Identification of a membrane protein from T84 cells using antibodies made against a DIDS-binding peptide ERIC J. SORSCHER, CATHERINE M. FULLER, ROBERT J. BRIDGES, ALBERT TOUSSON, RICHARD B. MARCHASE, B. R. BRINKLEY, RAYMOND A. FRIZZELL, AND DALE J. BENOS Departments of Physiology and Biophysics, Cell Biology, and Medicine, Division of Hematology and Oncology, University of Alabama at Birmingham, Birmingham, Alabama 35294 Sorscher, Eric J., Catherine M. Fuller, Robert J. Bridges, Albert Tousson, Richard B. Marchase, B. R. Brinkley, Raymond A. Frizzell, and Dale J. Benos. Identification of a membrane protein from T84 cells using antibodies made against a DIDS-binding peptide. Am. J. Physiol. 262 (CeLL Physiol. 31): C136-C147, 1992.-The outwardly rectified chloride channel of secretory epithelial cells is inhibited by disulfonic stilbene (DS) compounds such as 4,4’-diisothiostilbene-2,2’-disulfonic acid (DIDS) [R. J. Bridges, R. T. Worrell, R. A. Frizzell, and D. J. Benos. Am. J. Physiol. 256 (Cell Physiol. 25): C902-C912, 19891. A 13-amino acid peptide (P49) corresponding to the putative DS binding site region of the murine anion exchange protein was synthesized, and polyclonal antibodies were generated against it and then purified over a P49 affinity column. The resulting monospecific antibodies reacted on Western blots with a 95- to 100-kDa protein from human erythrocytes and a 55- to 60-kDa protein from the human colonic tumor cell line, T84. The reaction with T84 protein did not appear to represent recognition of an anion exchanger because anion efflux from T84 cells was independent of external Cl-. In addition, monoclonal antibodies raised against human band 3 recognized the band 3 protein in human red cell ghost preparations but recognized nothing in T84 cell membrane preparations. In T84 cells, DIDS protected the 60-kDa protein from antibody binding. The anti-P49 antibody blocked outwardly rectified Cl- channels incorporated into planar lipid bilayer membranes from rat colon. Immunocytochemical data reveal specific binding of the anti-P49 antibody to perinuclear cytoplasmic vesicles. Forskolin caused these antibody-labeled vesicles to migrate from the perinuclear region to the plasma membrane under conditions and with a time course identical to that seen for stimulation of Cl- transport in these cells. Our results suggest that the protein may be a part of a chloride channel complex of secretory epithelial cells. 4,4’-diisothiocyanostilbene-2,2’-disulfonic nel; vesicle migration

acid; chloride

chan-

STILBENES (DS) have been used to inhibit electroneutral anion exchange processes in erythrocytes and other cells (18, 19, 34). In addition, many investigators have found 4,4’-diisothiostilbene-2,2’-disulfonic acid (DIDS) inhibition of Cl-selective ion channels. DIDS was shown to block Cl- channels from electroplax incorporated into planar lipid bilayers (41) and a Clchannel from a toad kidney cell line (A6) in patch-clamp studies (42). Bridges et al. (9) recently reported the ability of disulfonic stilbene (DS) compounds, including DIDS and 4,4’-dinitrostilbene-2,2’-disulfonic acid (DNDS), to block secretory Cl- channels from rat colon plasma membrane vesicles reconstituted into planar lipid bilayers. Inhibitory constants were 2-4 PM for DNDS and were similar to those observed for conductive anion uptake into membrane vesicles isolated from T84 cells DISULFONIC

