THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No .6 , Issue of February 25, pp. 2839-2843,1986 Printed in U.S.A.

Photoaffinity Labeling of the Epithelial Sodium Channel* (Received for publication, June 27, 1985)

Thomas R. KleymanS, Teresa Yulo, Cameron Ashbaugh, Donald Landry, Edward Cragoe, Jr.& Arthur Karlin, and &ais Al-Awqati From the Departments of Medicine, Physiology, Neurology, and Biochemistry, College of Physicians and Surgeons, Columbia University, New York, New York 10032 and CMerck, Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486

Sodium enters tight epithelia across the apical plasma membrane through a sodium channel, a process inhibited by submicromolar concentrations of amilor- ide and benzamil. Using membrane vesicles from bo- vine kidney cortex, we found that sodium transport through the sodium channel was inhibited by benzamil with an ICs0 of 4 nM. Amiloride (ICs0 = 400 nM) was a weaker inhibitor of sodium transport. [3H]Benzamil bound to the vesicles at a single class of high affinity binding sites with a &of 5 nM, the similarity of which to the ICso suggests that these binding sites are associ- ated with the sodium channel. Amiloride displaced bound [3H]benzamil with a &of 2,500 nM. Bromoben- zamil is a photoactive amiloride analog with potency similar to benzamil in inhibiting sodium transport (ICs0 = 5 nM) and binding to the sodium channel (&= 6 nM). [3H]Bromobenzamil was specifically photoincorpo- rated into three molecular weight classes of polypep- tides with apparent M,values of 176,000,77,000, and 47,000. The photoincorporation of [3H]bromobenzamil into these three classes of polypeptides was blocked by addition of excess benzamil and by amiloride in a dose- dependent manner. These data suggest that these poly- peptides are components of the epithelial sodium chan- nel.

The plasma membrane of epithelial cells contains distinct apical and basolateral domains, whose series arrangement allows vectorial transport of solutes across the cell. In a “tight” epithelium, sodium passively enters the cell across the apical plasma membrane through a sodium-selective channel, which is inhibited by submicromolar concentrations of the diuretic amiloride and is electrophysiologically and pharmacologically distinct from the voltage-gated sodium channel. Sodium exits the cell across the basolateral membrane through the Na,K- ATPase (1). The amiloride-sensitive sodium channel is pres- ent in epithelia from a variety of sources, including the kidney cortical collecting tubule, distal colon, trachea, skin, and urinary bladder (1, 2). In these tight epithelia, the rate of net transepithelial sodium transport is influenced by a variety of factors including the activity of sodium in the medium bathing the apical plasma membrane (3), the intracellular sodium activity (4) and calcium activity ( 5 ) , cell metabolism (6), and

* This research was supported by Grants AM20999 and AM34742 from the United States Public Health Service. The costs of publica- tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Supported by a Clinician-Scientist Award from the American Heart Association with funds provided in part hy the New York Heart Association.

the hormones aldosterone and vasopressin (1, 7, 8). Changes in net transepithelial sodium transport are due, at least in part, to changes in the number and/or probability of opening of sodium channels in the apical plasma membrane (3,643).

To gain further insight into the mechanisms of regulation of the sodium channel, we must identify and eventually purify its protein components. In this paper, we used amiloride analogs to characterize the sodium channel present in mem- brane vesicles obtained from bovine kidney cortex and used a photoactive amiloride analog to identify components of the channel. A preliminary report of this work has appeared (9).

EXPERIMENTAL PROCEDURES

Materials-Amiloride, benzamil, bromobenzamil, [ben~yl-~HIben- zamil, and [benzyl-3H]bromobenzamil were prepared at Merck, Sharp and Dohme Research Laboratories by methods previously described (10). Carrier-free **NaCl was obtained from Amersham Corp. All other compounds used were reagent grade.

