Meigiu Lin?
`Katrina MacLeod®
`Sandra Guggino? »
`
`Division of Gastroenterology,
`Departmentof Medicine, and
`Department of Neuroscience,
`Johns Hopkins University,
`Baltimore, Md., USA
`
`Key Words
`Diarrhea
`Channel
`T84 cells
`Kinase
`Intestine
`
`Original Paper
`
`eee
`
`Cell Physiol Biochem 1995;5:23-32
`
`Heat-Stable Toxin from
`Escherichia coli Activates Chloride
`Current via cGMP-Dependent
`Protein Kinase
`
`Oa eeccaccccconaeenscces 00000 000000000000 06 OOOO OCC OS OODOODOSEEEDOOOEEE
`
`Abstract
`Heat-stable toxin (STa) increases cyclic GMP (cGMP)in iso-
`lated intestinal cells and in T84 cells, a colonic secretory cell
`line. Whole-cell current recordings from patch clamp experi-
`ments show identical properties for currents activated by
`either STa or the cystic fibrosis transmembrane conductance
`regulator (CFTR) channel. STa-activated currents display a
`linear current-voltage relationship and a relative permeability
`sequence of Br > Cl > I. STa or 8-Br-cGMP-activated currents
`remain when 20M Walsh inhibitor, a blocker of protein
`kinase A (PKA),
`is added in the pipette, suggesting that
`cGMP-dependent protein kinase (PKG) activates the cur-
`rents. Intracellular addition of Rp-8-Br-cGMP,an agent that
`activates PKGII and inhibits PKGI and PKA, causes induc-
`tion of a chloride conductanceidentical to that stimulated by
`STa. We conclude that STa activates CFTR by phosphoryla-
`tion with cGMP-dependentprotein kinase.
`Cooeecccscccoccce
`
`Introduction
`
`toxin (STa) produced by
`Heat-stable
`Escherichia coli is responsible for traveller’s
`diarrhea and is a major cause of death in
`youngchildren in developing countries. STa
`and the endogenousintestinal peptide guany-
`lin [1] bind to a luminal intestinal receptor
`with a guanylate cyclase activity [1] causing
`
`formation of cGMP [2]. Elevation ofintracel-
`lular cGMP accompanying the occupation of
`the STa receptor coincides with increased
`fluid secretion, suggesting the two are coupled
`(3, 4]. Our goal was to determine how STa
`increased cGMP levels to stimulate intestinal
`chloride channels,
`that result
`in increased
`chloride secretion.
`
`Accepted:
`October 7, 1993
`
`Dr. Sandra Guggina
`Division of Gastroenterology, Department of Medicine
`Johns Hopkins University
`929 Ross Building, 720 Rutland Avenue
`Baltimore, MD 21205 (USA)
`
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`T84 cells are convenient for the study of
`STa-mediated chloride secretion, because
`they express an apical STa receptor and dis-
`play net transepithelial chloride secretion [5].
`Transepithelial chloride transport can be
`measured by the short-circuit current
`that
`is caused by transepithelial
`ion movement,
`which in T84 cells is carried by chloride. In
`addition, the patch clamp technique can be
`used to measure the magnitude and properties
`of whole-cell chloride currents activated by
`STain individual cells.
`T84 cells have a 10 pS chloride channel [6,
`7] exhibiting ion selectivity (Br > Cl > 1 > F)
`that is identical to that of cystic fibrosis mem-
`brane conductance regulator (CFTR)[8-10].
`Since T84 cells express mRNA coding for
`CFTR,
`these channels are likely identical
`[11]. Addition of the catalytic subunit of pro-
`tein kinase A (PKA)[7] activates the 10 pS
`chloride channel, in excised patches of T84
`cells. Further, cGMP and ATP added to a
`bath including 10pAf Walsh inhibitor (a
`cAMP-dependent protein kinase inhibitor),
`also cause chloride channel activation in ex-
`cised patches [7]. Since these experiments
`were performed in the absence of added ki-
`nase, this suggests that a particulate cGMP-
`dependentprotein kinase endogenousto the
`excised patch (PKGII) phosphorylates the
`channel, causing it
`to open. Particulate
`PKGII is expressed in intestinal tissues [12]
`whereas soluble PKGI is expressed in the
`cytosolofothertissues,i.e. the lung,heart, liv-
`er andplatelets [13].
