Gut 1993; 34:1331-1338

Properties of a potassium channel in cultured humangastric cells (HGT-1) possessing specific omeprazolebinding sites

G I Sandle, G Fraser, K Fogg, G Warhurst

AbstractThe HGT-1 human gastric cell line is similar toacid secreting parietal cells in that it possessesH2 receptors, histamine sensitive adenylcyclase, and Cl- channels, which are activatedby histamine by a cyclic adenosine mono-phosphate (cAMP) dependent mechanism. Todiscover if HGT-1 cells have additional prop-erties found in parietal cells, [3H]omeprazoleand patch clamp recording techniques wereused to evaluate specific omeprazole bindingsites and K+ channels in the plasma mem-brane. HGT-1 cells exhibited [3H]omeprazolebinding in the non-stimulated state, whichincreased 100% in the presence of 1 mMhistamine. High conductance (about 155 pS)K+ channels were active spontaneously in 17%ofcell attached or excised inside out patches innon-stimulated subconfluent HGT-1 cells. Ininside out patches, channel activity increasedfivefold during depolarisation, ion substitutionexperiments confirmed that the channels werehighly selective for K+, and channel activitywas almost abolished by removal of Ca2+ oraddition of 5 mM Ba2+. In quiescent cellattached patches, 0.1 mM dibutyryl cAMPfailed to activate K+ channels. In contrast, 6-7,tM A23187 (a Ca2+ ionophore) increasedintracellular Ca2+ concentration from mean(SEM) 14 (3) nM to 248 (30) nM and activatedK+ channels in 21% ofpatches. It is concludedthat the plasma membrane of HGT-1 cellspossesses (a) specific 3H-omeprazole bindingsites, which may reflect the omeprazole sensi-tive H+,K+-ATPase present in gastric parietalcells; and (b) Ca2+-activated K+ channels,which may be located in the basolateral mem-brane ofhuman gastric parietal cells and play apart in acid secretion triggered by Ca +-mediated secretory agonists.(Gut 1993; 34: 1331-1338)

Manchester EpithelialMembrane ResearchCentre and Departmentof Medicine, Universityof Manchester, HopeHospital, ManchesterG I SandleG FraserK FoggG WarhurstCorrespondence to:Dr G I Sandle, Department ofMedicine, Hope Hospital,Eccles Old Road, SalfordM6 8HD.

Accepted for publication23 February 1993

Hydrochloric acid secretion by gastric parietalcells entails complex ion transport events at theapical (secretory) membrane. Movement of Cl-across the apical membrane of stimulatedparietal cells seems to be mediated by Cl-channels. In addition, apically located H+,K+,ATPase exchanges one H+ ion for one K+ ion,and is therefore dependent on the presence ofluminal K+.3-5 Although the precise mechanismby which K+ moves from cell cytosol to thelumen ofthe gastric gland is unclear, a number ofmechanisms that link apical K+ and Cl- trans-port have been proposed namely - solely electro-neutral KCI cotransport,6 electroneutral KCIcotransport in parallel with a large Cl- conduct-

ance,7 K+ movement by a population of K+-specific channels in parallel with apical Cl-channels,'8 and simultaneous K+ and C1- move-ment mediated by an apical cotransporter withpartial conductive properties.' Gastric parietalcells also possess a basolateral K+ conductivepathway9 10 in parallel with Na+,K+-ATPasepump units, which mediate basolateral K+uptake. This pump - leak system allows K+ ionsto recycle across the basolateral membrane,promotes hyperpolarisation of the parietal cell,and thereby contributes to the maintenance ofthe electrochemical gradient that favours Cl-exit at the apical membrane during acid secre-tion.A human gastric cell line (HGT-1) is now

available, the properties of which have beenpartially characterised. Similar to gastric parietalcells, the plasma membrane ofHGT-1 cells bearsH2 receptors and intracellular cyclic adenosinemonophosphate (cAMP) concentrations rise inresponse to histamine." The cells also possessoutward rectifying, voltage sensitive, low con-ductance Cl- channels, which are activated byhistamine by a cAMP dependent process,'2 andmay represent the apical Cl- channel activated inparietal cells during acid secretion. The aim ofthis study was to establish whether HGT-1 cellshave additional features in common with gastricparietal cells. The results show that the plasmamembrane of HGT-1 cells contains (a) specificbinding sites for omeprazole that probablyreflect omeprazole sensitive H+,K+-ATPaseunits; and (b) high conductance K+ channelsthat may represent the K+ channels present inthe basolateral membrane of gastric parietalcells.

Methods

[3H]OMEPRAZOLE BINDING SITESMonolayers ofthe HGT-1 human gastric cell line(and the HT29-19A human colonic cell line)were grown to confluency in 12-well plasticculture plates, and maintained in Dulbecco'smodification of Eagle's minimum essentialmedium, supplemented with 2mM L-glutamine,100 IU/ml penicillin, 100 [tg/ml streptomycin,and 10% (vol/vol) fetal calfserum (heat treated at55°C for 30 minutes). Monolayers were grown ina humidified atmosphere of 95% air/5% CO2 at37°C. Culture medium was removed, and the celllayers washed with a buffered NaCl solutioncontaining (mM): Na+, 140; K+, 4 5; Cl -, 149;Ca2+, 1 2; Mg2+, 1-2; glucose 10; and 4-(2-hydroxyethyl,- I -piperazine ethanesulphonicacid (HEPES) 10, titrated to pH 7*4 with NaOH,

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Sandle, Fraser, Fogg, Warhurst

and incubated with [3H]omeprazole (1[25 nmol/well; equivalent to about 3 x 105 counts per min)in the presence or absence of 1 mM histamine for30 minutes at 37°C. Histamine was used at aconcentration of 1 mM because previous studieshave shown this concentration is required toachieve maximal stimulation of adenylate cyclasein the HGT-1 cell line,'3 which is largely (77%)inhibited by 10-4 M cimetidine, an H2 receptorantagonist." Non-specific binding was assessedby incubating identical wells with [3H]omepra-zole in the presence of 10 FtM unlabelledomeprazole. Incubation was stopped by rapidwashing (4x2 ml over 10 s) with ice cold buffer.The cell layers were dissolved in 1% TritonX-100, sonicated, and samples taken for scintil-lation counting and protein determination."'Specific binding was calculated as the differencein cell associated counts between incubationswith [3H]omeprazole alone, and [3H]omeprazoleplus unlabelled omeprazole. Results are expres-sed as pmol omeprazole bound/mg cell protein.

PATCH CLAMP STUDIESMonolayers of HGT-1 cells were maintained asdescribed above, and cells were subculturedunder identical conditions at 7 day intervals.Cells from passages 45-65 were grown onuncoated plastic coverslips and used 4-15 daysafter subculture. Subconfluent monolayersremained attached to coverslips, which wereplaced in a small chamber (vol 2 ml) andvisualised on the stage of a Nikon (Diaphot)inverted microscope with Hoffmann modulatedoptics (x400 magnification). The microscopewas mounted on a vibration isolation table.Single channel recordings were obtained frommembrane patches in cell attached and excised

inside out configurations as described pre-viously.'5 In all experiments the bath solution(temperature 20-220C) initially contained (mM):K+, 145; Cl-, 149: Ca2+, 1[2; Mg2+, 1[2;glucose 10; and HEPES 10, titrated to pH 7*4with KOH (high K+ solution), and the pipettecontained the NaCl solution (see above). In someexperiments with inside out patches, singlechannel recordings were obtained after replacingthe high K+ solution with a low K+ solutioncontaining (mM): K+, 22 5; Na+, 122-5; CI-,149; Ca2+, 1 2; Mg2+, 1I2; glucose 10; andHEPES 10, titrated to pH 7-4 with NaOH. TheGoldman-Hodgkin-Katz (GHK) current andvoltage equations"" were used to calculate theK+:Na+ permeability ratios (PK:PNa) and thereversal potentials (Erev) respectively. Pipetteresistances varied between 2-15 MQ and mem-brane seal resistances between 5-40 GQ. Thebath electrode was a silver chloride coated silverwire.

Single channel currents were recorded with apatch clamp amplifier (List Electronics, Darm-stadt, Germany, model EPC-7), and stored onvideo tape after pulse code modulation (Sony,Japan, model PCM 701ES). Stored currentswere low pass filtered (-3 dB, 4-pole Butter-worth) at 1 kHz and loaded into computermemory (IBM PC 286-XT, sampling frequency4 kHz) through a Labmaster TLI interface andTM40 A/D converter (Axon Instruments, FosterCity, Ca, USA). Voltages applied to the mem-brane patches (Vcom) were referenced to theinterior of the patch pipette. With high K+solution in the bath, cell membrane voltage wasassumed to be zero, and Vcom was thereforeequivalent to the potential across the membranepatch in both the cell attached and inside outconfigurations. Current-voltage (I-Vcom) rela-

A

+60 mV.

+40 mV

+20 mV p N

0 mV 1

-20mV -__ - _

- 10 pA1 s

B

I (pA)

-100 -80 -60 -40 -20Vcom (mV)

0 +20 +40 +60

Figure 1: K+ channel in a cell attached patch ofan HGT-I cell (high K+ bath solution, NaCl pipette solution). (A) showsrecordings at different holding potentials (Vcom) referenced to pipette interior. Dashed lines show zero current values. Upwardcurrent deflections show outward K+ flowfrom cell to pipette. In this patch, channel openings were infrequent at Vcom valuesbetween -20 mV and -60 mV. (B) shows current/voltage (I/Vcom) relation ofthe recordings in (A). Datafit and reversalpotential (Erev) obtained using the Goldman-Hodgkin-Katz (GHK) current and voltage equations.

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Properties ofa potassium channel in cultured human gastric cells (HGT-1) possessing specific omeprazole bindingsites

oB

+20mVL [L, +40mV

0 mv W*w IKIKT

C I (pA)+14,

+20 mV -

-20 mV . |

-40 mV __ _ OmV-0 MV

-60 mV ,_ _ _ -20 mV _

5 pA1 s

|1 pA1 s -100 -80 -60 -40 -20 0

Vcom (mV)

Figure 2: K+ channel in an excised inside out patch ofan HGT-1 cell. (A) and (B) show recordings at different holding potentials (Vc m) referenced to pipetteinterior (filled with NaCl solution). Recordings were obtained first with high (145 mM) K' solution (A), then with low (22S mM)K solution (B) in the bath.Dashed lines show zero current values. Upward current deflections show outward K+ flowfrom bath to pipette. (C) shows currentlvoltage (IIVCom) relations ofthe recordings in (A) (145 mM bath K+, closed circles) and (B) (22-5 mM bath K+, closed squares). Data fits and reversal potentials (Erev) obtained using theGHK current and voltage equations.

tions were constructed by plotting the singlechannel current (equal to the difference betweenthe open and closed channel currents) at eachvalue of Vcom. Single channel slope conductancewas calculated at two values of Vcom: (a) at -40mV, the cell membrane voltage measured inrabbit gastric glands with conventional micro-electrodes and in rabbit parietal cells duringwhole cell current clamp studies,'8"9 and (b) atinfinite voltage (corresponding to the linearportion of the I-Vcom curve) where channelconductance was maximal.

Single channel open probability was deter-mined using an analysis programme written inQuick Basic 4 0 (Microsoft, USA). A transitionbetween the fully closed and fully open currentlevels occurred when the current crossed athreshold set halfway between these two states.Single channel open probability (PO) was calcu-lated as:

Po=(2:ntn)/Nwhere N is the maximum number of channelsseen to be open simultaneously during therecording (lasting 20-30 s), n represents the stateof the channels (0=closed, 1 =one channel open,etc), and tn is the time spent in state n. Channelkinetics were determined using pClamp software(Axon Instruments) and recordings from mem-brane patches in which only one channel wasseen to be active. As channel activity usuallyoccurred in bursts (Figs lA and 2A), mean openand mean closed times, and open and closedtime distribution histograms were obtained byrestricting the analyses to individual burstsduring 20-30 s of continuous recording. In mostcases the first bin was ignored when fitting thedistribution histograms (see Fig 4).The sensitivity of K+ channels to Ca2+ ions

was determined in excised inside out patches byrecording (Vcom=O mV) first in the presence ofthe Ca2+ containing high K+ bath solution, andthen in the presence of a Ca2+ free high K+solution containing the Ca2+ chelator ethylene-glycol-bis(,3-aminoethyl ether) N,N' tetra-aceticacid (EGTA, 5 mM).The effect of Ba2+ ions, which block K+

channels in a variety of epithelial cell types,20-25was assessed in excised inside out patches (Vcom =0 mV) by replacing the high K+ bath solutionwith a similar solution containing 5 mM BaCl2.

Additional experiments were performed todetermine the effects of increasing the intra-cellular concentrations ofcAMP and Ca2+ on K+channel activity. With NaCl solution in the bathand high K+ solution in the pipette, recordingswere obtained from cell attached patches (Vcom=0 mV) before and after the addition of 0 1 mMdibutyryl cAMP (a permanent analogue ofcAMP) or 6-7 ,uM A23187 (a Ca2+ ionophore).After the addition of agonist, 30 s segments ofdata were recorded at 1 minute intervals for 10-15 minutes.

MEASUREMENT OF INTRACELLULAR FREE CALCIUMCONCENTRATIONIntracellular free calcium concentration ([Ca2+],)was measured in HGT-1 cells loaded with theCa2+ sensitive dye Fura-2.26 Cells grown on glasscoverslips were loaded by incubating withculture medium (DMEM supplemented with15 mM HEPES, 2 mM glutamine, 50 tig/mlpenicillin, 50 [tg/ml streptomycin, and 10% fetalcalf serum) containing 10 FM Fura-2/AM for 90minutes in a humidified atmosphere of 5% C02/95% air at 37°C. Coverslips were washed withbuffered NaCl solution, mounted in a Sylgardlined chamber on the stage of a Nikon Diaphotmicroscope, and bathed in NaCl solution at roomtemperature (20-220C). Groups of about 5 cellswere illuminated alternately with light of 350 nmand 380 nm wavelengths by means of a motordriven filter change wheel. The emitted fluoresc-ence (510 (10 nm)) with each excitation wave-length was monitored by photon counting(Newcastle Photometric Systems, Newcastle,UK). Fluorescence was corrected for back-ground before calculation of the 350 nm/380 nmemission ratio (R). [Ca2+]i was calculated withreference to a standard calibration curve relatingCa2+ concentration to R, which was constructedin vitro using Ca2+-EGTA buffers (MolecularProbes, Eugene, USA) yielding known Ca2+

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Sandle, Fraser, Fogg, Warhurst

concentrations (0-39- 8 FM) and containing greater in the presence than in the absence of5 FM Fura-2 (free dye). R values were measured 1 mM histamine (4 17 (0 45) pmol/mg cellin 40 RI droplets of each solution on a glass protein v 2'08 (0 48) pmol/mg cell protein,coverslip. p

Properties ofa potassium channel in cultured human gastric cells (HGT-l ) possessingspecific omeprazole bindingsites

150 A

Open timet=5.28 (0-16) ms

75

iC 0> 0 20 405)

0 30011 Bz A Onen time

150-

0

0

Ir-\tl'= 26-63 (0 67) ms

380

190

3301

165

oh

120

Closed timet, = 0-65 (0-03) mst2= 4-62 (1 87) ms

6 10 20

Closed timeti = 0 36 (0 05) mst2 = 1-25 (0-36) ms

240 0 5 10

Time (ms)Figure 4: Open time and closed time histograms ofK+ channels in cell attached (A) and excisedinside out (B) patches ofHGT-1 cells at V...=0 mV (high K+ bath solution, NaCI pipettesolution in both configurations). Numbers ofeventsfor different open times and closed times areshown. Open time and closed time data were bestfitted by single and double exponentialsrespectively. Time constants (mean (SEM))for the open and closed states are shown in theFigure.

