Journal of Physiology (1995), 489.3, pp.745-754

The actin filament disrupter cytochalasin D activates therecombinant cystic fibrosis transmembrane conductance

regulator Cl- channel in mouse 3T3 fibroblasts

Horst Fischer, Beate Illek and Terry E. Machen

Department of Molecular and Cell Biology, University of California,235 Life Science Addition, Berkeley, CA 94720-3200, USA

1. Cytochalasin D (CD; 5/M) readily stimulated cystic fibrosis transmembrane conductanceregulator (CFTR) Cl- channel activity in cell-attached and whole-cell patch recordingsfrom 3T3 fibroblasts expressing recombinant CFTR but not in mock-transfected cells.CD-stimulated currents were indistinguishable from those evoked by forskolin stimulation.Kinetic analysis of CFTR gating showed identical channel behaviour independent of theagonist used.

2. To elucidate the mechanism of action of CD we tested its effects on cAMP, protein kinase A,and the CFTR itself during CD stimulation. In contrast to forskolin treatment, CD did notincrease cellular cAMP content.

3. A direct interaction of CD with the CFTR was ruled out because CD showed no effect onCFTR in excised inside-out patches.

4. CD effects were fully blocked when the cellular protein kinase A was inhibited bytreatment of cells with RpcAMPS in cell-attached patches or when protein kinase inhibitorpeptide was dialysed into cells in whole-cell experiments.

5. Addition of G-actin to excised patches had no effects on CFTR.

6. We conclude that the stimulatory effect of CD is cAMP independent, but needs a functionalprotein kinase A in order to activate the CFTR. We propose that cytochalasin D activatesCFTR by releasing a cellular inhibitor, e.g. a phosphatase, that is held in place by F-actin.

The CFTR Cl- channel (cystic fibrosis transmembraneconductance regulator) is thought to be mainly regulated bythe intracellular cAMP-protein kinase A (PKA) signallingsystem leading to a phosphorylation of the channel (Cheng,Rich, Marshall, Gregory, Welsh & Smith, 1991), which isthen gated by ATP (Venglarik, Schulz, Frizzell & Bridges,1994; Baukrowitz, Hwang, Nairn & Gadsby, 1994). Wehave previously described the activation of CFTR by theadenylate cyclase activator forskolin in the cell-attachedpatch clamp configuration and presented a detaileddescription of its gating behaviour, including voltagedependence and two distinct gating modes (Fischer &Machen, 1994).

In addition to the regulation of the CFTR byphosphorylation, activity of the CFTR-mediated Cl-currents has also been proposed to be modulated by rates ofexocytosis and endocytosis of CFTR-containing vesiclesinto the apical plasma membrane (Bradbury, Jilling, Berta,Sorscher, Bridges & Kirk, 1992). After long-term treatmentwith cytochalasin D (CD), cAMP-stimulated Cl- secretion

was blocked (Hug, Koslowsky, Ecke, Greger & Kunzelmann,1995), consistent with an interruption of the pathway ofvesicle fusion. We were interested in the potential role ofthe cytoskeleton in regulating membrane traffic, and wetested the effects of CD. It quickly became obvious thatimmediate effects of CD were to stimulate, not inhibit, theCFTR, and this aspect of the action of the drug wasinvestigated in more detail.

The cytochalasins are fungal metabolites that are able topermeate cell membranes, bind to actin and alter itspolymerization. Although cytochalasins A and B havemultiple effects, CD is thought to be very specific for theactin cytoskeleton (Cooper, 1987). Functionally, CD bindsto the fast-growing end of actin filaments, arresting actinturnover at that end, which often leads to filamentshortening.

In this report we show that CD activates the CFTR inintact cells in a way that is indistinguishable from forskolinactivation, and it depends on a functional PKA, but CDdoes not raise cAMP. Direct effects of CD or actin on the

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H. Fischer, B. Illek and T E. Machen

CFTR were experimentally ruled out. A likely explanationfor the presented data is that an actin-associated factor,e.g. a phosphatase, is dislocated by actin disruption, whichleads to an activation of the CFTR through basal PKAactivity in intact cells.

METHODSCellsThe continuous mouse fibroblast NIH 3T3 cell line stablytransfected with the wild-type CFTR or the vector only (mock-transfected) was originally generated in the laboratory of Dr R. C.Mulligan, MIT, Cambridge, MA, USA (for procedure see Andersonet aL 1991) and obtained through Dr W. W. Reenstra, Children'sHospital, Oakland, CA, USA. Cells were cultured with standardtechniques as described (Fischer & Machen, 1994). For patch clampexperiments cells were seeded at low density on glass coverslipsand were used after 1-2 days for whole-cell recordings and within1-4 days for single channel recordings. For cAMP measurements,cells were grown on polycarbonate filters (Transwell, Costar,Cambridge, MA, USA) to confluency.

Patch clampingAll measurements and manipulations were performed on thestage of a microscope in an open, constantly perfused chamber at37 'C as described before (Fischer & Machen, 1994). Briefly, forrecordings in the cell-attached and excised patch clamp mode,patch pipettes were pulled from thick-walled borosilicate glass(Corning 7052, World Precision Instruments, Sarasota, FL, USA)to yield, after filling and fire polishing, resistances of -20 MQ.After seal formation, currents (I) were continuously recorded on acomputer hard disk (sampled at 2 kHz, filtered at 500 Hz). In thispaper, current activation refers to either positive or negativeincreasing currents dependent on the applied potential. Mostrecordings in this report contained multiple channels. The samecurrent signal was, after highpass filtering (0 03 Hz to remove theDC current component) and lowpass filtering (400 Hz) and furtherx 10 amplification of the resulting noise signal, fed into a secondcomputer that performed on-line Fourier transforms of thesignal. The covered bandwidth was 0-25-400 Hz. Ten to twelveconsecutive time intervals (4 1 s) were Fourier transformed andthe resulting average spectrum was saved for analysis. Spectrawere fitted off-line with two Lorentzian components (Fischer &Machen, 1994) resulting in estimates for the corner frequencies (fl,fch) and the Lorentzian amplitudes (S01, Soh). The indices '1' and 'h'denote the low and high frequency Lorentzian, respectively.

For standard whole-cell measurements a thin-walled borosilicateglass was pulled and polished to yield resistances of 5 MQ2 afterfilling. For the experiments where the PKA inhibitory peptide(PKI) was present in the pipette solution, pipettes with resistancesof -1 MIQ were used, yielding a total access resistance ofapproximately 2-3 MQi. The membrane potential was clamped to-30 mV and pulsed every 10 s for 1 s to -20 or -25 mV as stated.Whole-cell currents are reported as total cell membrane currentswithout correction for cell capacitance, which was, owing to thespindle-like shape of the fibroblasts, not a simple exponential.

Potentials are reported according to the standard electro-physiological convention. In the cell-attached mode, potentials aregiven as the negative pipette potential, -Vp. Note that-Vp is nomeasure for the absolute potential, which is unknown in the cell-

attached mode. No corrections were applied to measured currentsor applied potentials. For display in some figures, the samplingfrequency was numerically reduced by averaging adjacent samples,or a Gaussian filter algorithm was used for further filtering of data.

Cyclic AMP measurementsCyclic AMP levels were kindly measured by Dr J. H. Widdicombe(University of California San Francisco), as part of an extendedseries of measurements of cAMP levels after treatment withdifferent agonists (Illek, Fischer, Santos, Widdicombe, Machen &Reenstra, 1995). Determination of cAMP was performed underblinded conditions with the tissues labelled with a numbered code.3T3 cells expressing the CFTR were grown to confluency and,after stimulation for given times with agonist, were placed inice-cold trichloroacetic acid. Levels of cAMP were determined by25I--radioimmunoassay and normalized to total cell proteincontent, determined with bicinchoninic acid, as described(Hartman, Kondo, Mochizuki, Verkman & Widdicombe, 1992;Illek et al. 1995).