`Demonstrating the role played by particu-
`late PKGII in STa-mediated secretion should
`increase our understanding of how cGMP
`stimulates chloride secretion in intestinal
`cells. Thus one purpose of our study was to
`determine whether cGMP participates in a
`signal transduction pathway leading to chlo-
`ride current activation in STa-stimulated
`cells. A second purpose was to determine
`
`whether more than onesignal transduction
`pathway activates the CFTR chloride chan-
`nel.
`
`Methods
`
`Cell Culture
`T84 cells obtained from Dr. Doug Jefferies (Tutt’s
`University, Boston, Mass.. USA) were grown in Dul-
`becco’s modified Eagle medium containing 25 mA/
`NaHCO;(Gibco, Gaithersburg. Md., USA) supple-
`mented with 10% fetal bovine serum (Hyclone, Logan,
`Utah. USA), 50 unitsofpenicillin and 50 g/mlstrep-
`tomycin. Cultures were maintained at 37°C in an
`atmosphere of 5% CQ. and 95% air. For single-chan-
`nel recording, celis from passages 28-42 were seeded
`onto small squares of Thomas microcover glass
`(Swedesboro, N.J., USA) and used 3-6 days fromseed-
`ing. For whole-cell recordings, cells trom passages 28-
`42 were grown in T25 flasks for 5-10 daysafter seeding.
`Cells were trypsinized with 0.025% trypsin in Ca?*,
`Me?*-free Hanks’ solution for less than 15 min, dis-
`persed bytrituration 3-4 times, then allowedto settle
`onto the glass bottom experimental chambertor !0-
`15 min. This protocol consistently yielded STa-stimu-
`lated whole-cell currents.
`
`Channel Recordings
`Fabrication and use ofpipettes for single-channel
`recording were performedas previously described (7).
`Whole-ce!l recordings were performed using pipettes
`fabricated trom 1.2 mmdiameterglass capillary tubes.
`The pipettes were pulled twice on a Kopf (Tujunga,
`Calif., USA) puller and fire polished on a microforge
`(Narishige MF 83). Anisolated cell was touched from
`above, gentle suction was applied to torm a tight seal
`andfinally, abrupt strong suction broke a connection
`between the pipette and the cell interior. In expceri-
`ments involving anion substitutions, a 150 mAs KCI
`agar bridge was connected betweenthe bath andsilver
`pellet at ground. Ail recordings were done at 25°C.
`
`Data Acquisition and Analysis
`Whole-cell currents were amplified on an EPC-7
`patch clamp amplifier (List Electronics, Darmstadt,
`FRG) without capacitance compensation, visualized
`on a Nicclet digital oscilloscope (Nicolet Instruments,
`Madison, Wisc., USA) and stored on a VCR tape
`through aSony PCM-601! digital audio processorsetat
`44 kHz. The whole-cell currents were stimulated using
`voltages generated and currents measured on ‘P clamp’
`software version 5.1 (Axon Instruments, Foster City,
`
`24
`
`Lin/MacLeod/Guggino
`
`Heat-Stable E. coli Toxin Activates
`Cl Current
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`Calif., USA) on an AST IBM-PC compatible 386 com-
`puter. The membranepotential was held at 0 mV, then
`depolarized or hyperpolarized for I's in 20 mV steps
`(between -100 and +100 mV) with a 5 second pause
`between each pulse control.
`
`Solutions
`For whole-cell recordings the bath solutions con-
`tained (in mAZ): 115 NaCl, 40 N-methyl-D-glucamine
`glutamate, 5 K glutamate. 2 MgCl.
`| CaCl, Hepes.
`pH 7.2, with NaOH. The pipette solutions contained
`(in mV): 75 N-methyl-D-glucamine Cl, 40 CsCl, 25 N-
`methyl-D-glucamine glutamate,
`| EGTA, 0.1 CaCh.,
`2 MgCl. 5 Hepes, 2 ATP, 0.5 GTP,pH 7.2, with gluta-
`mate. Measurements indicated about 100n‘/ free
`Ca?* using fura-2: Cs was addedto the pipette solution
`in block potassium channels. In addition, potassium
`was removed and replaced by a more impermeant cat-
`ion in an effort to make chloride currents predominate.
`For anion substition experiments NaC]in the bath was
`replaced with Nal, NaBr or NaF.Therelative perme-
`ability ratio (Px/PCl) was calculated using the Gold-
`man-Hodgkin-Katz equation [14]. for different bath
`solutions.
