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Physiology

Convergent and parallel activation of low-conductance potassium channels by calcium and cAMPE-dependent protein kinase

(livercells/patchclamp/signaltransduction)

STEVEN D. LIDOFSKY

Departmentof Medicine and the LiverCenter,UniversityofCalifornia,SanFrancisco,CA94143 CommunicatedbyRudiSchmid, UniversityofCalifornia, SanFrancisco, CA,April 21, 1995 ABSTRACT K+ channels, which have been linked to

regulation ofelectrogenic solute transport as well as Ca2+

influx, represent a locus in

hepatocytes

for the concerted actions of hormones that employ Ca2+ and cAMP as intra- cellular messengers. Despiteconsiderable study, the single- channel basis forsynergistic effects ofCa2+ and cAMPon hepatocellular K+ conductance is not well understood. To addressthisquestion, patch-clamprecordingtechniqueswere applied to a model liver cell line, HTC hepatoma cells.

Increasing thecytosolicCa2+concentration

([Ca2+]J)

inHTC cells, either by activation ofpurinergicreceptorswith ATPor by inhibition of intracellularCa2+ sequestration with thap- sigargin, activated low-conductance (9-pS) K+ channels.

Studieswith excised membranepatches suggestedthatthese channels were

directly

activatedby Ca2+. Exposure of HTC cells to a permeant cAMP analog,

8-(4-chlorophenylthio)-

cAMP, alsoactivated 9-pS K+ channels butdidnot change

[Ca2+]1.

Inexcised membranepatches,cAMP-dependentpro- tein kinase (thedownstream effector of

cAMP)

activated K+

channelswith conductance andselectivity identicaltothoseof channels activated by Ca2 . In addition, cAMP-dependent protein kinase activated a distinct K+ channel type (5

pS).

These data represent the differential regulation of low- conductanceK+ channelsbysignaling pathwaysmediatedby Ca + and cAMP. Moreover, since low-conductance Ca2 - activated K+ channels have beenidentifiedina

variety

of cell types,thesefindingssuggest thatdifferentialregulationof K+

channels byhormones with distinctsignaling pathways may provideamechanism for hormonal controlofsolute transport andCa2 -dependentcellular functions in theliveraswellas othernonexcitable tissues.

Signaling pathwaysthatemploy

Ca2+

andcAMPas intracel- lular messengersactinconcert toregulate cellularprocessesas diverse as glucose metabolism (1, 2), exocytosis (3, 4), and electrolyte transport (5). Although acceleration of cellular

Ca2+

entry by cAMP represents a possible basis for these concertedactions(3, 4, 6-8), the responsible mechanismsare not fully understood. In hepatocytes,measurement oftrans- membrane K+ flux hassuggested that synergy between sig- naling pathways mediated by

Ca2+

andcAMP occurs, in part, through activation ofK+channels (9).Thesechannelssetthe electrical driving forcefor

Ca2+

entry; moreover, certain K+

channel types, including those in hepatocytes (10),are regu- lated bychangesin cytosolic

Ca2+

concentration

([Ca2+],)

(11).

It isuncertain, however, whether such K+ channels are also activated by cAMP or whether cAMP activates a distinct populationof K+channels.

The HTC hepatomacell line, which has been employed as amodelsystemfor thestudy of hepatocellular metabolism (12, 13),possesseselectrophysiologiccharacteristics of hepatocytes inprimary culture(14-16).Inthis study, HTC cells have been Thepublication costs of this article were defrayed in part by page charge payment. Thisarticlemusttherefore be hereby marked "advertisement" in accordancewith 18 U.S.C. §1734 solely to indicate this fact.

used to investigate whether there is dual activation of K+

channelsbyCa2+ and cAMP. Thisreportprovidesevidence for one K+ channel type that is a common target ofCa2+ and cAMP (through cAMP-dependent protein kinase, PKA)and another that isselectively activated by PKA.

MATERIALS AND METHODS

Cell Culture. HTC cells were grown in minimal essential mediumsupplementedwith 5% fetalbovineserumand 2mM glutamine, aspreviously described(14), and plated eitheron glass coverslips (for measurement of

[Ca2+]1)

or on plastic tissueculture dishes (forpatch-clamp recording).