cl36

0363-6143/92

$2.00

Copyright

0 1992

or rat colonic epithelium (8, 44). Valdivia et al. (48) report that DIDS inhibits a Cl- channel from bovine trachea apical membranes incorporated into planar lipid bilayers. Kopito and Lodish (35) and Demuth et al. (14) have isolated cDNA clones coding for the murine erythrocyte and human nonerythroid anion exchange (band 3) proteins and deduced their primary structure from sequence analysis of the cDNA. The transport domain of the band 3 protein is thought to contain 12 membrane-spanning regions; 7 of these are amphipathic and may orient to form an anion-translocating pore. One of the amphipathic regions is thought to contain the putative DIDS binding site (minimal amino acids 558-561; see Ref. 35). One of two lysines within this site may covalently interact with DIDS, although requirement of the lysines for DIDS binding, covalent labeling, or inhibition may not be absolute (3, 4, 21). The murine amino acid sequence is highly homologous to the cloned regions of the human erythrocyte anion exchanger in the region predicted to be important for stilbene binding. Although this predicted DIDS-binding site sequence is not identical among all band 3 and band 3-like proteins, of the few nonidentical amino acids that do exist, most represent conservative substitutions so that a high degree of homology is evident among these proteins and also between species (3, 4, 21, 31). Based on high-affinity DS inhibition of both anion exchange and Cl- channel currents, we assumed that these anion transport proteins share structural similarities, specifically at a DS binding site. Accordingly, we synthesized a peptide consisting of amino acids comprising the putative DS binding site of murine erythroid band 3 and generated polyclonal antibodies against this peptide. The approach of generating probes for proteins by raising antibodies to synthetic peptides corresponding to limited regions of these proteins has been successfully applied to the interleukin-2 receptor system (36), the corticotropin, transferrin, and epidermal growth factor receptors (6, 7), the voltagedependent Na+ channel (23), and other regions of band 3 (32). The antibodies generated against the synthetic peptide described here have recently been used to identify a band 3-like anion exchange protein in Chinese hamster ovary (CHO) cells (16). The specific objectives of our work were fourfold. First, we characterized and defined conditions in which the stilbene disulfonates inhibit the adenosine 3’,5’-cyclic monophosphate (CAMP)-activated Cl- conductance pathway in T84 cells. Second, we generated polyclonal antibodies against a synthetic peptide whose sequence the American

Physiological

Society

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DIDS-BINDING

PROTEIN

ANTI-P4

0 Control l Forskolin A Forskolin+DIDS

90

Cl37

FROM T84 CELLS

9 ANTIBODY

RBC Ghosts

5

Time

(minutes)

Fig. 1. 12Y uptake into T84 cell monolayers. T84 cells were pretreated for 80 min in NaCl-containing buffer containing 100 KM ouabain and then transferred to ““I--containing Na+ gluconate buffer for l-30 min in presence or absence of 1 PM forskolin. Each point represents mean of 2 determinations. Results are representative of 10 experiments. Time course of 12’1- uptake and peak of overshoot vary between experiments, precluding presentations of average data.

Fig. 4. Western blot analysis of human erythrocyte ghost membrane protein overlaid with anti-P49 (50 kg membrane protein loaded, see METHODS). X Remaining

‘.

t

2

1

Time

in the absence of DIDS, recognizes a 60-kDa polypeptide in T84 cells. Fourth, we used this antibody to show that vesicular movement to surface membrane is induced by CAMP-mediated agonists. Our results suggest that this 60-kDa protein may be a component of a Cl- channel in Cl- secretory epithelial cells.

\

(minutes)

Fig. 2. Effect of forskolin on 12’11efflux from either T84 (solid circles) or Chinese hamster ovary (CHO; open circles) cells. Results are representative of 6 experiments. In this experiment, basal efflux rates in CHO and T84 cells were not markedly different, but in a larger series (see Fig. 3) basal efflux rates differed by a factor of -2.5.

METHODS

Cell Culture

T84 cells were obtained from K. Dharmsathaphorn and J. McRoberts (University of California at San Diego) at passage 12 and studied up to passage65. The cells were grown in a 1:l mixture

mcl-lo

Dulbecco’s

minimal

essential

medium

and Ham’s F-12

plus 5% bovine calf serum. Cells were fed twice weekly and subculturedjust prior to reaching confluence. CHO cells were obtained from Dr. J. Esko, Department of Biochemistry, Univ. of Alabama at Birmingham, and cultured as describedpreviously (17).

30 0.4

‘ii % P 9 2 0.2

Isotopic Flux Assays

i Ei 0.0 Control

Cl-free

DrnS

Cl-free

DLS Fig. 3. The effect of Cl--free medium (gluconate replacement) and/or 4,4’-diisothiostilbene-2,2’-disulfonic acid (DIDS) (100 PM) on lz511 efflux from T84 and CHO cells. Efflux rate constant was reduced from 0.55 to 0.20 s-l by Cl- removal or DIDS in CHO cells; basal (nonforskolin stimulated) T84 efflux was unaffected.

corresponds to the suggested DS binding site of the murine anion exchange protein and showed the specificity of this antibody. Third, we used Western blot analysis to show that this antibody, following forskolin treatment

We used radioisotopic anion uptake or efflux measurements to correlate antibody binding with CAMP-regulated anion transport asa function of the experimental conditions. In most of the flux assays,“‘1 was employed as the tracer for anion conductanceor exchangepathways. Our studiesfocusedon two cell lines, T84 and CHO (line Kl). T84 cells are known to express CAMP-stimulated secretory Cl- channels, and CHO cells expressan anion exchangepathway that hasbeen previously characterized (17). Previous single-channel measurementsindicate that the permeability of T84 Cl- channelsto I- is 1.7-fold greater than that of Cl- (20). In addition, I- is a poor substrate for Na+-K+2Cll cotransport processes(43). I- traversesthe anion exchanger of red blood cells at -l/260 the rate of Cl- (13). The results of critical experiments were confirmed using “Cl-.