`
`cGMP Accumulation
`cGMP accumulation was measured between days|
`and 7, after trypsinization and at 22 or 37°C to deter-
`minetheeffect of cell confluence and handling proce-
`dures on the STaactivity. T84 cells seeded in 35-mm
`dishes, 3 per determination, were grown according to
`procedures described above and held at 37°C until
`just before STa in maximal doses of 1-2 pg/ml was
`added. cGMP accumulation was measured at 37 or
`22°C,as indicated, in the presence of Hanks’ medium
`containing | mf isomethylbutylxanthine. At the end
`of an incubation, buffer was removed and | ml of
`0.1 N HCI was added for 30 min at room temperature
`to lyse the cells. CGMP was measured with !?1-labeled
`cGMP using an Amerlcx-M magnetic separation kit
`distributed by Amersham Corp. (Arlington Heights.
`Ill, USA).
`
`Materials
`STa was obtained from Dr. Donald C. Robertson,
`University of Kansas. In some experiments, STa was
`purchased from Sigma Chemical Co. (St. Louis. Mo..,
`USA). This STa was about 4-fold less potent than that
`provided by Dr. Robertson. H8, N-[2-(methylamino}
`ethyl]-5-isoquinolinesulfonamide, was obtained from
`Sigma Chemical Co. Rp-8-Br-cGMP (a diastercomer
`of 8-Br-cGMP phosphorothioate) and Rp-8-Br-cAMP
`were obtained from Dr. Hugo de Jonge, Erasmus Uni-
`
`versity, Rotterdam. The Netherlands. The Walsh in-
`hibitor was kindly provided by Dr. Richard Huganir,
`Johns Hopkins University.
`
`Results
`
`STa-induced cGMP accumulation was
`measured in cells with or without trypsiniza-
`tion (fig. 1a), at 22 or 37°C (fig. 1b) or after
`increasing days in culture (fig. Ic) in order to
`determine whether cGMP levels werealtered
`under these conditions. Trypsinization did
`not affect cGMP accumulation, as depicted in
`figure la. In contrast, lowering temperature of
`the assay from 37 to 22°C dramatically de-
`creased basal cGMP levels (no STa), from 21
`+ 2.4to 1 + 0.2 pmol/mgprotein, respective-
`ly, and also decreased STa-mediated increase
`in cGMP (fig. 1b). A most important factor in
`cGMP accumulation was found to be the
`numberof days ofcell culture. At 7 days after
`seeding, the capacity to generate cGMP in-
`creased 5-fold at 60min and 10-fold at
`90 min (fig. 1c). We found that maximal lev-
`els of cGMP occurred in cells that were cul-
`tured for at least 7 days, a time which coin-
`cides with confluence. Although trypsiniza-
`tion, used to release cells for patch clamp
`experiments, was less successful in terms of
`cell viability for confluent versus preconfluent
`cells, the older cells were used becauseoftheir
`favorable cGMP accumulation.
`Short-circuit currents are activated by STa,
`forskolin and in some experiments by high
`doses of cGMP [15]. Therefore. in order to
`better understand how STaactivates cellular
`chloride currents, we measured whole-cell
`currentsstimulated by these agents. Using the
`whole-cell patch clamp technique in the ab-
`sence of chemical stimuli (before STa), the
`current at 100 mV was very small (14.6 + 1.8
`pA; n = 9). Extracellular bath addition of STa
`(1 g/ml) activated a linear whole-cell current
`
`25
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`

`Trypsin
`--& Control
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`Day 3
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`
`Fig. 1. a Effect oftrypsinization on cGMP accumu-
`lation. Cells were trypsinized or not trypsinized (con-
`trol), then cGMP accumulation was measured on the
`two populationsofcells at 37°C. Cells were used on
`day 7 after seeding. Data were collected from 3 dishes
`tor cach time point and experiments repeated on 3 dif-
`ferent culture passes. Data are shown as means + SE
`(n = 3). b Effect of temperature on CGMP accumula-
`tion. Assays at 22 and 37°C were comparedfor cells
`used 7 days after seceding. cGMP accumulation at
`37°C is significantly greater than that at 22°C at
`90 min (p < 0.1 using Student's t test). e CGMP accu-
`mulation of cells cultured for increasing numbers of
`days. cGMP accumulationinereases after culturing for
`7 days. This coincides with confluence. cGMP accu-
`mulation on day7 is significantly different from that of
`day 2 (p < 0.02 using Student'sttest).