Solutions and chemicalsThe composition of the bath solution was (mM): 141 NaCl, 4 KCl,1 KH2PO4, 1 MgCl2, 1*7 CaCl2, 10 Hepes and 25 glucose; pH 7*4.Pipette filling solution for single channel recordings was (mM): 147N-methyl-D-glucamine chloride (NMDG-Cl), 1-7 CaCl2, 10 Hepesand 25 glucose; pH 7 4, and for whole-cell recordings CaCl2 wasreplaced by 1'7 MgCl2, 0-1 EGTA and 3 MgATP. This solution wasalso used as the bath solution for excised patches. Forskolin(Calbiochem), the adenylyl cyclase activator, was made up as a100 mm stock in dimethyl sulphoxide (DMSO) and was used at1 /M. Cytochalasin D was made as a 10 mm stock in DMSO andused at 5 FM. Colchicine, the microtubule disrupter, was made upas a 10 mm stock in DMSO and used at 10 #M. The Rp-isomer ofcyclic AMP thioate (RpcAMPS; Calbiochem), the competitivesubstrate inhibitor of PKA, was made up fresh in standard NaClbath solution at 0 5 mm, and cells were incubated for 2-4 h. Thecatalytic subunit of PKA (Promega, Madison, WI, USA) wasdissolved to approximately 60 nm in NMDG-Cl bath solution. ThePKA inhibitory peptide (PKI; Promega) was made up in thepipette solution at 10 /M plus 04 mg ml-' bovine serum albumin(BSA). Actin (from porcine heart, lyophilized from a low ionicstrength buffer; Sigma) was dissolved in water to a concentrationof 4 mg ml' actin (containing (mM): 0-2 CaCl2, 2 Tris-HCl, 0-2ATP and 0 5 fl-mercaptoethanol; pH 8 0) immediately before useand added into the bath chamber to yield final actin concentrationsof 0 4 mg ml-' in NMDG-Cl solution. During actin addition thebath perfusion was stopped.

Statistical analysisData are given as original values or as mean values + S.E.M.Standard statistical testing (Student's t test, simple regressionanalysis) was employed.

RESULTSCytochalasin D activated the CFTRFigure 1 shows recordings from cell-attached patches on3T3 fibroblasts expressing the recombinant CFTR (Fig. 1A)or the vector only (Fig. 1B). The recording in Fig. 1A (andmost recordings from CFTR-transfected cells in this study)contained multiple channels per patch. Addition of CD ledto an immediate current stimulation through activation of

746 J Physiol.489.3

Cytochalasin D activates CFTR

CFTR Cl- channels. Current activation was transient, andchannels inactivated in spite of the continuous presence ofagonist. Figure 1B shows a cell-attached recording from a

mock-transfected cell as a control: CD showed no effect on

channel activity in cell-attached patches (n = 3), and alsodid not affect the Cl- conductance of cells in whole-cellrecordings (n = 3, not shown).

Figure 2 further characterizes the CD-activated, cell-attached channel in CFTR-transfected cells. Theconductance was 10 3 + 0 5 pS (n = 6) in the positive, linearvoltage range, and decreased at negative voltages, probablydue to the lower Cl- concentration inside the cell (Fig. 2A).The cell-attached reversal potential was close to zero

(-0 3 + 3X5 mV, n = 6). With Cl- as the only significantion in the pipette, negative currents in the I-Vrelationships are indicative of Cl- movement out of the cell(given that NMDG+ is not conducted).

We applied noise analysis to CD-activated currents (Fig. 2B)to describe channel gating because most recordings in thisstudy were from multichannel patches. Spectra containedtwo Lorentzian noise components, a low frequencyLorentzian with f,j = 1 1 + 0-12 Hz (n = 22), and a highfrequency Lorentzian that showed clear voltage dependence.At negative potentials fch was 86-4 + 8-9 Hz (n = 6,recordings at -Vp = -80 and -90 mV were pooled). Atpositive, depolarizing potentials fch was significantly slower(fCh=36-4+4-3Hz, n=12, P<0-01, -Vp=+80 and+90 mV pooled), and the Lorentzian amplitude was also

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reduced (Fig. 2B). The channels also exhibited the CFTR-typical high open probability (P.) mode (as we havedefined it previously, Fischer & Machen, 1994), which is a

physiological locked open state of channel pairs (arrow inFig. 2C). Since the CD-stimulated channel was present inCFTR-transfected cells only, and showed gating andconductance characteristics identical to those we haveshown for the forskolin-stimulated CFTR (Fischer & Machen,1994), the channel stimulated by CD was identified as theCFTR. Because the CFTR stimulated by both CD andforskolin (Fischer & Machen, 1994) behaved identically, wespeculated that both kinds of stimulation eventually leadto the same type of signal to activate the CFTR.

Figure 3 shows experiments in which CFTR-expressing3T3 cells were treated with combinations of CD andforskolin. In the whole-cell recording shown in Fig. 3A,CD elicited a transient current stimulation. Subsequentaddition of forskolin in the presence of CD caused thecurrent to increase again transiently. During the secondstimulation in Fig. 3A forskolin activated maximally, andaddition of CD had no further effect. Stimulations witheither CD or forskolin were generally transient andinactivated despite the continued presence of agonist.Figure 3B shows a similar stimulation protocol in the cell-attached mode. CD and further application of forskolin ledto two current transients that were similar to the whole-cellmeasurement in Fig. 3A. An absolute quantification ofcurrents was not practical owing to the transient nature of

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Figure 1. Activation of CFTR by cytochalasin D in 3T3 cellsA, CD (5 /M) activated multiple channels in a cell-attached experiment on a CFTR-expressing 3T3 cell.-Vp = 80 mV, sampled at 50 Hz. CD was added at time zero. Experiment typical of twelve comparablecell-attached runs. B, control on a mock-transfected 3T3 cell. No channel activity was detected in this cell-attached experiment after incubation with CD. Typical control experiment for three cell-attached andthree whole-cell experiments.

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748 H. Fischer, B. Illc

the responses to both agonists. Current stimulated by CDalone or in combination with forskolin was quite variable.In most cases the two agonists showed no significantadditivity (as shown in Fig. 3), but in two of five cell-attached and in two of seven whole-cell stimulationsaddition of forskolin on a CD-stimulated cell (or vice versa)more than doubled currents.

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Colchicine had no effect on CFTRFor comparison, Fig. 4 shows the effect of the microtubuledisrupter colchicine. Colchicine did not activate any CFTRs,although this patch contained a large number of channels,as shown by the subsequent addition of forskolin. Also,since colchicine did not appear to hinder forskolinactivation of the CFTR, microtubules are probably notinvolved in the regulation of CFTR.

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Figure 2. Characteristics of the cytochalasin D-activated CFTRA, current-voltage (I-V) relationship. In the cell-attached mode the I-V relationship rectified, probablybecause of an appreciable Cl- gradient. The conductance in the positive voltage range was, on average,10-3 + 0 5 pS (n = 6) and the reversal potential, -0 3 + 3-5 mV. Pipette contained NMDG-Cl solution,bath contained NaCl solution. B, current noise spectra of cell-attached recordings. - Vp = +80 mV (0)and -80 mV (0). Spectral densities (S) were fitted with a double Lorentzian of the formS = Sol/(1 + (f/fcl)2) + Soh/(1 + (f/fch)2). As described in detail for the forskolin-stimulated CFTR(Fischer & Machen, 1994), the fast Lorentzian was strongly affected by the applied voltage, i.e. theLorentzian corner frequency (eh) and its power (SOO) increased at negative voltages. Fit results for theshown typical spectra were, at +80 mV (0): fcj = 0-68 Hz, fch = 44 0 Hz, So1 = 6-7 x 10-25 A2 s,SOh = 5-2 x 10-28 A2 s, and for -80 mV (0): fj = 0-80 Hz, fch = 72-0 Hz, SO1 = 1-67 x 10-25 A2 s,SOh = 1-14 x 10-27 A2 s. On average, fc, was 1 ±i 0 12 Hz (n = 22, all potentials pooled). fch wassignificantly higher at negative potentials (86-4 + 8-9 Hz, n = 6, - Vp = -80 and -90 mV pooled)compared with fch = 36-4 + 4-3 Hz (n = 12, P< 0-01) at positive potentials (-Vp = +80 and +90 mVpooled). Fits were performed over a limited frequency range to omit increasing non-specific noise at highfrequencies. C, CD activation induced the low PO mode and the high P0 mode. The two gating modes arecharacterized by independently gating channels with a maximal P.0 0 5 (low P. mode), and by co-operative, locked open channels with P0 t 1 (high PO mode). Note the prolonged opening of a pair ofchannels at the arrow where these channels switch from the low P. mode to the high P. mode. Otherchannels remained in the low P0 mode. The spectra shown in B describe the low PO gating mode onlybecause channels in the high PO mode are beyond the analysed frequency range. CD was added 15 s beforethe start of the trace. -Vp, = 90 mV, sampled at 200 Hz. Typical of three other observations. Note thatthe high P0 mode can be identified only in patches with few enough channels that allow visual distinctionof gating.

J Physiol.489.3 Cytochalasin D activates CFTR

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Figure 3. Activation of CFTR by cytochalasin D and forskolin in 3T3 fibroblast

A, whole-cell recording. Drugs were applied as indicated by the bars. Membrane potential is clamped to-30 mV with superimposed +5 mV pulses. Pipette contained NMDG-Cl solution and 3 mm MgATP, bathcontained NaCl solution. B, cell-attached recording. -V = -80 mV.

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Figure 4. Effects of colchicineThe microtubule disrupter colchicine (10 sM) showed no effect in cell-attached recordings on a CFTR-transfected cell, while forskolin (1 /M) stimulated readily. - Vp = 90 mV, sampled at 20 Hz. Typical of

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Figure 5. Cytochalasin D did not affect cAMP levelsTime course of total cellular cAMP in forskolin-treated (1 /tM, *)or CD-treated (5 /tM, 0) CFTR-transfected 3T3 cells for the showntimes. Mean values + S.E.M., the number of measured cultures foreach point is given.

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On the mechanism of CFTR activation bycytochalasin DStimulation of the CFTR is mediated through thecAMP-PKA pathway, while the only known effect of CD isthe depolymerization of the actin cytoskeleton. We

ACell attached

therefore investigated possible interaction between thecytoskeleton and cAMP-PKA by testing the effects of CDon intracellular cAMP levels, on PKA, and on the isolatedCFTR. Since CD application probably increases the pool ofG-actin in a cell, we also tested the effects of G-actin on

isolated channels.

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Figure 6. Cytochalasin D had no effect on the isolated CFTRA and B show two patch experiments from two different cells. A, first trace: cell-attached recording froma CFTR-expressing 3T3 cell was silent, -Vp = -75 mV; second trace: excision of the patch (arrow) intoNMDG-Cl solution with 1 mm MgATP; third trace: addition of the catalytic subunit of PKA (-60 nM)activated multiple channels (n > 5) in this patch; fourth trace: wash-out of PKA (while keeping MgATPpresent) inactivated CFTR; fifth trace: addition of CD (5 /M) did not further activate the isolated, excisedCFTR; recording typical of two other experiments. Each trace shows a 10 s interval from one continuousrecording, times between shown traces are (top to bottom): 35, 250, 150 and 105 s; membrane potential(Vm) = -75 mV, filtered at 200 Hz, line on the right of each current trace shows all-closed state. B, firsttrace: a patch excised from an unstimulated CFTR-expressing 3T3 cell shows little activity; second trace:addition of CD did not significantly affect channel activity (CD was present for 125 s without affecting theactivity of CFTR); third trace: addition of PKA stimulated multiple channels (n > 8) after CD treatment.Recording typical of four other experiments. Traces were taken from one continuous recording. Timesbetween traces are 60 and 110 s. Conditions as in A.

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-4Figure 7. Effects of G-actin addition on CFTRFirst trace: current recording from an inside-out patch excisedfrom a resting CFTR-expressing 3T3 cell into NMDG-Clsolution containing 1 mM ATP, in which CFTR activity isvery low. Second trace: recorded immediately after G-actinaddition to the bath, final concentration of G-actin is-0 4 mg ml-', in the presence of 1 mm ATP. Flow of solutionwas halted during actin addition. Third trace: recorded after170 s in presence of actin. No significant change in channelactivity was observed during presence of actin for 235 s in thisexperiment. Fourth trace: subsequent perfusion of the bathwith the catalytic subunit ofPKA in presence of 1 mm ATPreadily stimulated channel activity. This patch probablycontained exactly one channel. Recording typical of five otherexperiments. Traces were taken from one continuousrecording. Times between traces are (top to bottom): 45, 115and 505 s. Conditions as in Fig. 6A.

Figure 5 shows the effects of CD and forskolin treatment oncellular cAMP levels. Treatment with forskolin (@) but notCD (0) caused a time-dependent increase of cellular cAMP.Thus, CD-dependent stimulation of CFTR did not requireincreases of cellular cAMP concentrations.

Next, we tested the effect of CD directly on the CFTRchannel. Figure 6A shows an experiment in which a silentcell-attached patch on a CFTR-expressing 3T3 cell (firsttrace) was excised into ATP-containing solution (secondtrace). ATP alone did not activate the CFTR (as shownbefore, Anderson & Welsh, 1992). Subsequent addition ofthe catalytic subunit of PKA activated channels in thisexcised patch (third trace), and channels inactivated tovery low levels after wash-out of PKA (fourth trace).Addition of CD to this patch in the continued presence ofATP did not stimulate CFTR activity (fifth trace),indicating that CD had no direct effects on CFTR (or onpossible regulatory factors in its immediate environment).Addition of CD can be expected to depolymerize patch-associated F-actin to G-actin. Since the CFTRs containedin the patch remained inactive after CD treatment, itappeared that actin depolymerization alone did not activate

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CFTR. Figure 6B shows the reversed protocol. The excisedpatch shows some low level activity in this patch in thepresence of 1 mm ATP (first trace). Addition of CD did notsignificantly affect CFTR activity (second trace) butprobably depolymerized any patch-associated actin. Furtheraddition of PKA activated a large number (n > 8) ofchannels in this patch (third trace). CD again showed no

effect directly on the CFTR, indicating that CD had no

direct effects on CFTR and that CD treatment did notdeprive the patch of any possible unknown cofactorsnecessary for stimulation with PKA.

Possible effects of G-actin were further tested by directaddition of G-actin to the excised patches. Figure 7 showsan excised patch from a CFTR-expressing 3T3 cell. Theunstimulated patch showed very little activity in thepresence of 1 mm ATP (first trace). Addition of G-actin tothis patch did not alter channel activity. The second traceshows channel activity immediately after G-actin addition,and the third trace after 170 s in the presence of actin.Since G-actin should polymerize in the high ionic strengthbath solution (Cantiello, Stow, Prat & Ausiello, 1991), neitherG-actin nor, presumably, short actin filaments altered

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Figure 8. Cell-attached recording from a RpcAMPS-treated CFTR-expressing cellBlock of PKA with RpcAMPS (0 5 mM) led to a total block of both CD (5/M) and forskolin (1 SM)stimulation of CFTR. Cell was incubated for 2 h in 0 5 mm RpcAMPS. - Vp = -90 mV. Recordingtypical of three other experiments.

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CFTR activity in this patch. Actin did not stimulate theexcised, inactive CFTR in five similar experiments, nor didaddition of actin affect the activity of stimulated CFTRs(n = 3, not shown). Therefore, it appeared that neitheractin depolymerization nor G-actin itself (both of whichwere expected to occur during CD treatment of an intactcell) had any influence on the activity of CFTR in excisedpatches and could not explain the effects of CD in intactcell preparations.

Then we tested whether CD was able to activate CFTRwhen PKA had been blocked. We used RpcAMPS, thecompetetive inhibitor of the endogenous substrate cAMPin activating PKA, and PKI, a peptide that specificallybinds and inhibits the catalytic subunit of PKA. Theseexperiments tested the hypothesis that the cAMP-PKApathway was independent of the CD pathway in activatingthe channel. Figure 8 shows a cell-attached current tracefrom a CFTR-expressing 3T3 cell that had been pre-incubated with RpcAMPS. Under these conditions activationof the CFTR by CD was totally blocked. Also, the furtheraddition of forskolin did not activate CFTR.

Since the experiment shown in Fig. 8 had no internalcontrol testing for the presence of CFTR in the patch, weperformed this series of experiments on RpcAMPS-treatedor untreated cells in an alternating fashion. In four of fouruntreated cells (i.e. controls) CFTR was activated by bothCD and forskolin (one of which is shown in Fig. 3B). Fourof five RpcAMPS-treated cells were fully inactivated bythis treatment, as shown in Fig. 8. In one RpcAMPS-treated cell, however, CFTR was activated normally byboth CD and forskolin. Since forskolin activation in any

Figure 9. Protein kinase A inhibitor blocks CFTRstimulation by cytochalasin DA, whole-cell recording from a CFTR-expressing 3T3 cellwith 10 #M PKI and 0 I mg ml-' BSA present in theNMDG-Cl pipette solution. After establishment ofwhole-cell conditions, solutions were allowed toequilibrate for 5 min before stimulation. CD stimulationwas fully blocked by PKI. Vm = -30 mV, pulsed to-20 mV. Recording typical of two other experiments.B, control experiment. Pipette contained 0 I mg ml-'BSA in NMDG-Cl solution, and the same equilibrationtime was allowed. All conditions as in A. Recordingtypical of one other experiment.

case relies on PKA, in this experiment RpcAMPS wasprobably ineffective in blocking PKA owing to its poorability to permeate membranes.

