Biohimia et Biophysia Ata 1370 1998 169 174 Cu reveals different binding sites of amiloride and CDPC on the apial Na hannel of frog skin M.L. Flonta b,), J.De Beir-Simaels a, D. Mesotten a, W. Van Drieshe a a Laboratorium Õoor Fysiologie, K.U.LeuÕen, Campus Gasthuisberg, B- 3000 LeuÕen, Belgium b Dept. Physiology and Biophysis, Faulty of Biology, UniÕersity of Buharest, Splaiul Independentei 91 95, Buharest 76201, Romania Reeived 8 August 1997; revised 4 November 1997; aepted 4 November 1997 Abstrat The effet of Cu ions, present in the muosal bathing solution, on the transepithelial short-iruit urrent Ž I. and ondutane Ž G. t and on the bloker-indued noise of apial Na hannels, as studied on the isolated ventral skin of the frog Rana temporaria. Cu effets ere onentration-dependent, the full effet being reahed at 50 mmolrl. Cu inreased I and G ; this effet as eliminated by high onentrations of amiloride Ž 30 mmolrl. t and of CDPC 150 mmolrl. Cu markedly redued the orner freueny Ž f. of the Na hannel noise, hile having virtually no effet on the f of CDPC-indued noise. Cu redues the assoiation rate onstant of amiloride to the Na hannel to one third; this effet is interpreted as indiating ompetition beteen Cu and amiloride for the same Ž negatively harged. binding site on the hannel, hile CDPC appears to bind on a different site. 1998 Elsevier Siene B.V. Keyords: Frog skin; Copper; Na hannel; Bloker-indued noise; Amiloride; CDPC 1. Introdution The apial Na hannel of frog skin is a member of a nely identified ion-hannel family, hih is haraterized by sensitivity to amiloride. The primary struture of an epithelial Na hannel Ž ENaC. as first eluidated in 1994 x 1. The hannel is thought to be omposed of three homologous but nonidential subunits, named a, b and g x 2. The a-subunit alone an form fully funtional amiloridesensitive Na hannels, hereas the b- and g-subunits annot x 3. In turn, the presene of b-org-subunits ) Corresponding author. Fax: 40-1-2233933; E-mail: flonta@bio.bio.unibu.ro inreases the amiloride-sensitive urrent ; 10 fold. In spite of muh ne moleular information on the ENaC, a number of uestions need further investigation. Speifially, hat regions of the a-, b-, or g-subunits ontain the ondution pore, hat regions are involved in hannel gating, and hat regions interat ith regulatory effetors or knon blokers? A ay to obtain information hih an be used to predit some strutural features of the hannel is to investigate the binding affinities of the effetive blokers and ho these are influened by other interating agents. With this idea in mind e have studied the effet of Cu on the apial Na hannel of amphibian skin. This tissue is a good model of tight epithelia in general, it an be easily isolated ith 0005-2736r98r$19.00 1998 Elsevier Siene B.V. All rights reserved. PII S0005-2736 97 00261-7
170 ( ) M.L. Flonta et al.rbiohimia et Biophysia Ata 1370 1998 169 174 minimal damage and it is stable for many hours in vitro. Copper is a trae element essential for life x 4, hose defiieny or exess entail serious pathologix 5. Some years ago, Fereira 6 8x shoed that Cu stimulates the short-iruit urrent al onseuenes Ž I. through amphibian epithelia. Hoever, I is a omposite measure of the funtion of ioni hannels hose population is multifarious on both apial and basolateral membranes x 9 and of the basolateral Na rk pumps, so that the effet of Cu on I has to be disseted into its elementary omponents. Using a noninvasive method, Na hannel harateristis ere derived from the analysis of bloker-indued lorentzian noise, produed by to pyrazine derivatives, amiloride and CDPC Ž6-hloro-3,5-di- aminopyrazine-2-arboxamide.. Here e report data shoing that Cu ompetes ith amiloride for the same binding site