`
`accumulation,
` cGMP
`
`Time, min
`
`(fig. 2a) resulting in a linear current-voltage
`relationship (fig. 2b), similar to that activated
`by 10 y'/ forskolin or 500 yA/ 8-Br-cGMP,as
`shownin figure 2a. As the STa-mediated cur-
`rent
`increased,
`the reversal potential ap-
`proached 0 mV(the chloride equilibrium po-
`tential), because chloride concentrations were
`equalin thecell and bath (fig. 2b). When the
`bath solution was replaced with Nal in the
`presence of STa, the iodide current was less
`than the chloride current, and the reversal
`
`potential was more positive (fig. 2b). As
`shown in table |
`the reversal potentials for
`different ion replacements had a sequenceof
`Br<Cl<I. Therefore the relative ion perme-
`abilities (Px/PC1) have a sequence of PBr/PCI
`> PCV/PCI> PI/PC1.
`A representative example of the times
`required to reach peak conductance (at
`100 mV)
`is shown in figure 2c. Figure 2d
`showsthe average peak conductancefor each
`treatment. The 10forskolin-activated
`
`26
`
`Lin/MacLeod/Guggino
`
`Heat-Stable E. coli Toxin Activates
`Ci Current
`
`MSN Exhibit 1039 - Page 4 of 10
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`

`

`8,000
`
`
`0
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`0
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`potential reversed toslightly positive since the channel
`is less permeable to |
`than Cl. When chloride was
`returnedto the bath, the reversal potential reversed to
`the equilibrium potential for Cl (not shown). b Cur-
`rent-voltage relationships in chloride or iodide me-
`dium. STa with Cl in the bath or STa-treated cells with
`| replacing Cl in the bath all yiclded linear currents.
`Vm = Membranepotential. ¢ Activation of whole-cell
`conductance with time. Cells were stimulated with
`forskolin 10 wM, STa (1 pg/ml: Sigma) or 500 nM 8-
`Br-cGMP. Three individualcells trypsinized from the
`same culture are shown. d Average peak whole-cell
`conductances from several experiments. Forskolin
`(10 wf), low- and high-potency STa (1 g/ml) and low
`(100 or 200 pA/) 8-Br-cGMP or high (500pA/ to
`{ mM) 8-Br-cGMP each caused increased chloride
`conductance.Each barrepresents the data from 9 indi-
`vidualcells trom different days and cultures. The lower
`baron the left ofeach bar represents the data from con-
`trols before adding any agents. Cell capacitances were
`uniformly about 20 pF.
`
`27
`
`
`
`MSN Exhibit 1039 - Page 5 of 10 .
`MSNv. Bausch - IPR2023-00016
`
`a Forskolin
`a STa
`& cGMP
`
`wa
`a 6,000
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`
`5 4,000
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`ooO
`= 2,000
`©
`
`100 mV;
`-100 mvE
`
`Control—_—
`
`STa
`
`
`
`200 ms
`
`Control
`—e 57a with NaC!
`—a— STa with Nal
`
`“1, pA
`
`-100
`
`100
`
`/
`-100-
`
`-300 +
`
`300 =
`
`
`100
`Vinv AV
`
`Fig. 2. a Activation of whole-cell chloride currents
`with STa. cGMP orforskolin. The bath surrounding
`the cells was exchanged with the same type of bath
`solution containing either | pg/ml STa. 10 pA¥ forsko-
`lin or 500 pAf 8-Br-cGMP. Undercontrol conditions
`the reversal potential was negative, when STa was add-
`ed the reversal potential approached zero as predicted
`for a chloride current. when Nal bath was added the
`
`

`

`Table 1. Composition ofbath
`RP +SE,mV
`Br
`Ir
`NMDG Clr
`K*
`=Na*
`Bath
`in ion substitution experimentsin
`
`the presence of STa——smMNaNIan90—010000
`
`
`NaCl 1156§ 40 121 -6.341.1 (n=9)
`
`
`Nal
`115
`6§
`40
`6
`115
`+12.80.5 (n = 5)
`NaBr
`115)
`6§
`40
`6
`-17.841.8(n=5)
`
`115
`
`NMDG = N-methyl-D-glucamine. For ion substitution experiments
`bath chloride was substituted with anions as above (in mA/) . Minor com-
`ponents, pH and pipette solution are as in Materials and Methods. Under
`control conditions in the presence of 121 mf chloride in the bath butin
`the absenceof STa the reversal potential (RP) was -99.1 + 2.8 mV (n = 9).