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DIDS-BINDING

Cl38

PROTEIN

FROM T84 CELLS

of monolayer “‘I- accumulatedper milligram protein. All flux values representthe mean of duplicate determinations. Isotopic anion efflux. For the efflux studies,cells were seeded and grown asdescribedabove. The efflux protocol is described in detail (49). Briefly, monolayers were loadedto “‘I- equilibrium and efflux was followed by a sample-replaceprocedure. ““II efflux was treated as a first-order processby plotting log cellular “‘I- as a function of time. The slope of this relation provides the first-order efflux rate coefficient, which is useful for quantitating the effects of agonists or inhibitors. The primary advantageof the efflux protocol is that it permits control and experimental data to be collected from a single dish of cells, whereasthe uptake assayrequires several dishesof cells for a complete time courseunder a single experimental condition.

55+

Crude Membrane

+DIDS -DIDS Fig. 5. Western blot of detergent-solubilized T84 high-speed crude membrane homogenates (50 pg) overlaid with anti-P49 antibody. Prior to homogenization, half of cultures were exposed to 100 WM DIDS under conditions known to lead to irreversible block of anion transport (see METHODS). T84 cells were pretreated with ouabain and forskolin added in Cl--free medium following identical protocol used in ““Itracer uptake experiments (see legend to Fig. 1). Isotopic anion uptake. All experiments were conducted at 37°C. For the anion uptake measurements, five 35-mm cultured dishes were seeded with 0.5 x 10” cells per ml 5-6 days before the experiment and fed once in the intervening period. Dishes

were usedwhen they were -80% confluent. The uptake conditions were similar to those of Garty, Rudy, and Karlish (22), and, under theseconditions, T84 cellsexpressconductancesfor both Cl- and K’ (12). The outwardly-directed K’ gradient maintains a cell-negative membranepotential, which tends to dissipatean electrical driving force for IL’II uptake established by an outwardly directed Cll gradient. Accordingly, cells were preincubatedwith ouabain for 60 min to replacecell K’ by Na’ and minimize the influence of K’ conductanceson maximum velocity. Cellswereremoved from the incubator, and the growth mediumwasreplacedby a NaCl buffer containing (in mM) 140 NaCl, 1 CaS04, 1 MgS04, 10 glucose,10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (pH 7.4), and 0.1 ouabain.After a brief wash,the cellswere exposedto an uptake buffer containing (in mM) 140 Na-gluconate, 1 CaSO+ 1 MgS04, 10 glucose,10 HEPES (pH 7.4), and 0.1 &!i ‘>‘,I-. The uptake buffer was removed at timed intervals, and the plates were washedfour times with ice-cold isotonic mannitol. One milliliter 10% tricarboxylic acid (TCA) was added and plates were refrigerated overnight. The TCA extract wasremovedand counted. Cells remaining in the dish were solubilized in 1 ml 1 N NaOH, and protein was determined using the Bio-Rad protein assaykit. Results are expressedas counts per minute

Preparation

Twenty loo-mm dishesof T84 cells (at 70-90% confluence) were scrapedinto 60 ml of isolation medium, consisting of 250 mM sucroseand 10 mM triethanolamine HCl, pH 7.6, and a mixture of the following proteaseinhibitors: aprotinin (25 pg/ ml), leupeptin (10 Fg/ml), pepstatin (10 pg/ml), phenylmethylsulfonyl fluoride (PMSF) (175 hg/ml), and deoxyribonuclease(DNase) (2 pg/ml). The scrapedmaterial was homogenized on ice with a motor-driven Teflon pestle (10 strokes). The homogenatewasspunfor 10 min at 5,000rpm in an SS34rotor. The resulting pellet was discarded, and the supernatant was centrifuged for 60 min at 20,000 rpm. The pellet was resuspended in ice-cold solubilization medium containing 150 mM Na2HP04, 1 mM EDTA, 10 mM 3-[(3-cholamidopropyl)dimethylammoniol-1-propanesulfonate, pH 7.4, 0.5% (wt/vol) eggphosphatidylcholine, 175 pg/ml PMSF, and 20% (vol/vol) glycerol and wasconcentrated to 1.5 mg/ml, supplementedwith 0.005%NaN: