk indicates average ratio is significantly differ- F F F
higher amiloride-sensitive I .
ent from 1.0. sc
To verify that the ENaC subunits expressed in trans-
ney cortex. When cDNA that had been prepared in the fected MDCK cells contained the FLAG epitope, two se-
same manner from parental MDCK cells (induced ries of immunoprecipitation experiments were per-
overnight with butyrate and dexamethasone) was am- formed. First, proteins were immunoprecipitated from
plified with the same primers, only a faint band of the (cid:3) (cid:4) (cid:5) MDCK cell lysates with the M2 antibody. The im-
F F F
expected size for (cid:3)ENaC was obtained even with 40 cy- munoprecipitated proteins were visualized on subse-
cles of amplification, but there was no amplification of quent Western blots using the same subunit-specific an-
(cid:4)- or (cid:5)ENaC (unpublished data). The apparent low (or tibodies as above. Bands of the appropriate molecular
absence of) expression of significant (cid:4)ENaC protein or weights (Fig. 3 A) confirmed the presence of the FLAG
mRNA for any of the three subunits is consistent with in each of the ENaC subunits, but with the presence of
the very low or absent amiloride-sensitive Na(cid:2) transport a doublet for the (cid:4) subunit. As further confirmation, a
activity observed previously in this MDCK cell line (Ish- second series of immunoprecipitation experiments was
76 cAMP Increases ENaC Membrane Density
D
o
w
n
lo
a
d
FMigDuCrKe c4e.lls Saps eac fiufinc cbtiionnd ionfg a onft i1b25o-Id-lya bcoelnecde nMtr2a atinotnib. oCdeyll sin w (cid:3)erFe(cid:4) Fi(cid:5)nF- ed fro
m
duced overnight with butyrate and dexamethasone, and the exper- h
ttp
iments were performed in DMEM. M2 antibody was added at ://ru
seven different concentrations to the apical membrane only. The p
re
dwaetiag hwteedre l efiats tt osq uthaer eMs (icr h(cid:14)a e0l.i9s-5M, Pen (cid:8)te 0n. 0r0e1la)t.i oEnstsihmipat ebsy o nf omnalixnimeaarl, ss.org
sawpgeeercse i ffi7rco.4 ma (cid:7)n ste i0vbe.o7nd fyem xbopilenerdsi/mincemgn 2it nsa wnfimdth o7 l1./92 c6(cid:7)m i n21 s.(e9Br tmnsa.Mx), arensdp eacftfiivneiltyy. A(kv0e.5r)- /jgp/article-p
d
f/1
2
0
/1
/7
1
Specific Binding of 125I-labeled M2 Antibody to (cid:3)(cid:4)(cid:5) /12
F F F 17
MDCK cells 41
7
/jg
M2 antibody labeled with 125I was used to measure the p1
2
0
apical membrane surface density of FLAG. In initial ex- 17
1
periments we found appreciable binding of this anti- .pd
body to the (cid:3)F(cid:4)F(cid:5)F MDCK cells and culture inserts that f by gu
Foifg (cid:3)uFr(cid:4)eF(cid:5) 3F .MDImCmK ucnelolsp rinedciupciteadt iownit ho fb uEtNyraaCte saunbdu ndietxs afmroemth alyssoantees. pbeelresids tMed2 eavnetnib iond tyh eo rp rthesee nFLceA oGf panep etxidcee.s sI to wf tahs en uencelas-- est on 0
(A) Monoclonal anti-FLAG (M2) antibody was used to immuno- sary to measure this nonspecific binding for each data 4 Ja
precipitate ENaC subunits. Three aliquots of the immunoprecipi- point by adding a 100-fold excess (by weight) of FLAG nua
tcaloten awl,e srueb usunbits-sepqeuceinfitcl ya nvtiis-EuaNliazCed a nitnib oimdimesu anso pbrloobtse su. (siBn)g Tphorelye- peptide. (The ratio of specific to nonspecific binding, ry 20
2
aliquots of cell lysates were separately immunoprecipitated with i.e., the signal-to-noise ratio, varied from 3:1 to 4:1, de- 3
polyclonal, subunit-specific anti-(cid:3), -(cid:4), and -(cid:5) antibodies. M2 anti- pending on the antibody concentration.) Thus, the
body was used as the probe in immunoblots. specific binding of 125I-labeled M2 antibody was de-
fined as the portion of the total binding displaced by
performed in which each ENaC subunit was initially the competing peptide.
immunoprecipitated from (cid:3) (cid:4) (cid:5) MDCK cell lysates us- Fig. 4 presents the results of seven experiments with
F F F
ing the subunit-specific anti-ENaC antibodies. Immu- (cid:3) (cid:4) (cid:5) MDCK cells in DMEM in which specific binding
F F F
noprecipitated proteins were processed for Western was measured as a function of the 125I-labeled M2 anti-
blotting and visualized with the M2 antibody shown in body concentration. The specific binding showed satu-
Fig. 3 B. Using this approach, the doublet for (cid:4) sub- ration with increasing antibody concentration as would
F
unit was absent, although, as noted above, the presence be expected for a typical receptor ligand interaction; in
of this band was variable. Together, the results (Figs. 2, this case, the binding of 125I-labeled M2 antibody to the
A and B, and 3, A and B) demonstrate that all three FLAG present in the ectodomain of the ENaC subunits
flagged ENaC subunits are expressed in the (cid:3) (cid:4) (cid:5) in the apical membrane. Because less than 1% of the
F F F
MDCK cells. M2 antibody added to the apical medium was bound to
77 Morris and Schafer
the membrane surface at equilibrium, we fit the data in
Fig. 4 according to the Michaelis-Menten equation.
Nonlinear least squares fitting gave estimates of k and
0.5
B of 7.9 (cid:7) 1.9 nM and 7.4 (cid:7) 0.7 fmol/cm2, respec-
max
tively.2 The k thus obtained is not significantly differ-
0.5
ent from the k of (cid:2)3 nM reported by Firsov et al.
0.5
(1996) using the same FLAG-M2 antibody pairing in
the Xenopus oocyte expression system.
We also performed three control-binding experi-
ments on MDCK cells transfected with rat wild-type (cid:4)-
and (cid:5)ENaC subunits together with a rat (cid:3)ENaC subunit
that was modified to contain an unrelated epitope
(hemagglutinin, [HA]: YPYDVPDYA) in the same ec-
todomain region as the FLAG epitope in the construct
of Firsov et al. (1996). Using a single 125I-labeled M2 an-
tibody concentration (1.7 nM) revealed an insignifi- D
o
w
cant amount of binding that could be displaced by the n
lo
flag peptide (n (cid:14) 3, 0.04 (cid:7) 0.01 fmol/cm2). ad
e
d
Finally, we conducted four experiments in which we fro
m
measured the specific binding of 125I-labeled M2 anti- h
body applied to the basolateral surface of the (cid:3)F(cid:4)F(cid:5)F ttp://ru
MDCK monolayers in the presence and absence of pre
FLAG peptide. There was no significant specific bind- ss.o
rg
ing, however, it would have been difficult to discern /jg
p
scpael csuifircfa bcien (dii.ne.g, (cid:8)at 2t hfme loolw/cemr l2e)v belesc oabusseer ovef dth oen h tihgeh alepvi-- /article-p
d
els of nonspecific binding when the antibody was f/1
2
0
added to basolateral side. We attributed these higher /1
/7
nonspecific counts to antibody binding to the mem- 1/1
2
brane support because we found similar levels of non- 17
4
1
specific binding to membrane inserts in the absence of 7/jg
p
cells. The absence of significant ENaC activity in the 1
2
0
apical membrane was also supported by the absence of 17
1
any change in V or I when amiloride was added to Figure 5. Effect of Cl(cid:15) on the response of short-circuit current .pd
T sc f b
the basolateral membrane (see below). (Isc) to cAMP stimulation. (A) Time course of Isc response to cAMP y g
Electrophysiological Characteristics tarceraotsms emnot ninol aDyMerEs Mof a(cid:3)nF(cid:4)dF (cid:5)chF lMorDidCeK-f rceeel lsm ienddiuucmed. Ioscv ewransi gmhet awsuitrhe d2 uest on
mM butyrate and 1 (cid:6)M dexamethasone. A mixture of 20 (cid:6)M CPT- 04
Parental MDCK cells exhibited very little evidence of cAMP and 200 (cid:6)M IBMX was added at the time indicated, fol- Ja
n
awmithil opraidreen-steanl sMitiDveC KN ac(cid:2)e ltlsr atnhsapt owret.r eIn n otht riened uexcepde,r itmhee natvs- lsocrwiebded b iyn 2A0. S(cid:6)ixM e xapmeriliomriednet.s w(Be)re A cvoenrdaguec teIsdc iinn DeMxpEeMrim anend t2s 0d ien- uary 20
chloride-free medium. Basal I was measured just before, and the 23
erage baseline open-circuit VT and Isc in DMEM were, re- cAMP I just after the additionsc of 20 (cid:6)M CPT-cAMP plus 200 (cid:6)M
spectively, (cid:15)1.2 (cid:7) 0.3 mV and 0.8 (cid:7) 0.1 (cid:6)A/cm2. In five IBMX, asct the point of maximal response, 5–10 min in DMEM and
10–20 min in chloride-free medium. The final measurement was
made 1–3 min after the addition of 20 (cid:6)M amiloride.
2Plots of specific binding as a function of antibody concentration are
presented and analyzed using the Michaelis-Menten equation as de- experiments with parental cells that had been induced
scribed in materials and methods. This approach is used in prefer-
overnight with butyrate and dexamethasone, baseline
ence to a Scatchard plot (bound/free versus free antibody concentra- V and I were, respectively, (cid:15)7.1 (cid:7) 2.1 mV and 3.7 (cid:7)
tion) because the Michaelis-Menten plot shows the actual data ob- T sc
tained and its variation, and because the nonlinear regression of the 0.3 (cid:6)A/cm2. After the latter cells were treated with 20
data according to the Michaelis-Menten relationship avoids the bias (cid:6)M 8-CPT-cAMP plus 200 (cid:6)M IBMX, I decreased by
sc
introduced into least-squares fitting by the linearized Scatchard rela- only 1.8 (cid:7) 0.3 (cid:6)A/cm2 upon addition of 20 (cid:6)M
tionship. Nevertheless, the results of Scatchard analysis were quite
amiloride to the apical solution. In many other groups
comparable with those obtained from nonlinear regression using the
of parental cells, treated with both induction and cAMP,
Michaelis-Menten equation. In the case of Fig. 4, linear regression of
the Scatchard plot gave k0.5 and Bmax estimates of, respectively, 9.2 (cid:7) there was no change whatsoever in Isc upon the addition
2.2 nM and 8.0 (cid:7) 0.8 fmol/cm2 (r (cid:14) 0.90, P (cid:8) 0.001). of amiloride to the apical solution. Thus, while there
78 cAMP Increases ENaC Membrane Density
may be a small amiloride-sensitive I in some groups of TABLE I
sc
parental cells after overnight induction, it was small in Effect of Amiloride on Transepithelial Voltage and Resistance in (cid:3)(cid:4)(cid:5)
F F F
comparison with the transfected cells. MDCK Cells
Table I documents the effects of 20 (cid:6)M amiloride on Treatmenta V b R c Eq. I d
T T SC
the open-circuit V and R that develop across conflu-
T T mV k(cid:10)·cm2 (cid:6)A/cm2
ent monolayers of (cid:3) (cid:4) (cid:5) MDCK cells after overnight
F F F Basal (cid:15)27.7 (cid:7) 1.2 2.2 (cid:7) 0.1 12.5
induction with both dexamethasone and butyrate. Ad-
dition of 20 (cid:6)M amiloride to the apical medium signifi- Amiloride (cid:15)6.4 (cid:7) 0.3e 2.8 (cid:7) 0.1e 2.2
cantly reduced V from (cid:15)27.7 (cid:7) 1.2 mV to (cid:15)6.4 (cid:7) 0.3 aAfter overnight induction with 2 mM butyrate and 1 µM dexamethasone,
mV (n (cid:14) 95, P (cid:8)T 0.001), and significantly elevated R triple-flagged ((cid:3)F(cid:4)F(cid:5)F) MDCK cells on either cyclopore or transwell mem-
T brane inserts were incubated in DMEM. Basal transepithelial voltage (V,
from 2.2 (cid:7) 0.1 to 2.8 (cid:7) 0.1 k(cid:10)·cm2 (n (cid:14) 90, P (cid:8) 0.001). T
apical to basolateral) and resistance (R ) were measured during the first
T
We also examined the dose response of the transfected 20–30-min incubation with no additions to the medium. Amiloride (20
cells to amiloride. Based on the percent inhibition of (cid:6)M) was then added to the apical solution and the same parameters were
I , the k for amiloride in the absence and presence of measured when they reached a nadir 1–3 min after amiloride addition.