Figure 9 shows the effect of PKI in whole-cell experimentsin a CFTR-expressing 3T3 cell. When PKI was includedin the pipette solution, CD did not stimulate currents(Fig. 9A). For the PKI experiments very low resistancepipettes (< 1 MQ) were used and, after break-in into thewhole-cell mode, solutions were allowed to equilibrate for3-5 min. A control experiment for this procedure (Fig. 9B)shows that the CD stimulation is maintained under thoseconditions. Therefore, inactivation of PKA by eitherRpcAMPS or PKI blocked the ability of CD to stimulateCFTR-dependent currents, indicating that the action of CDon CFTR stimulation involved a step that was dependenton a functional PKA.

DISCUSSIONIn this report we have shown that the actin filamentdisrupter CD activated the CFTR Cl- channel in intactcells. The likely target of CD, actin, appears not to be adirect modifier of CFTR activity because G-actin added toexcised patches had no effect on the activity of CFTR.Although actin and its state of polymerization have beenshown to regulate several other channels (e.g. Cantiello etal. 1991; Rosenmund & Westbrook, 1993), we have shownhere that the CFTR Cl- channel appeared to be independentof actin (also described by Hug et al. 1995).

Addition of CD to excised patches also produced no effectson CFTR activity, ruling out any direct action of CD on the

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J Physiol. 489.3 Cytochalasin D activates CFTR 753

CFTR as a possible direct channel opener or on putativepatch-associated factors necessary for activation. CD showedits stimulatory action on CFTR only in intact cells (in thecell-attached and the whole-cell mode), indicating that theactivator (i.e. PKA) needed for CD stimulation of CFTR islost after patch excision.

The cAMP-PKA system has been described as the majoractivator of the CFTR (Cheng et al. 1991), and it appearsthat multiple phosphatases can inactivate or regulate thechannel (Hwang, Horie & Gadsby, 1993; Berger, Travis &Welsh, 1993; Fischer, Illek & Machen, 1995). We haveshown here that a functional PKA is necessary to mediatethe effects of CD on the CFTR, because blocking PKA alsoblocked the effects of CD (Figs 8 and 9). Interestingly, CDdid not significantly affect cAMP levels of the cell,indicating that during CD activation even a low, baselinelevel of cAMP and PKA is adequate for CFTR stimulation.Given that in an unstimulated cell the basal PKA activityis well balanced by the activity of phosphatases (therebykeeping the CFTR inactive), block of phosphatase(s)regulating the CFTR will lead to the activation of theCFTR by offsetting the balance of PKA and phosphataseactivity. In fact, blocking phosphatases activates the CFTRin intact, resting cells (Becq et al. 1994; Fischer et al. 1995).To our knowledge there are no reports that CD mightaffect cellular phosphatases, although this would be thesimplest explanation of our data. A more likely scenario isthat CD, by attacking actin, dislocates an actin-associatedphosphatase that is held in place in the close vicinity of thechannel. This idea is consistent with all results presented inthis report and discussed above and provides a linkbetween actin dissociation and activation of the CFTR byPKA. It is also consistent with the rapid onset of currentactivation (within 30-60 s) after CD application, indicatingthat only actin closely associated with the membrane isinvolved.

A direct, regulatory interaction between actin and theCFTR (without the need for activating PKA) has beenproposed for CFTR-transfected mouse mammary adeno-carcinoma cells (Prat, Xiao, Ausiello & Cantiello, 1995),similar to what has been shown by the same investigatorsfor interactions between the actin cytoskeleton and theepithelial Na+ channel (Cantiello et al. 1991). In the presentstudy, direct application of actin neither activated silentCFTRs nor interfered with active CFTRs in excisedpatches. Similarly, depolymerization of filamentous actin inexcised patches by CD had no effect on the CFTR, nor didit hinder subsequent stimulation with PKA. Therefore,actin does not appear to have a direct role during activationof CFTR in 3T3 cells. Similarly, Hug et al. (1995) recentlynoted that addition of G-actin to patches excised from abronchial epithelial cell line did not activate silent patches.

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754 H. Fischer, B. Illek and T E. Machen J Physiol. 489.3

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AcknowledgementsWe are extremely grateful to Dr J. H. Widdicombe, University ofCalifornia San Francisco, for performing the cAMP measurements.We thank Drs K. Pagh Roehl and B. Burnside, Berkeley, forhelpful discussions. This study was supported by the CysticFibrosis Foundation (grant F633 to H. F. and grant Z441 toT.E.M.), the Deutsche Forschungsgemeinschaft (grant 11-28 toB.I.), and the Cystic Fibrosis Research Inc. (grant 7461 to T.E.M.).

Received 15 November 1994; accepted 31 July 1995.