Measurement of

[Ca2+1,. [Ca2+],

was measured in single HTC cells, 24-48 hr after plating on glass

coverslips,

by dual-excitation-wavelength microfluorimetry with the Ca2+- sensitivefluorescentdyefura-2. Cells wereloaded for 1 hr at roomtemperaturewiththe cell-permeantacetoxymethylester of fura-2 (5,uM fura2-AM; MolecularProbes) in thepresence of0.025% Pluronic F-127. Coverslips weretransferred to a specially designed perfusion chamber housedonthestageof a microscope interfaced with a computer-controlled dual- excitation-wavelength fluorimeter (Spex Industries, Edison, NJ) (17). Cellswere perfused at room temperature with a bufferedelectrolyte solution that contained 135mMNaCl,5 mMKCl, 0.8mMKH2PO4,0.8mMMgC12, 1.2mMCaCl2, 5 mM glucose, and 10 mM Hepes (pH 7.4). Agonists were introduced by superfusion.In situ calibration of fura-2fluo- rescence was performed as described

(17).

Agonist-induced changes in

[Ca2+]1

were calculated from the difference be- tweenthe maximal

[Ca2+],

value within 60safterexposureto agonist and

[Ca2+],

just priortoagonistexposure.Inthisway, each cell servedasitsowncontrol.

Measurement ofSingle-Channel Currents. Single-channel currents were measured in cell-attached and excised mem- branepatches of HTCcells,24-48 hrafterplatingonplastic tissue culturedishes(1-ml volume), by patch-clamp recording techniques (14). Data were acquired on a computer during bursts ofsingle-channel openings andsubsequentlyanalyzed for determination ofsingle-channel current amplitudes and opentimeswith PCLAMPsoftware (AxonInstruments, Foster City,CA). In each of therecording configurations employed, downward deflections in the current tracing indicate the outward flowofpositivecurrent.

Forstudiesemploying cell-attachedmembrane patches, the pipette and bathing solutions were identical to the above solution used formeasurementof

[Ca2+]j.

Forstudiesemploy- ing excised membrane patches, the pipette (extracellular) solution was also identical to the above solution, and the

bathing (cytosolic)

solutioncontained 10 mM NaCl, 130 mM KCl,2 mMMgCl2,0.5 mMCaCl2,1mM EGTA, and 10 mM Hepes(pH 7.3; free

Ca2+

concentration -50 nM); a freeCa2+

Abbreviations:

[Ca2+],,

cytosolicCa2+ concentration; PKA, cAMP- dependent protein kinase; CPT-cAMP, 8-(4-chlorophenylthio)- cAMP.

(2)

Proc. Natl. Acad. Sci. USA 92 (1995) concentration of 1

,tM

wasachievedby bringingthe total

Ca21

concentration in this bathing solution to 0.95 mM by the addition of S ,ul of 90 mM CaCl2. Free Ca2+ concentration was calculated according to published algorithms (18).

For measurements ofsingle-channelcurrents ineither cell- attached or excised membrane patches, all reagents, including ATP, thapsigargin (Calbiochem), 8-(4-chlorophenylthio)- cAMP(CPT-cAMP;Sigma), and thecatalyticsubunit of PKA (Promega),wereintroducedbythe addition of a concentrated aliquot (5-10 ,ul) to the dish. All measurements were per- formed at roomtemperature.

RESULTS

Activation ofK+Channels byCa2+.Todetermine whether K+channels in HTC cells were affected byCa2 , [Ca2+]1was increased by two independent means. In one set of experi- ments, cells were exposed to ATP, which binds topurinergic receptors and increases

[Ca2+]i

by activation of the phospha- tidylinositolcascade(15).Inanothersetofexperiments, cells were exposed to thapsigargin, which increases

[Ca2+]i

by inhibition ofCa2+sequestrationwithinintracellularorganelles (19).Under restingconditions,

[Ca2+]i

was 176 +39nM(mean +

SE,

n = 15). Exposure to 100 ,MATP

(a

concentration chosen tomaximally saturate purinergic receptors and ensure anincrease in[Ca2

]j)

increased

[Ca2]i

by346 + 94 nM(n = 5; Fig. 1) and activated low-cociductance ion channels'that carried outwardcurrent atthe resting membrane potentialin 11 of 24 initially quiescent cell-attached membrane patches (Fig. 2). Exposure to 200nM thapsigarginproduced a slower increase in

[Ca2+1]

ofsimilar

magnitude (A[Ca2+1]