with 22 5 mM K+ in the bath solution were allsignificantly (p35 ms) seen between burstswould probably provide a third closed timeconstant, but there were insufficient for histo-gram analysis. It should be noted that the meanopen time was significantly longer and the meanclosed time was significantly shorter in the insideout patches than in the cell attached patches(30 4 (8-1) ms v 6-8 (0 7) ms, p

Sandle, Fraser, Fogg, Warhurst

recordings from one patch in which Ba2+inhibited K+ channel activity, while channelactivity returned almost to the initial level afterwashing out Ba2 . In three patches, Ba2+decreased PO from 0-392 (0-081) to 0 030 (0 024)(p

Properties ofa potassium channel in cultured human gastric cells (HGT-J)possessingspecific omeprazole bindingsites

800 -

600-

77 400-

200-

0

0 2 4 64-bromo-A23187 (,M)

Figure 8: Effect of4-bromo-A23187 on intracelltconcentration ([Ca2+]) in HGT-1 cells. Results Xmean (SEM) (n=4 forO, 3, 6 7, and 10pM 4-bA23187).

K+ channel activity in cell attached paFig 7) by increasing [Ca2+], to similar v

Discussion

EVIDENCE FOR H+,K+-ATPASE IN HGT-1HGT- 1 cells share a number of charawith acid secreting gastric parietalexample, H2 receptors, histamineadenylate cyclase, cAMP mediaichannels), but previous studies Ievaluated their H+,K+-ATPase conte[3H]omeprazole as a probe for H+,K+we found that HGT-1 cells exhibitedant degree of specific [3H]omeprazolewhich increased 100% in the presencemine. Although histamine stimulincorporation of H+,K+-ATPase riplasmic tubulovesicles into the apical nin parietal cells,30 it is unclear whetheiphenomenon occurred in HGT-1 cellhave been shown to be devoid oforganelles." It is worth noting at tihowever, that although the HGT-1shows morphological heterogeneity, se)cell lines each with homogeneous mchave been identified. 3 Two of therexhibited activities of histamine inducproduction that were 2-3 times grethose in the other clones or in the parerline despite the absence of interclonalin the affinity of the clones for histamirit seems that the HGT-1 cell linesubpopulations of epithelial cells, twomay have a comparatively high densreceptors or a particularly efficient iibetween the H2 receptor and adenylatThese two clones (clones 3 and 6, see rebear the closest resemblance to gastriicells and contain secretory organelhtheir cytoplasm, which become inc4into the cell membrane in the prehistamine. Furthermore, histamineshown to trigger cAMP dependent photion of a 27 kDa phosphoprotein,restricted to the same low speed frparietal cells that contains increasedATPase activity after histamine stirnThe ability of histamine to increase [3H

zole binding in HGT-l cells may therefore reflectthe presence of H+,K+-ATPase units withinthe plasma membrane whose affinity for[3H]omeprazole is increased by cAMP depen-dent phosphorylation of the enzyme protein.

HGT-1 CELLS EXPRESS K+ CHANNELSThere is strong evidence that K+ channels existin both the apical and basolateral membranesof gastric parietal cells. In frog stomach andisolated rabbit gastric glands, external K+ is an

8 o0 absolute requirement for acid secretion.33 36 Atthe external (luminal) surface of the apical mem-

ular Ca2+ brane, K+ ions participate in a H+,K+-ATPaseare shown as mediated exchange with H+ ions, althoughPromo- whether the initial apical K+ exit step is electro-

neutral or channel mediated remains controver-sial. l 6- If it is assumed that basolateral Na+-K+pump activity increases during acid secretion,

tches (see K+ entering the cell must recycle across theralues. basolateral membrane to maintain intracellular

K+ concentration, and a basolateral conductancehas been shown in Necturus and frog oxynticcells.9' 10253738 This study shows that HGT-1 cellsexpress K+ channels, and we now consider their