Preparation of Bovine Kidney Cortical Membrane Vesicles-Fresh bovine kidneys were obtained from a kosher slaughterhouse and kept on ice. All subsequent steps were performed at 4 “C. Cortical tissue was removed, finely minced, and washed twice in a buffer containing 100 mM KC1, 1 mM EDTA, 10 mM KPO, (pH 7.4), 0.1 mM phenyl- methylsulfonyl fluoride, and 1.0 pg/ml pepstatin A (homogenizing buffer). The tissue was suspended 1 : l O (w/v) in homogenizing buffer and homogenized with 10 strokes in a Teflon-glass homogenizer at 800 rpm. The homogenate was spun at 1,000 X g for 10 min to pellet unbroken cells and nuclear debris. The supernatant was spun at 6,000 X g for 5 min to pellet mitochondria and again at 15,000 X g for 20 min. The supernatant and loose white outer pellet were then spun at 40,000 X g for 1 h. The pellet was suspended in homogenizing buffer and spun at 40,000 X g for 30 min. The final pellet was resuspended in the same buffer and stored at -20 “C. Aliquots were thawed and used for binding studies.

To measure sodium influx, we prepared vesicles using a procedure similar to that described above with the following modifications. The buffer used contained 55 mM NaCl, 87.5 mM sucrose, 2.5 mM EDTA, 12.5 mM imidazole (pH 7.0), 0.1 mM phenylmethylsulfonyl fluoride, and 1.0 pg/ml pepstatin A (transport buffer). Following the spin at 1,000 X g for 10 min, the supernatant was spun at 10,000 X g for 5 min. The supernatant and loose white outer pellet were spun at 30,000 X g for 1 h. The pellet was resuspended in transport buffer and spun again at 40,000 X g for 30 min. The pellet was suspended in buffer, stored at 4 “C, and used within 24 h for transport studies.

Transport Assay-Sodium transport in bovine kidney cortical membrane vesicles was measured with a modification of the method of Garty et al. (ll), as described in the legend of Fig. 1.

Binding Studies-binding of [3H]benzamil (4.7 Ci/mmol) to bovine kidney cortical membrane vesicles was assayed by equilibrium dialysis with 12,000-14,000 molecular weight cutoff dialysis tubing. Mem- brane vesicles, containing approximately 250 pg of protein, and [3H] benzamil were placed in the dialysis tubing, which was then placed in a test tube with 7.5 ml of homogenizing buffer containing the same concentration of [3H]benzamil. The tubes were stirred on a flatbed rotary mixer (160 rpm) for at least 16 h at 4 “C, by which time equilibrium had been achieved (data not shown). Aliquots were re- moved from the dialysis bag and dialysate to determine the [3H] benzamil concentrations. Protein determinations were performed by

2839

2840 Photoaffinity Labeling of the Epithelial Sodium Channel the method of Bradford (12), using bovine serum albumin as the standard. Nonspecific binding was determined in parallel experiments in which 1 p~ unlabeled benzamil was added to the vesicles and dialysate.

Photoaffinity Labeling-Bovine kidney cortical membrane vesicles were diluted to a protein concentration of 0.125 mg/ml with homog- enizing buffer containing 20 nM [3H]bromobenzamil (21.2 Ci/mmol) and incubated for greater than 1 h at 4 "C. The vesicle suspension (2 ml) was stirred at room temperature and irradiated for 15 min with light from a mercury arc lamp (Zeiss HBO, 50 watts) filtered through 1345-nm long pass and 300-400-nm band pass filters (Oriel Corp., Stratford, CT). Two filters were used to eliminate all wavelengths <300 nm. The vesicles were then diluted to 10 ml with homogenizing buffer and spun at 40,000 x g for 30 min. Nonspecific incorporation of the photolabel was determined with the addition of benzamil or amiloride. The pellets were resuspended in 0.6 ml of homogenizing buffer at 4 "C; 5.4 ml of acetone was added, and after 5 min precipi- tated protein was collected with a 200 X g 5-min spin. The protein pellet was washed again with 3 ml of acetone and dried with a stream of argon. The acetone not only precipitated protein but also extracted low molecular weight labeled products, which otherwise ran at the dye front on SDS-PAGE.'

SDS-PAGE-Protein samples (100 pg) were incubated at 90 "C for 1 min in 62.5 mM Tris-C1, pH 6.8, 3% SDS, 5% (v/v) 2-mercaptoeth- anol and subjected to SDS-PAGE on an 8-15% gradient acrylamide gel (13). To visualize labeled proteins, gels were soaked for 30 min in Amplify (Amersham Corp.), dried, and exposed for 9 days. Protein samples were also subjected to 8.75% SDS-PAGE and then sliced. The slices were incubated for 16 h at 37 "C in Econofluor with 10% Protosol (New England Nuclear) and counted.

RESULTS

Benzamil-sensitive Sodium Channel-When sodium-loaded vesicles are passed down a cation exchange column, a large outwardly directed concentration gradient for sodium is gen- erated; however, only the vesicles which contain a sodium channel will develop a membrane potential inside negative. Hence, "Na added to the extravesicular medium will accu- mulate in sodium channel-containing vesicles. "Na uptake into vesicles was measured as a function of time and was found to increase over 10-15 min, the rate being nearly linear with time for the first 4 min (Fig. 1, a and b (Nain)). To demonstrate that "Na uptake was potential dependent, the membrane potential was collapsed with the addition of 5 mM KC1 and 5 WM valinomycin to the extravesicular space. The initial rate of 22Na uptake was reduced by 75%. To examine cation selectivity of the channel, vesicles were loaded with a buffer in which 55 mM choline c1 or 55 mM LiCl replaced the NaC1, and 22Na uptake was measured. LiC1- and NaC1-loaded vesicles had similar rates of "Na uptake, whereas the rate of uptake into choline C1-loaded vesicles was reduced by 70% (Fig. lb) . This cation selectivity was similar to that reported in electrophysiological studies of the channel in intact epithe- lia (14). When the transmembrane sodium gradient was re- duced by addition of extravesicular NaC1, the initial rate of "Na uptake was maximally reduced (Fig. la).

Benzamil inhibited the initial rate of "Na uptake (Fig. la). However, two sites of transport inhibition were observed (Fig. 2a). Up to concentrations of 100 nM, benzamil inhibited the initial rate of 22Na uptake between 30 to 50%. This high affinity site had an IC50 of 4 nM (Fig. 2b), similar to the ICbo of 10 nM found for inhibition of sodium transport by benzamil in frog skin (15). At concentrations of benzamil greater than 1 WM, further inhibition occurred with an ICso between and M (Fig. 2a). The sodium flux inhibited at higher concentrations of benzamil is probably due to Na'/Na' ex- change, driven by the outwardly directed sodium gradient,

' The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

Noin

o .,= Cholinei,

5 IO

[ min)

FIG. 1. "Na uptake in bovine kidney membrane vesicles. a, membrane vesicles were prepared in transport buffer and incubated overnight with (0) and without (0, A) 1 p~ benzamil to allow equilibration of the drug across the vesicle membrane. All steps were performed at 4 "C. The vesicle suspension (200 pl, 1 mg/ml) was passed through Dowex 50-X8 cation exchange resin (Tris form), packed in Pasteur pipettes (5 X 0.5 cm), to bind the extravesicular sodium and benzamil. The vesicles were eluted with 1 ml of 175 mM sucrose. The columns were washed with 1.5 ml of 175 mM sucrose containing 25 mg/ml bovine serum albumin prior to use. Eluted vesicles (400 pl) were diluted into 400 pl of 175 mM sucrose (0), 175 mM sucrose plus 2 pM benzamil (a), or transport buffer containing 55 mM NaCl (A). Vesicles were incubated for 30 s, and uptake was then initiated with addition of 20 pl(4 pCi) of "NaC1. After incubation for varying time intervals, 100 pl of vesicles were removed, passed down a second cation exchange column, and eluted with 1.5 ml of 175 mM sucrose. The eluate was counted, and the "Na uptake was expressed as a percentage of the total counts present in the transport assay. b, the protocol was the same as in a, except that following isolation, the membrane vesicles were washed twice in transport buffer containing either 55 mM NaCl (0) or 55 mM choline C1 (M) which replaced the NaC1, and then resuspended in the same buffer at a protein concentration of 1 mg/ml.

[BENZAMIL] (MI [INHlBlTOR] ( M I

FIG. 2. Inhibition of 22Na uptake by benzamil and amilor- ide. a, the initial rate of "Na uptake was measured, as described in the legend of Fig. 1, in the presence of increasing concentrations of benzamil. Results were expressed as a percentage of the initial rate in the absence of inhibitors. Both a high affinity site of inhibition of "Na transport, representing transport through the sodium channel, and low affinity site were observed. b, the initial rate of "Na uptake was measured in the presence of increasing concentrations of benza- mil (O), bromobenzamil (O), or amiloride (0) to examine transport through the sodium channel in greater detail.

either through the Na'/H' exchanger, the Na+/Ca2+ exchan- ger, or the Na+,K'-ATPase. All of these transport processes have been shown to be inhibited by amiloride or benzamil at concentrations of M or greater (16-19).

Bromobenzamil inhibited "Na uptake through the high affinity site with an ICbo of 5 nM. Amiloride inhibited "Na transport at the high affinity site with an ICs0 of 400 nM (Fig. 2b), in reasonable agreement with studies in isolated perfused

Photoaffinity Labeling of the Epithelial Sodium Channel 2841

rabbit cortiGa1-collecting tubules that have shown amiloride inhibition of sodium transport with a Ki of 100 nM (20). Using electrophysplogical studies in the toad urinary bladder and frog skin, ye and others (15) also found that amiloride is a weaker inbibitor of the sodium channel than benzamil or bromobenzbil.

Benzamit;b,inding Sites-Scatchard analysis of binding of [3H]benzaMil to bovine kidney cortical membrane vesicles showed a siijgle class of high.affinity binding sites with a K d

of 5 f 1 nu (+S.D., n = 4, 'Fig. 3), in good agreement with the ICso of'4.nM observed.in transport studies, suggesting that benzamil is binding tq the putative sodium channel. The number of binding sites was 130 f 22 pmoljmg of protein (n = 4). [3H]Bmmobenzamil'binding to the membrane vesicles was also studied, and Scatchard analysis showed a single class of high affiqity sites yith,a Kd of 6 nM. The nuinber of binding sites was 80.pmol/mg,of protein (n = 1). The ratio of specific to nonspecific binding was 3 to 1 (at a cohcentration of free [3H]bromobenzamil of 15 nM). We compared .the relative affinities of bromo4enzamil and amiloride for binding to the putative sodium channel by competition with [3H]benzamil. The Ki (the concentration giving.50% displacement of specific [3H]benzamil bindhg) for bromobenzamil was 4 +: 2 nM (n = 3), and the Ki of amilorid6 'was 2500, f 1000 IIM f n = 4, Fig. 4, Table I). We used a nonlinear least squares method to test whether a two-site model fit .the amiloride data"bebter tKan a single-site model. It-.did not; -however; a :Hill piot of the inferred amiloride binding had a Hill coefficient of 0.7.

As sodium transport in ,intact' bovine renal epithelia has not been studied, it is difficult to,predict the expected number of binding sites/mg of protein. Further, recent evidence sug- gests that both open and closed sodium channels may bind amiloride (21). It is possible, therefore, that closed channels may contribute to the large number of binding sites observed in the bovine kidney cortical membrane vesicles. We..have prepared membrane vesicles from rat kidney.cortex and toad urinary bladder, and with Scatchard analysis of [3H]benzamil binding, found a single class of high affinity sites, (22) with affinities similar to that found in the bovine kidney cortical membrane vesicles (Kd values of3 and 11 nM, respectively). The numbers of binding sites (19 pmol/mg of protein and 23 pmol/mg of protein, respectively), however, were considerably

I A

0 5 I O I 5 20 FREE [ 'HI BENZAMIL (nM1

25 50 7 5 100 125 I50

FIG. 3. A, binding of increasing concentrations of [3H]benzamil to bovine kidney cortical membrane vesicles was measured with the method of equilibrium dialysis, as described under "Experimental Procedures." Results from one experiment are shown. Points are means of duplicates. 0, total bound [3H]benzamil; 0, nonspecific bound [3H]benzamil, determined in the presence of 1 PM unlabeled benzamil; 0, specific bound [3H]benzamil. B, Scatchard analysis of specific binding of [3H]benzamil to the membrane vesicles. A disso- ciation constant of 5 k 1 nM (+S.D., n = 4) was obtained. The number of binding sites was 130 & 22 pmol/mg of protein. A similar number of high affinity binding sites (90 pmol/mg of protein) was found in membrane vesicles prepared for transport assays.

0

ZJ 7 5 -

0 m

$ 50 a

-

N z 25 -

n I ." u I I I I I I I

10-9 10 -8 10-7 1 0 - 6 10-5 10-4

[INHIBITOR] (M)

FIG. 4. Inhibition of [3H]benzamil binding to bovine kidney cortical membrane vesicles by benzamil (O), bromobenzamil (a), and amiloride ( 0 ) . Binding was measured as described under "Experimental Procedures." Results are expressed the percentage of [3H]benzamil bound in the absence of inhibitors.

TABLE I Inhibition of transport and binding to the putative sodium channel by

amiloride analoes

Amiloride Transport Binding analog ( I C W ) K; -e SD. K d & S.D.

IZM

Benzamil 4" 5 + 1 ( n = 4 ) Bromoben- 5" 4 + - 2 ( n = 3 ) 6 (n = 1 )

Amiloride 4OOb (n = 3) 2500 ? 1000b (n = 4) zamil

n = 1, triplicates. Individual amiloride experiments were plotted on the same graph

and the IC50 and Ki were estimated from the combined results. The S.D,, however, was obtained from analysis of the individual experi- ments.

less than that found in bovine kidney cortical membrane uesicles.

Photoaffinity Lubeling-When frog skin,was irradiated with UV in the presence,of bromoamiloride, sodium transport was irreversibly inhibited (23). These data suggested that bro- moimiloride ( k d bromobenzamil) 'mai be useful as photoaf- finity lab'els for the epithelial ,sodium channel. Bromobenza- mil 'is a potent inhibito:r, of the sodium channel and binds to the 9utative.channel with sufficiently high affinity (see above) -to allow its use 'as a photoaffinity label. When 50 n~ [3H] bromobenzamil was photolyzed in' the presence of bovine kidney cortical membrane vesicles, incorporation of tritium intci trichloroacetic acid-precipitable protein increased with the duration of photolysis. After 15 min of photolysis, 10.7 f 0.2 X lo3 cpm was bound per 0.1 mg of protein (fS.D.; n = 3). The presence of 1 p~ benzamil reduced photoincorporation by 7.5 & 0.1 x lo3 cpm. This reduction of counts-was not due to absorbance of light by 1 p~ benzaqjl, which was only about 0.02 ( E ~ ~ ~ ~ ~ ~ ~ ~ (360nml = 20,000 M-' cm"j. .. . .

Membrane vesicles Were photolyzed with 20 nM [3H]bro- mobenzamil and then sbbjected'to SDS-PAGE. Three major bands of radioactivity were observed (Fig. 5), with. apparent M, values of.176,000 +; 9,000, 77,000 f 3,000, and 47,000 +i 2,000 (+S.D., n = 6). Counts were also. fioted at the tQp of the gel and remained at the tap of 5-15% gradient gels (data not shown). Photoaffinitylabeling was performed with the addi- tion of 200 nM benzamil or 200 nM amiloride. Incorporation of the photolabel into the three classes of polypeptides with apparent M, values of 176,000, 77,000, ,and 47,000 was. inhib- ited by 200 nM benzami1,'whereas minimal inhibition was noted in the presence of 200 nM amiloride, (Fig. 5, Table 11). At a concentration of 200 nM, amiloride inhibition of [3H,].

2842 Photoaffinity Labeling of the Epithelial Sodium Channel

1 2 3

-top

- 1 1 6 K - 9 7 K

- 6 6 K

- 4 5 K

- 2 9 K

FIG. 5. SDS-PAGE of bovine kidney cortical membrane vesicles photolabeled with [3H]bromobenzamil. Membrane ves- icles were prepared and photoaffinity labeled with: lane I, 20 nM ['HI bromobenzamil plus 200 nM benzamil; lane 2, 20 nM [3H]bromoben- zamil plus 200 nM amiloride; lane 3, 20 nM [3H]bromobenzamil; and subjected to 8-15% SDS-PAGE followed by fluorography as described under "Experimental Procedures." Migration of molecular weight standards is shown to the right of the fluorogram. Results from one experiment are shown.

TABLE I1 Photoincorporation of rH]bromobenzamil into M, 176,000, 77,000,

and 47,000 polypeptides; inhibition by benzamil and amiloride Inhibitor

None Benzamil Amiloride

(*O0 nM) 200 nM 1000 nM 5000 nM

specific cprn" 176,000 Da 244 67 204 149 76 77,000 Da 446 86 363 226 83 47,000 Da 500 126 421 208 104 Photoaffinity labeling was performed with 20 nM [3H]bromoben-

zamil and unlabeled amiloride analogs as described under "Experi- mental Procedures," followed by 8.75% SDS-PAGE. Gels were sliced and incubated in Econofluor with 10% Protosol. Counts at M , 176,000, 77,000, and 47,000 were obtained, and counts from parallel experiments with 1 p M benzamil were subtracted (159, 228, and 182 cpm at M , 176,000, 77,000, and 47,000, respectively) to determine the specific cpm at each molecular weight region. The average of counts from 2 experiments is shown.

benzamil binding to the sodium channel and inhibition of "Na transport was slight (Figs. 2b and 4). However, 200 nM benzamil produced nearly complete inhibition of binding and transport. Further inhibition of photoincorporation of the photolabel was noted in the presence of 1000 and 5000 nM amiloride, in a dose-dependent manner (Table 11).