= 285 + 42

nM, n =5;Fig.1) and also activated ion channels that carried outward current at theresting membrane potentialin 9 of 16

600r

2400

-

+

0 200[

ATP

+I

L

600

2 400

N

o 200

0

600 r

400

200

CPT-cAMP

40

- I1 I

0 50 100 150

Time(sec)

FIG. 1. Effects ofATP,thapsigargin,andCPT-cAMP on [Ca2+]i (mean ± SE ofgroupsof5)inHTC cells. At thearrows, cells were exposedto ATP(100 ,uM),thapsigargin (200nM),orCPT-cAMP(100

ILM).

initially quiescentcell-attached membranepatches(Fig. 2). By contrast,exposure to vehicle alone had no effect (n = 8; data notshown). The conductances of channels activatedbyATP andthapsigarginwerealso similar(10.1±0.9pS,n=7, versus 11.9 ± 1.0pSforthapsigargin-activatedchannels, n = 5). This suggests that ATP andthapsigarginactivated acommontype of ion channel.Moreover,thecurrent-voltagerelation(Fig. 2) suggests that these channels were K+-selective, as theirpolarity was estimated to reverse at a potential at least 30 mV more negativethan the restingmembrane potential (estimated re- versal potential of -43 ± 6 mV relative to the resting membranepotential,n = 7, for ATP-activated channels and -33 ± 5 mVrelative to therestingmembranepotential,n = 5, forthapsigargin-activated channels). If we assume a resting membrane

potential

of -55 mV in HTC cells

(15),

the estimated reversal potentials for these channels would be approximately -98 mVand -88mV, respectively.

To examine moredirectlythe mechanismsresponsiblefor channelactivation,studieswereperformedwith excisedmem- branepatches.Increasingfree [Ca2+]in the fluidbathingthe

cystosolic

face of the membrane

patch

from 50 nMto1,uMled totheopening oflow-conductancechannels in 10 of 18initially quiescent patches (Fig. 3).The conductance of these channels

(9.0

±1.5pS,n=

10)

wassimilartothose ofchannelsactivated by ATP and thapsigargin, as was their estimated reversal potential.The estimated reversalpotential (-83± 6mV,n= 10, extrapolatedfrom the linearregionof thecurrent-voltage relation-i.e., correspondingtopotentialsmorepositive than +20

mV)

indicated selectivity for K+

(permeability

of Na+

relative to K+ was -0.04 by the Goldman-Hodgkin-Katz relation). This suggests that activation of K+ channels in response to ATP andthapsigarginresulted from direct acti- vationbyCa2+.

Activationof K+ChannelsbycAMP. K+channels werealso activated

by

cAMP. Exposure to the cell-permeant cAMP analogCPT-cAMP (100 ,uM) activated low-conductance ion channels that carried outwardpositive current at theresting membranepotentialin 7of 12initially quiescent cell-attached membrane patches (Fig. 2).The current-voltage relation for thesechannels

(single-channel

conductanceof11.0 ± 1.6

pS,

n =

6)

wasidenticaltothatofchannels activatedbyATPand

thapsigargin,

and the estimated reversal potential (-42 ± 8 mVrelativetothe

resting

membranepotential,n = 6, andin absolute terms, approximately -97 mV, for a resting mem- brane

potential

of -55

mV)

is consistent withK+selectivity

(Fig. 2).

This suggests that cAMP activated a K+channel type identicalto thatactivatedbyCa2+.

K+ channel activation by cAMP appeared to occur inde-

pendently

of

changes

in

[Ca2+]i,

asCPT-cAMP hadnosignif- icanteffecton

[Ca2+1] (A[Ca2+]i

= 2 + 12nM, n =5; Fig. 1).

Rather, studieswith excisedmembranepatches suggested that channelactivationbycAMPoccurredthroughitsdownstream effector PKA. Exposure of the cytosolic face of excised membrane patches to the catalytic subunit of PKA (200

units/ml)

in the presence of 100 ,uM ATP activated low- conductanceK+channelsin 9of 15initiallyquiescentpatches

(Fig. 3).

Exposureofthe cytosolic face to 100 ,uM ATPalone had no effect (n = 8; data not shown). Analysis of the current-voltage relations

revealed

that PKA activated two channeltypes, one with a

conductqnce

(8.4 ± 1.1pS, n = 6)

statistically

indistinguishablefrom that of the

Ca2+-activated

channel seen in excised membrane patches (Fig. 3), aswell as a5-pS channel

(single

channelconductance of 5.4 ± 0.5pS,n

=

3)

notseenwithexposure to

Ca2+

(Fig. 3).On thebasis of

extrapolated

reversal potential (-82 ± 16 mV for the 9pS channeland-67 ± 8 mVfor the5 pS channel, eachderived from the linear

region

ofthe

current-voltage

relationcorre-

sponding

to

potentials

more

positive

than +20

mV)

and the

Goldman-Hodgkin-Katz relation,

the

permeability

of the

9-pS

channelfor Na+relativetoK+wasestimatedtobe

0.04,

7116

Physiology: Lidofsky

F

(3)

A

Thapsigargin

--r_r- _MIMWfr , o illymvWjim InpRI-

CPT-cAMP

1.0

<0-8

)-

a) 0 0.6 0)

0.4h

-40 -20 0 20 40 60

Voltage (mV)

FIG. 2. ActivationofK+ chan- nels in HTC cells by Ca2+ and cAMP.(A)Activationof K+ chan- nels by ATP, thapsigargin, and CPT-cAMP. Single-channel cur- rents were measured in cell-at- tached membrane patches at the resting membrane potential in HTC cells exposed to ATP (100

,M), thapsigargin (200 nM),

or

CPT-cAMP(100 ,uM).Downward deflections in the current tracing indicate theopening of ion chan- nels that carry outward positive current. The large deflection (ar- row) results from theaddition of an aliquot oftheagonisttotheculture dish and isfollowedbyalag time due todiffusion.(B)Effect on sin- gle-channel currents of voltage across the membranepatch(rela- tive to the resting membrane po- tential) in acell exposedtoCPT- cAMP.Similar effects were seenin cells exposed toATPand thapsi- gargin. (C) Current-voltage rela- tion forchannels activatedbyATP 80 (n = 7), thapsigargin (n = 5), or

CPT-cAMP(n = 6).

and that of the5-pSchannelwasestimated tobe 0.07. These data indicate that these channelsarehighly selective forK+.

Collectively, these findings suggest that Ca2+- and cAMP- mediatedsignaling pathwaysconverge to activateacommon

typeofK+channel and that cAMP activatesadistinct type of K+ channel aswell.

Single-ChannelKinetics.Analysisofsingle-channel kinetics in excised membrane patches provided support for the con-

clusion given above. Duringbursts of channel openings, the

openprobability

(PO)

of 9pS Ca2+-activated K+ channelswas

0.35 ±0.15,meanopentime

(to)

was12.6± 3.3ms,andmean closed time (tc)was57.9 ± 33.1 ms (1185 events; transmem- branevoltage, +40 mV). At thesametransmembranevoltage, channelkinetics for9-pS K+ channels activated byPKAwere

notsignificantly different:

PO

was0.33 ±0.10, towas8.0± 1.2 ms, andtcwas23.7 ± 25.9ms(1934 events). Bycontrast,for 5-pS PKA-activated K+ channels,

PO

was0.68 ± 0.05, towas

22.9 ± 7.3 ms, and tc was 12.4 ± 7.3 ms (1688 events;

transmembranevoltage, +80 mV). These datasuggestthatthe higherPOof 5-pSchannels in comparison with9-pSchannels is due in parttolonger channel openings.

DISCUSSION

This study provides evidence for differential regulation of low-conductance K+channels in liver cells by Ca2+and cAMP.

Twoprincipalfindingssupportthis conclusion. First, the data suggest that Ca2+ and cAMP activate a common (9-pS) K+

channel type, as evidenced by the similarity of the conduc- tance, K+ permeability relative to Na+, atid gating kinetics

between channels activated by Ca2+ and those activated by PKA. Second, the data suggest that PKA activates an addi- tionalK+channel, distinct in conductance (5pS),relativeK+

selectivity, and kinetics of channel gating. Further inferences regarding mechanisms of channel gating by Ca2+ and PKA cannotbedrawn, because single-channel analysiswasderived only from data acquired during periods of burst activity.

Differentialregulation of K+ channels hasconsequencesfor hormonal control ofhepatocellulartransportandmetabolism.