csAcMP trieatment (20 (cid:6)M 8-CPT-cAMP plus 200 (cid:6)M bn (cid:14) 95.
cn (cid:14) 90.
IBMX) was, respectively, 0.48 (cid:7) 0.14 and 0.48 (cid:7) 0.18 D
(cid:6)erMal. mAdemdibtiroann eo fh a1d0– n1o0 0s i(cid:6)gnMifi acmanilto erfidfeec tt oo nth eei tbhaesro tlhate- cdeDaTnhiftef e( “Pree (cid:8)qnuc 0iev. a0bl0ee1tnw)t e”b esynh p obaraitrs-ecadilr actn utdeits atc.murirloenritd (eE vqa.l uIsecs) oisf VcaTl caunlda tReTd aarse V sTig/nRiTfi.- ownload
e
d
basal VT, or on Isc in the presence or absence of cAMP from
treatment. These observations demonstrate the pres- h
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st(cid:3)iuFmr(cid:4)ieFn(cid:5) gFc oIMsuc DrisnCe K UI cssces ilonlsfg i nrce hDparMmeEsbeMenr sta.a ntFidvig ec. he5lxo Apri edcreoi-mmfreepnea trmse sew dtihitahe, a(cid:2)m3Ti h(cid:6)loeAr ib/dicpem ht2ao.s itch ree asppoicnasle s tiod ec AsiMgnPi fiinc athnetl yp rreesdeuncceed o Ifs cC tlo(cid:15) /article-pdf/12
0
and the average results of several experiments are pre- in the bathing solution has been described previously /1/7
sented in Fig. 5 B. In DMEM, basal Isc was significantly for MDCK and similar epithelia. It has been interpreted 1/12
stimulated from 8.1 (cid:7) 1.0 to a peak value of 18.9 (cid:7) 2.2 to reflect at least two components of I : active Cl(cid:15) secre- 174
(cid:6)A/cm2 by the addition of 20 (cid:6)M 8-CPT-cAMP plus 200 tion and active Na(cid:2) transport via ENasCc (Chalfant et al., 17/jg
(cid:6)M IBMX to the apical and basolateral media. There 1993; Kleyman et al., 1994; Letz and Korbmacher, 1997; p12
0
was rapid spike in Isc followed by a broader peak, which Morris et al., 1998), both of which are stimulated by 171
tdheec aaypeidc aol vseorl u3t0io mn irna. pAiddldy itrieodnu ocef d2 0Is c(cid:6) tMo (cid:2)am5 il(cid:6)oAri/dcem t2o. ccAAMMPP. pUrnoddeurc eshs oar rt-acpiridcu iint ictioanl dinitciorenass ein i nth Cesle(cid:15) esepcitrheetiloian, .pdf by gu
The effect of amiloride was statistically significant and followed by a slower activation of Na(cid:2) transport. Fur- est o
confirmed the presence of amiloride-sensitive, cAMP- thermore, as observed in Fig. 5 A, after cAMP stimula- n 0
sscthoimouwrusnela tionefd F2 Ni0g a.(cid:6)(cid:2) 5 Mt rA a8n, -CsipnPo Tcr-htc liAonMr (cid:3)idPFe(cid:4) p-Ff(cid:5)lrueF seM 2mD00eC dK(cid:6)iu Mcme l IlsBt.h MAe Xst ai mlascoe- tictihos anan bignsee stnh ciene . p tBrheeesc edanuecnsees oiotfyu Corl f(cid:15)o ,Eb INjsec acftCailv lessu mwbauosrn etito rs a cipnoi rdtrlheyle at htaeap naic nianyl 4 January 20
2
3
membrane with the amiloride-sensitive I , we used a
sc
chloride-free medium in the remainder of these studies
3Drs. J.K. Bubien and Z.-H. Zhou in our department were kind to reduce any variability that might be introduced by dif-
enough to conduct preliminary patch clamp studies of the cells used
ferences in the time course of the I response to cAMP.