The cpm specifically incorporated into the 3 major bands was 1190 (see Table 11). This represents incorporation of 0.8 pmol of [3H]bromobenzamil/mg of protein or approximately 1% of the benzamil-binding sites. Consistent with this low level of incorporation, we were unable to detect an effect of

photoincorporation of bromobenzamil on sodium transport or [3H]benzamil binding. Neither in the presence nor in the absence of 20 nM bromobenzamil did photolysis of vesicles for up to 8 min reduce the initial rate of "Na transport. Moreover, photolysis for 15 min did not reduce the number of high affinity [3H]benzamil-binding sites in the presence or absence of 20 nM bromobenzamil (n = 5). Addition of 20 nM bromobenzamil and 1000 nM benzamil to vesicles prior to photolysis for 15 min did not significantly reduce the number of binding sites. Thus, the decrease in the number of sodium channels following photoaffinity labeling appears to be too small to be detected in our transport and binding assays.

DISCUSSION

A sodium conductive pathway was found with transport studies in membrane vesicles from bovine kidney cortex. Sodium transport was through an electrogenic pathway that was conductive to sodium and lithium, but poorly conductive to choline, and was inhibited by benzamil, bromobenzamil, and amiloride with ICs0 values for a high affinity site of 4, 5, and 400 nM, respectively (Table I). These are well known characteristics of the epithelial sodium channel (14, 15, 20).

The Kd (or Ki) for binding of both benzamil and bromoben- zamil to the membrane vesicles is in excellent agreement with the ICs0 found in transport experiments with these drugs, strongly suggesting that benzamil and bromobenzamil are binding to the putative sodium channel (Table I). The Ki of 2500 nM for amiloride is greater than its IC50 of 400 nM. Amiloride binding, however, is complex, as reflected in the Hill coefficient of 0.7. The interaction of amiloride with the sodium channel will require further study in a more homoge- neous system. A similar Hill coefficient has been obtained for the interaction of amiloride with the sodium channel from a variety of anuran epithelia, analyzed by electrophysiologic studies (24).

[3H]Bromobenzamil (20 nM) binds to the high affinity sodium transport site, or putative sodium channel, and cova- lently incorporates into three classes of polypeptides with apparent M, values of 176,000, 77,000, and 47,000. Benzamil, a t a concentration of 200 nM, inhibited the photoincorpora- tion of [3H]bromobenzamil into these proteins, whereas 200 nM amiloride did not. The correlation of the dose response of the amiloride analogs for transport inhibition, binding affinity (Table I), and inhibition of [3H]bromobenzamil photoincor- poration (Fig. 5, Table 11) strongly suggests that [3H]bromo- benzamil is labeling protein components of the putative epi- thelial sodium channel. However, only when the sodium chan- nel is purified and reconstituted can one be certain that these proteins are components of this transporter.

A number of other membrane transporters have been shown to be inhibited by benzamil or amiloride. These include Na'/ H+ exchanger (16), Na+/Ca'+ exchanger (17), Na+/K+-ATP- ase (18,19), Na+-alanine and Na+-glucose cotransporters (18), voltage-gated sodium channel,' and nicotinic3 and muscarinic acetylcholine receptors (25). However, concentrations of ben- zamil or amiloride required to achieve significant inhibition of these transporters were greater than M (16-19, 25). Only the epithelial sodium channel is inhibited by nanomolar concentrations of benzamil and amiloride. It is likely, there- fore, that binding and photoaffinity labeling with nanomolar concentrations of benzamil and bromobenzamil are specific for the epithelial sodium channel.

B. Rudy, J. Bennett, and J. Weiner, personal communication. A. Karlin, unpublished result.

Photoaffinity Labeling of the Epithelial Sodium Channel 2843

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