CELLS comparatively low prevalence, likely site ofacteristics origin, and characteristics.cells (for We saw K+ channel activity in 17% of cellactivated attached and inside out patches from HGT-1ted Cl- cells in the absence of acid secretory agonists.iave not This fairly low level of detection suggests thatnt. With either K+ channels were present at low density in-ATPase, the plasma membrane, or that K+ channels werea signific- limited to a small subpopulation of cells withinbinding, the HGT-1 cell line. It should be noted that K+of hista- channels were seen after four to eight days of

lates the culture when cells were subconfluent, but neverich cyto- beyond eight days when the cells were fullyaembrane confluent. This suggests that either the K+r a similar channel bearing cells were overgrown by otherIs as they cell types when true confluency was achieved, orsecretory confluency was associated with full polarisationiis point, of the cells and movement of K+ channels to thecell line basolateral membrane, which was inaccessible toven clonal the patch pipette. The fact that the prevalence of)rphology K+ channels in cell attached patches was similarse clones when A23187 stimulated cells were bathed ined cAMP NaCl solution (21%) and non-stimulated cellsater than were bathed in high K+ solution (17%), suggestsat HGT-1 that the high external K+ concentration depolar-variability ised the cells and caused a passive increase inie.'3 Thus [Ca2+]i.contains Our results show that the K+ channel expres-of which sed by HGT-1 cells is Ca2+ dependent, voltageity of H2 and Ba2+ sensitive, and possesses complexteraction gating characteristics with one open state and ate cyclase. least two closed states. Single channel conduct-f 13) may ance was high (about 155 pS) at infinite voltageLcparietal but considerably lower (about 60 pS) at -40 mV,es within the cell membrane voltage measured in rabbitorporated parietal cells.' 119 Basolateral K+ conductance is,sence of the main determinant of basolateral membranehas been voltage in Necturus oxyntic cells,37 and the baso-isphoryla- lateral membrane of frog oxyntic cells possesses awhich is substantial Ba2+-sensitive K+ conductance,*action of which increases in the presence of histamine.91'H+,K+- Single channel studies have shown two types of

iulation.'2 basolateral K+ channel (one activated by cAMP,[Qomepra- the other by Ca2+) in Necturus oxyntic cells.38

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1338 Sandle, Fraser, Fogg, Warhurst

Frog oxyntic cells also possess two types ofinward rectifying basolateral K+ channel (a) aCa2+ activated, 61 pS channel that is voltageindependent, Ca2+ sensitive and Ba2+ sensitive,and (b) a cAMP-activated, 30 pS channel that isCa2+ insensitve.25 In contrast, it is not clear if thebasolateral membrane voltage in mammalian(rabbit) parietal cells reflects Cl-, K+, or bothtypes of channel.'9 Although the properties ofbasolateral K+ channels in oxyntic cells seem tobe species dependent, K+ channels in HGT-1cells share some characteristics (for example,similar conductance within the physiologicalvoltage range, activation by Ca2+, and sensitivityto Ca2+ and Ba2+) with the 61 pS K+ channelidentified in amphibian oxyntic cells.2538

Acid secretion by gastric parietal cells isregulated by several receptor mediated neuro-humoral agonists including histamine, whichincreases the intracellular concentrations of bothcAMP and Ca2+.3 Indeed, concomitantincreases in intracellular cAMP (induced byadding dibutyryl cAMP) and Ca2+ (induced byadding carbachol) have a potentiating effect onacid accumulation in isolated canine parietalcells.41 It is now clear that HGT-1 cells resembleparietal cells in possessing H2 receptors andspecific omeprazole binding sites (presumablyH+,K+-ATPase units). The additional presenceof cAMP dependent Cl- channels and Ca2+dependent K+ channels in this gastric cell linemay provide an opportunity to study possibleinteractions between these two types of channelin response to a variety ofacid secretory agonists.

We are grateful to Dr C L Laboisse (Laboratoire de Biologie et dePhysiologie des Cellules Digestives, U 239 INSERM, Paris) forsupplying the HGT-l cell line, and to Astra Pharmaceuticals Ltdfor supplying radiolabelled omeprazole. The single channel analy-sis programme was a gift of Dr M Hunter (Department ofPhysiology, University of Leeds). This study was supported inpart by a grant from the Royal Society.

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