in these studies using methods they have described previously (Bu- sc
bien et al., 2001; Zhou and Bubien, 2001). In detached patches from
Correlation between ENaC Surface Labeling and I
(cid:3)(cid:4)(cid:5) MDCK cells, they observed one to three channels per patch sc
F F F
with conductances in the range of 5–10 pS, NPo products of 0.5–1.5, We used two different approaches to produce cell cul-
and the long open and closed intervals characteristic of ENaC chan-
tures that exhibited a wide range of ENaC expression
nels with or without the FLAG label (Firsov et al., 1996; Garty and
Palmer, 1997). In one inside-out patch from an (cid:3)(cid:4)(cid:5) MDCK cell, and, thus, of surface binding. In both sets of experi-
F F F
the I-V relationship was linear in the range of (cid:7)80 mV with a slope ments, the chloride-free medium was used. First, we pre-
conductance of 8.6 pS. In whole-cell patches of untreated (cid:3)F(cid:4)F(cid:5)F pared mixtures of varying proportions of parental (wild-
MDCK cells, there was a basal level of ENaC activity that was markedly type) to (cid:3) (cid:4) (cid:5) MDCK cells at the time of seeding onto
increased by cAMP. In whole-cell patch clamps of parental cells, F F F
cell culture inserts while keeping the total number of
there was no evidence of ENaC-like channel activity in the presence
cells constant. For each experiment, a tray of six cell cul-
or absence of cAMP treatment (Bubien, J.K., and A.L. Birmingham,
personal communication). ture inserts was seeded for each mixture of cells. This ar-
79 Morris and Schafer
D
o
w
n
Figure 6. Correlation between specific binding of 125I-labeled lo
a
M2 antibody and amiloride-sensitive short-circuit current (Isc). Figure 7. Specific binding of 125I-labeled M2 antibody in (cid:3)F(cid:4)F(cid:5)F ded
The M2 antibody concentration in the binding experiments was MDCK cells as a function of antibody concentration in the pres- fro
m
16 nM. Chloride-free medium was used for both the apical and ba- ence and absence of cAMP treatment. In each of six experiments, h
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p
bmwoiitlxh tk urearyet)is.o o( obf f)(cid:3) pTFa(cid:4)hrFee(cid:5)n F(cid:3) taaF(cid:4)nl Fdt(cid:5)o Fp (cid:3)aMFr(cid:4)eDFnC(cid:5)tFaK lM cMeDDlClsC KeK ict hceelellrsl siw n(esdrqiecu agatrievede dnian nt atoh pieno sdinyumtcs,-- I(aB(cid:2)ftMecrAX cM AwPMa)sP aa ntdrded aeotdnm ltyeo nt htt,he t ehv eeah piiniccslaeelr ttsoso wltuhhteieo ronet h coehfr i lohlenadelf - ih(nac laofn no tifrc oetlh )be. a 3ti0hn smaenritnds ress.org/jgp
tmioenth oasro wnee,r ea s iinndduiccaetde dw iinth t h2e smymMb oblu ktyerya. tTeh aen ldea/sot-rs q1u a(cid:6)rMes ldineexaar- smuertfahcoed lsa.b Sepliencgifi wcaalsl yc boonudnudc t1e2d5I -laasb edleesdc rMib2e da nitnib omdayt iesr pialoltst eadn ads /article
regression, indicated by the line, has a slope of 0.12 (cid:7) 0.03 fmol/ a function of the 125I-labeled M2 antibody concentration added to -pd
(cid:6)A (P (cid:8) 0.001) and intercept of 0.62 (cid:7) 0.33 (NS). The correlation the apical membrane. The data were fit to the Michaelis-Menten f/12
0
coefficient r is 0.82 (P (cid:8) 0.001). relationship by nonlinear regression (control: r (cid:14) 0.99, P (cid:8) 0.001; /1/7
(cid:2)cAMP: r (cid:14) 0.97, P (cid:8) 0.001). Asterisk denotes specific binding 1/1
2
was significantly higher in cAMP-treated than controls. 17
4
1
7
rangement typically resulted in a total of four trays, or /jg
p
1
24 inserts for each experiment. After reaching conflu- dard combination of dexamethasone and butyrate. As 20
1
7
ence, the cells were induced with butyrate and dexa- in the previous protocol, Isc was measured in the pres- 1.pd
methasone and the following day the cell culture inserts ence and absence of amiloride using the multi-insert f b
were placed in the multiinsert apparatus for rapid, con- apparatus. In nine experiments conducted in chloride- y gu
e
secutive measurement of Isc in each of the 24 inserts. free medium, the average AS-Isc values in uninduced st on
Tcahlceu almateildo riads et-sheen smitievaen s hpoaritr-ecidr cduiiftf ceurernrecnet b(AetSw-Iesce)n w Iassc MdeDxaCmKe cthelalss,o nane da ilno ncee,l lso rin dboutche da wgeitnht sb uwteyrrea,t er aelsopneec,- 04 Janu
mafteears uardeddi njugs t2 0b e(cid:6)foMre aamndil otrhidate mtoe atshuer eadp iwciatlh isno l3u tmioinn. (cid:6)tivAe/lyc,m 22.7 ((cid:7)ea c0h.7 ,m 8e.1a n(cid:7) s1ig.3n,i fi1c1a.8n t(cid:7)ly 0d.i7f,f earnedn t1 6fr.7o m(cid:7) t1h.6e ary 202
3
There was considerable variability in AS-I measured for other three by ANOVA).
sc
a given cell mixture, which probably reflects a variable In each of the two sets of experiments described
rate of growth of parental compared with (cid:3) (cid:4) (cid:5) MDCK above, the specific binding 125I-labeled M2 antibody was
F F F
cells among the different preparations of cells used to measured at an antibody concentration of 16 nM. This
plate the inserts. Nevertheless, this approach provided intermediate concentration was chosen because the ra-
inserts with the wide range of AS-I that was desired. tio of specific to nonspecific binding (i.e., the signal-to-
sc
In the second protocol, we varied the expression of noise ratio) decreased at higher antibody concentra-
ENaC among the four trays used for each experiment tions as specific binding sites became saturated. Spe-
by varying the induction method. In each experiment, cific binding was measured in the same inserts as I ,
sc
we seeded (cid:3) (cid:4) (cid:5) MDCK cells onto cell culture inserts and it was plotted against the AS-I as shown in Fig. 6.
F F F sc
resulting in four trays, or 24 inserts, per experiment. Linear regression demonstrated a significant correla-
The evening before the experiment, one tray was not tion between specific binding and AS-I (n (cid:14) 45, r (cid:14)
sc
induced, the second tray was induced with 2 mM bu- 0.82, P (cid:8) 0.001). The slope of this relationship was 0.12 (cid:7)
tyrate only, the third with 1 (cid:6)M dexamethasone only, 0.03 fmol/(cid:6)A, and the intercept (0.62 (cid:7) 0.33 fmol/
and the fourth tray of cells was induced with the stan- cm2) was not significantly different from zero.
80 cAMP Increases ENaC Membrane Density