`In most experiments reversibility was checked by perfusion of chloride
`back onto the cells in which case nearly the same chloride reversal potenti-
`al was obtained, suggesting that the alterations in reversal potential were
`duc to changes in ion composition.
`
`current had a peak of 3,180 + 670 pS (n = 9)
`at 100 mV. With low-dose 8-Br-cGMP (50-
`100 1.4) the peak currents was 1,080 + 150
`pS (n = 9), and with high-dose (500 pA7 to
`| mA4) 8-Br-cGMP the current was 2,540 +
`560 pS (n = 9). The 1-2 ug/ml maximal dose
`STa-stimulated peak current was 2,760 + 560
`pS (n = 9). These results indicate that STa or
`8-Br-cGMP stimulates a peak chloride cur-
`rent which is somewhat smaller than that acti-
`vated by forskolin.
`Activation of CFTR-mediated chloride
`currents is known to occur via cAMP-depen-
`dent protein kinase phosphorylation. We
`wished to determine whether the STa-in-
`duced conductance is activated by PKG or
`PKA in the presenceof high concentrations of
`cGMP. To addressthis question, we added to
`the pipette 20 Af Walsh inhibitor which
`blocks PKA-activated currents, while allow-
`ing activation of PKG. After a 10 min prein-
`cubation period, further addition of 10 pA
`forskolin produced a short current pulse,
`which decayed within 5 min to the control
`(preforskolin) current level. The average peak
`forskolin conductance was 260 + 40 pS (n =
`6), which wasnotsignificantly different from
`
`control 170 + 20 pS (n = 6) in the absence of
`any agents. The magnitudeofthis current was
`8%ofthe peak forskolin currentelicited in the
`absence of Walsh inhibitor. With the subse-
`quent addition of STa (1 g/ml) or 500 Ad 8-
`Br-cGMP to the bath, the current gradually
`increased for 20 min until a peak conductance
`of 620
`120 pS (n = 3) or 690 + 160 pS(n=
`3), respectively, was achieved. Shown in fig-
`ure 3a, a typical experiment, after a 10-min
`preincubation with Walsh inhibitor in the pi-
`pette, addition of 10 pAforskolin produced a
`300 pS conductance increase that decayed
`within 5 min to control
`level conductance,
`present before addition of forskolin. With
`subsequent addition of I yg/ml STa to the
`bath, the conductance gradually increased un-
`til a peak conductance of 1,000 pS was
`reached.
`In order to asses which kinase causes phos-
`phorylation of the channel, we used two types
`of kinase inhibitors. In the pipette, 50 .A/ Rp-
`8-Br-cGMP, an analogue which activates
`PKGII but inhibits PKA and PKGI, caused a
`linear conductance with a peak amplitude of
`870 + 140 pS (n= 3;fig. 3b). This current was
`moderately enhanced by the addition of STa
`
`28
`
`Lin/MacLeod/Guggino
`
`Heat-Stable £. cofi Toxin Activates
`Ci Current
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`1,200
`
`°SSs
`
`800
`
`Forskol
`‘OF:
`
`nv STa
`
`00
`
`0
`
`10
`
`20
`Time, min
`
`30
`
`40
`
`0
`
`10
`
`20
`
`30
`Time, min
`
`40
`
`50
`
`60
`
`4a
`
`2,000
`
`wa
`
` 3a
`
`600 4
`
`Cellconductance,pS
`
`Cellconductance,pS
`oO @
`
`Cellconductance,pS
`
`1,000
`
`
`sia
`Forskolin
`STa Y
`
`
`
`40
`
`50
`
`60
`
`®e&8
`
`23 =
`
`3
`
`2,000
`
`DU
`
`QoQoQo
`
`Fig. 3. a Whole-cell conductance measured in the
`presence of the Walsh inhibitor. Cells preincubated
`with 20 uf Walsh inhibitor, which blocks phosphory-
`lation by PKAin the pipette, show a small activation,
`then inactivation of currents when 10 pM forskolin is
`added. A subsequent addition of | pg/ml STa caus-
`es a gradual increase in whole-cell current. b Whole-
`cell conductance activated by Rp-8-Br-cGMP. Addi-
`tion of 50 1.4 Rp-8-Br-cGMP to thepipette caused the
`conductanceto increase over 50 min. then addition of
`1 ug/ml STa caused a further increase in conductance.