First, since K+ channels regulate hepatocellular membrane potential (20), processes influenced by membrane potential, such asthe transport of amino acids, HCO-, and bile acids (21-23), are modulatedby K+ channel activity. Thus, differ- ential control ofK+ channel activitymayprovideabasisfor differential regulation of such critical liver functions by hor-

monesthat employCa2+ andcAMPasintracellularmessen-

gers. Second, hepatocellular K+ channel activity has been linkedtocontrol ofCa2+ influx andCa2+-regulatedprocesses, such asglucose production (24-26). Concerted activation of K+ channels by Ca2+ and cAMP could thus facilitate Ca2+

influxand mayrepresent a mechanism forsynergybetween hormones that employ Ca2+ and cAMPasintracellular mes- sengers (2, 7, 8).

Although the role of low-conductance K+ channels in regulating liver functionsdescribedabovehasnotbeendefin- itively established, support forsuch a role comesfrom fluc- tuationanalysis of whole-cellcurrents(27). These datasuggest

thatlow-conductance K+ channlels in hepatocytesarelargely responsible for membrane hyperpolarization that occurs in

response to agonists that signal through increasesin [Ca2+]1.

lpA 30sec

l' :' 'I

B

V(mV)

C

+60

-4 1I

+20 I7 O

* ATP o Thapsigargin O CPT-cAMP

-20

S*

m

lpA

100ms O0

im- 1l w rwTTWP

off._- -! -_

2f I

ATP I

IlillatmopwF

1) lI

1

(4)

Proc.Natl.Acad. Sci. USA 92

(1995) A

1pA 30sec

ATP PKA

A PK

ATP PKA

B

V

(mV)

9

pS

+80 +40

C

1.2

5 pS

+120

+80 _

0~~~~~~~~~~~~~~~~~0

+40

1pAL

100ms

20 40 60 80 100 120

Voltage (mV)

FIG.3. Activation of K+ channels in excised membranepatches. (A)Effect ofincreasing freeCa2+ concentration (from50 nMto1

,.M)

inthe medium bathingthecytosolicfaceof themem- brane (top trace) or exposure to the catalyticsubunit of PKA(200units/ml) in the presence of 100,uMATP(free Ca2+, 50nM). Two typesof channels wereactivatedbyPKA (lowertraces), as canbeseenbytheir distinctcurrent

amplitudes.Membranevoltagewas40 mV. (B)Effectonsingle-channelcur- rentsofvoltageacrossthe membrane patch in excised membranepatchesex- posedtoPKAin the presenceof ATP.

The upperseriesoftracescorresponds to a single-channel conductance of 9 pS; the lower series of traces corre-

spondsto aconductance of 5 pS. (C) Current-voltage relation for channels activatedbyCa2+(n=10)orPKA(9-pS channels,n = 6;5-pS channels,n =3).

Indeed,twotypesoflow-conductanceK+channels inhepato- cytes have been previously reported, one of which is Ca2+- activated(10). The other typeappears to beCa2+-independent (28), but its potential regulation by phosphorylation (inpar-

ticularby PKA) hasnotbeenexamined.It ispossiblethatthese channeltypes representdistincttargetsforCa2+ andcAMP- mediatedsignaling pathwaysas described here.

Low-conductance Ca2+-activated K+ channels, similar in magnitude to those

delineated

in this report, have been described in avariety of cell typesinadditionto hepatocytes (11). Such channelsareimplicated in catecholamine secretion by chromaffin cells (29), Cl- secretion by respiratory epithe- lium

(30),

nitric oxide synthesis by endothelial cells (31), and cell volume regulation (32). These channels differ in tissue distribution and function from high-conductance Ca2+- activated("maxi") K+ channels, whichareregulatednotonly by Ca2+ but also by voltage (11). Although convergent acti- vation ofhigh-conductance K+ channels byCa2+ and cAMP has been described (33-37), this is the firstreportof which I

am aware that describes convergent activation of low- conductanceK+ channelsbyCa2+ and cAMP. Convergent and

parallel activation of low-conductance K+ channels

by Ca2+

andcAMP-mediated

signaling pathways

maybea

regulatory

mechanism

employed

not

only

inthe liver butinother tissues aswell.

Ithank L.Ruslim for technical assistance and E. T. Barfod, B. F.

Scharschmidt,andH. F.Yee,Jr., forhelpfuldiscussions. Thisworkwas supported in partby National Institutes of Health Grants DK01987 and DK26743(UniversityofCalifornia,SanFrancisco, LiverCenter) andaLiver Scholar Awardfrom the American Liver Foundation.

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