`
`Forskolin did not increase currents in the presence of
`Rp-8-Br-cGMP. Rp-8-Br-cGMP has a Ky, for PKGII
`of 3 Af [de Jonge, pers. commun.] and a K; for PKA
`and PKGI of 4 and 8 ‘7.respectively.
`Fig. 4a, b. Conductanceactivated in the presence
`of kinase blockers. Currents were not activated by
`pipette addition of Rp-8-Br-cAMP orafter a 10-min
`preincubation with 3 pf H8. Rp-8-Br-cAMP has a K;
`of 20. 8 and 7M for PKGI, PKAII and PKGII,
`respectively. H8 is known to block PKA, PKC and
`PKGwitha K, of 1.2, 15 and 0.48 1.7,respectively.
`
`whereas later addition of forskolin did not
`increase currents further. Ion replacement ex-
`periments showed that the ion selectivity for
`this current was Br > Cl > I > F (data not
`shown) which is the same as the sequence for
`CFTR. Pipette addition of 50-80 pM Rp-8-
`Br-cAMP (fig. 4a), an analogue of cAMP
`which inhibits phosphorylation by PKA,
`
`PKGI and PKGII, gave no current response
`for STa, 8-Br-cGMP or forskolin (n = 3),
`although a rectifying chloride conductance
`could be elicited (n = 2) by ionomycin (data
`not shown). In other experiments on cells
`incubated for 10-15 min in the presence of
`3 uM bath H8,an inhibitor of PKA, PKG and
`PKC,there was no current whether STa,fors-
`
`29
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`

`kolin or ionomycin were applied to the bath
`(fig. 4b), but when H8 was washed fromthe
`cells, a linear current was activated by STa
`(smallest) or forskolin (larger), or a rectifying
`current (largest) was activated by ionomycin
`(data not shown) [5].
`
`Discussion
`
`STa causes chloride secretion in both in-
`tact intestine and the colonic cell line T84, but
`the molecular mechanisms underlying the ac-
`tivation of this secretory pathway are only
`partially understood.
`In both systems STa
`bindsto an apical receptor which hasintrinsic
`membrane guanylate cyclase activity [2] and
`causes an accumulation of cGMP [5, 13]. The
`chloride-secretory pathway that is activated
`following cGMP accumulation was not pre-
`viously known.
`In T84 cells the whole-cell chloride cur-
`rents generated by STa and cGMP have a
`linear current-voltage relationship and rela-
`tive ion permeabilities of PBr > PC] > PI, like
`that of forskolin-activated currents [16]. This
`suggests that two stimulating signals, cAMP
`and cGMP. may converge on the samecon-
`ductive pathway - CFTR. The major bio-
`physical characteristics, a linear current-vol-
`tage relationship and permselectivity of chlo-
`ride overiodide, are like CFTR-induced chlo-
`ride currents in Xenopus oocytes [17]. On the
`other hand, the currents generated by STa or
`cGMP are not
`like the nonlinear currents
`through the outwardly rectifying chloride
`channel, which are activated by ionomycin
`[18], an agent that increases intracellular cal-
`cium. The STa-activated currentsare also dis-
`tinguished from currentsactivated by calcium
`which havea selectivity of iodide over chlo-
`ride. Finally,
`the currents elicited by STa
`(data not shown) or forskolin [16] are not
`blocked by 4,4’-diisothiocyanatostilbene-2,2’-
`
`disulfonic acid which readily blocks the out-
`wardly rectifying chloride channel
`[6]. Al-
`though there are occasionally small, baseline
`voltage and time-dependent currents, these
`do not increase with the same magnitudeafter
`cGMP asthelinearcurrents.
`The 10 pS single-channelfluctuations gen-
`erated by STa in cell-attached patches (data
`not shown) resemble the 8.7 pS fluctuations
`activated by cAMP in T84 cells [6] or by
`CFTRin reconstituted systems[9]. The char-
`acteristics of the single-channel fluctuations
`and the whole-cell currents are consistent with
`CFTRbeingthe secretory pathway stimulated
`by STa in the T84cellline.
`The peak amplitude of the whole-cell cur-
`rents in T84 cells mimics the pattern seen for
`short-circuit currents in the intestine [3]. STa
`and 8-Br-cGMP consistently activate smaller
`whole-cell currents than forskolin (fig. 2c).
`The short-circuit current
`in T84 cells also
`repeats the pattern of smaller currents for STa
`than for forskolin. This suggests that cAMP
`may activate more chloride channels or more
`fully activate a single population of chloride
`channels.
`Whatis the signalling pathway by which
`these chloride channels are activated? There
`are at least two potential pathways to increase
`chloride currents including cAMP-dependent
`phosphorylation by PKAII or cGMP-depen-
`dent phosphorylation by PKG I or II. Our
`experiments using Rp-8-Br-cGMP, which ac-
`tivates PKGII while inhibiting PKGI and
`PKA,is consistent with PKGII being at least
`part of the activation pathway in T84 cells.
`Thedata of Forteet al. [19] suggest that PKGI
`does not exist in T84 cells making it unlikely
`that this kinase is involved in chloride-secre-
`tory eventsin thiscell type.
`Forte et al. [19] found that high levels of
`cGMP. 1,600 pmol/mg protein (640.19,
`were generated when T84 cells were incu-
`bated with a | pMsaturating dose of STa.
`
`30
`
`Lin/MacLeod/Guggino
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`Heat-Stable E. coli Toxin Activates
`Ct Current
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`

`This means enough cGMP would be gener-
`ated to activate both PKAII and PKGII since
`the K, of cGMP for PKAII is 60 pf. The
`affinities of these kinases for CGMP are 5 nAf
`(PKGII), 110 nAv (PKGI) and 60 pAf (PKA).
`The affinity for cAMP is 2yA/ (PKGII),
`39 pAf (PKGI) and 80 nAf (PKA)[de Jonge,
`pers. commun.], but we found activation of
`chloride currents with 100 nAf intracellular
`cGMP or when the Walsh inhibitor, which
`blocks PKA,wasinjected into thecell before
`STa was addedto the bath. This suggests that
`low levels of cGMP can activate chloride cur-
`rents through PKGII. STa also generates
`900 pmol cGMP/mgprotein in ileal cells and
`400 pmol cGMP/mgprotein in colonic cells
`[19]. The PKGII-mediated phosphorylation
`is probably rapid in intact tissue becauselarg-
`er amounts of PKGII are present in thesetis-
`sues comparedto the amounts in T84cells.
`Guanylin (1 yA)
`[1] produces
`about
`100 pmol cGMP/mgprotein (assuming a pro-
`tein concentration of 0.3 mg/well). Thus less
`cGMP is accumulated in the presence of gua-
`nylin than STa. This suggests that the hor-
`mone-mediated pathway produces less cGMP
`
`than the toxin-stimulated pathway but can
`still stimulate chloride secretion via PKGII.
`CFTR is phosphorylated by both PKAII
`and soluble PKGI, at the same 7 sites on the R
`domain [21]. Calcium calmodulin kinase |
`not II also phosphorylates someofthese sites.
`Bergeret al. [8] have found that CFTR opens
`in the presence of PKA but not PKGI or CAM
`kinase II, whereas de Jonge [pers. commun.]
`finds that PKGII activates the channel. Our
`previous data suggesting that PKGI opens
`CFTR-like channels in T84 cells are compat-
`ible with the model of phosphorylation caus-
`ing channel activation. The data presented
`here implicate PKGII in the activation of
`chloride currents in T84 cells and suggest that
`PKGII directly mediates STa and guanylin
`regulation of these chloride currents.
`
`Acknowledgments
`
`The authors thank Dr. Hugo de Jonge for kinase
`inhibitors and timely discussions and Elizabeth Potter
`for editorial comments. This work was supported by
`the Meyerholl Digestive Disease Center, the Cystic
`Fibrosis Foundation and NIH grant DK44648.
`
`OOO e meee Rae e OOOO DAESAAD EEO OA OEEREEEEEOEEEDD ODO OHODOE OOOO EES DOSS SOOO SEER SOOSOSOSOS ESOS SOE OO CEOS OOO OD EEODEDESOOEDOEEOE
`
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`Cl Current
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