on the apial Na hannel, hile it has only minor affinity for the CDPC-binding site. This latter site thus appears to be distint from the amiloride-binding site. 2. Methods Frogs Ž Rana temporaria., eighing around 25 g, ere kept at 178C ith free aess to tap ater. The abdominal skin of doubly pithed animals as disseted and mounted in an Ussing-type luite ham10 x. The hamber en- ber, as previously deribed sured negligible edge damage and alloed the ontinuous perfusion ith fresh solutions of both the muosal and serosal m s sides of the epithelium, at a rate of 5 mlrmin. The tissue area in ontat ith bathing media as 1 m 2. The transepithelial potential as lamped to zero ith a lo-noise voltage lamp 11x and the short-iruit urrent Ž I. as reorded on a standard X-T reorder. The transepithelial ondutane Ž G. t as alulated from the urrent hanges aused by 256 ms voltage pulses of 5 mv amplitude, applied every 14 s. 2.1. Solutions All solutions ere freshly prepared ith highly purified ater ŽMilli-Q Water Purifiation System, Millipore.. In order to eliminate the ontribution of Cl y to the measured I and G t, Cl-free Ringer solutions, having SO 2y 4 as the major anion, ere used. Na 2SO4 Ringer solution, bathing both sides of non-depolarized skins, onsisted of Ž in mmolrl.: 115 Na, 58.5 SO 2y, 2.5 K, 2.5 HCO y,1ca 4 3.K2SO4 Ringer solution, used to depolarize the serosal side of the epithelium, ontained in mmolrl : 97.5 K, 20 Na, 58.5 SO 2y, 2.5 HCO y and 1 Ca 4 3. The ph of all solutions as adjusted to 8.0. The onentrations in the muosal ompartment of amiloride hydrohloride, Sigma. and of CDPC Ž6-hloro-3,5-diamino- pyrazine-2-arboxamide, Aldrih. respetively ranged from 2 to 40 mmolrl ŽAMIx. m and from 10 to 150 mmolrl ŽCDPCx. 12 x m. Copper as added as CuSO ; free Cu 4 onentrations are estimated by preliminary ondutane measurements, to be 30% loer than the values given in the ase of both amiloride and CDPC. 2.2. Noise analysis The flutuation in urrent as reorded and analyzed in the freueny domain, as previously de13 x. The tehniue exploits the fat that the ribed average maroopi Na urrent Ž ma. onsists of the random flikering of millions of independent hannels Ž pa. hih gives rise to flutuations Ž na. around the mean urrent. These data an be resolved into their freueny omponents using the Fourier transform. The urrent signal as high-pass filtered ith a 24 dbrotave Butterorth type filter ith a ut-off freueny of 0.3 Hz. Furthermore, to prevent aliasing, the signal as lo-pass filtered ith a 48 dbrotave Butterorth filter Žut-off freuenys 850 Hz.. Poer density spetra Ž PDS. ere alulated from Fourier transformed reords of 4096 points olleted over a 2 s period, yielding a fundamental freueny of 0.5 Hz. Averaged PDS of 2048 freuenies ere obtained from 50 reords. Freuenies above 760 Hz ere diarded in further analysis. Spetra ontaining relaxation noise ere fitted ith the sum of a lorentzian omponent and a bakground noise term Ž Arf a. aording to the folloing formula: So A SŽ f. s 2 a 1 Ž frf. f
( ) M.L. Flonta et al.rbiohimia et Biophysia Ata 1370 1998 169 174 171 The plateau value Ž S. o and the orner freueny Ž f. of the lorentzian funtion, determined by nonlin- ear regression analysis of the PDS, ere used for the alulation of single hannel urrent and hannel density, aording to a to-state model for hannel opening and losing 9,14 x. A represents the poer density at 1 Hz and a the slope of the 1rf noise omponent. The model predits a linear dependene of f on the onentration of the flutuation-induing bloker B: x 2p fsk01 B k10 Ž 1. here k and k respetively are the ON- Ž 01 10 assoiation. and the OFF- Ž dissoiation. rate onstants. It also allos alulation of the single hannel urrent Ž i. and the number of open hannels Ž N. Na o in the presene of the bloker, from the plateau value Ž S o. and the maroopi Na urrent Ž I.: 2 SoŽ 2p f. inas Ž 2. 4INak01 B NosI NariNa Ž 3. 3. Results The data in Fig. 1 shos the stimulatory effet of Cu on the transepithelial urrent and ondutane. The onset of dose-dependent inhibition of I and Gt by amiloride as sloed don after the addition of Cu on the apial side of the skin ŽFig. 1Ž A.., the time onstant of the exponential fall rising from about 1 s to more than 3.5 s after Cu addition. The sloing by Cu of the onset of CDPC blok of I as less obvious ŽFig. 1Ž B... In all experiments, Cu xm as 50 mmolrl, be- ause a preliminary investigation of the effet of Cu xm on bloker-indued noise of apial Na han- nels indiated this as the loest onentration giving a maximal f hange, 10 min after Cu addition Ž Fig. 2.. The effet of Cu on the spetrum of amilorideindued noise of the apial Na-hannel is illustrated in Fig. 3, shoing the derease of the orner freueny and the inrease of the plateau values of the Na Fig. 1. Effet of Cu Ž 50 mmolrl, in the muosal ompartment. on the short-iruit urrent and transepithelial ondutane of frog skin, in the presene of inreasing muosal onentrations of amiloride Ž AMI. Ž A. and of CDPC Ž B.. AMIxm as sues- sively: 2, 4, 6, 10, 20 and 30 mmolrl and CDPC xm: 10, 20, 50, 100 and 150 mmolrl. The reords are representative for ŽA, above. 5 and Ž B, belo. 7 experiments. reorded lorentzian spetra. Fig. 3 is representative for 6 experiments in idential onditions and for the general appearane of the rude noise data. In aord Fig. 2. Effet of muosal onentration of Cu on the orner freueny of the amiloride-indued noise of apial Na hannels. f as reorded 10 min after inreasing CuSO x 4 m, in the pres- ene of 20 mmolrl AMI x. m
172 ( ) M.L. Flonta et al.rbiohimia et Biophysia Ata 1370 1998 169 174 x Fig. 3. Effet of 50 mmolrl Cu m on the Na urrent poer density spetrum, reorded in the presene of 6 mmolrl AMI x. m ith E. Ž 1. above, the dependene of f on muosal bloker onentration as approximately linear, in the ase of both amiloride and CDPC Ž Fig. 4.. A lear-ut effet of Cu, namely to redue the slope of the f - blokerx m dependene, as observed hen the bloker as amiloride ŽFig. 4Ž A.., but not hen the bloker as CDPC ŽFig. 4Ž B.., hile the interepts on the ordinate are less influened by Cu in either ase. In order to have an isolated apial membrane subjeted to Cu effets, the basolateral membrane as depolarized ith high K Ringer solution in the serosal ompartment. The dependene of f on amiloridexm as then studied in ontrol onditions and in the presene of 50 mmol Cu rl at the muosal side. The ensemble of bloker kineti onstants Ž k and k. 01 10 and of apial Na hannel harateristis Ži and N, alulated ith Es. Ž 2. and Ž 3. above. Na 0 Fig. 4. Effet of 50 mmolrl Cu xm on the dependene of noise orner freueny on bloker onentration, in the ase of 6 amiloride-treated frog skins Ž A, above. and of 7 CDPC-treated frog skins Ž B, belo.. are given in Table 1, for non-depolarized frog skins treated ith amiloride and CDPC, and for depolarized frog skins treated ith amiloride, both ithout and in the presene of Cu. In order to further haraterize the binding of Cu on the Na hannel, the influene of transepithelial voltage on the f of the noise indued by 20 mmolrl amiloride as studied, in ontrol sulphate Ringer and in the presene of 50 mmolrl Cu ŽFig. 5.. In ontrol f inreases at positive or negative Table 1 Effet of 50 mmolrl Cu xm on bloker ONrOFF rate onstants of amiloride and CDPC, and on the parameters of frog skin apial Na hannel, as revealed by bloker-indued noise. k - assoiation Ž ON. onstant, k - dissoiation Ž OFF. 01 10 onstant, ina - single hannel urrent and N0 - open hannel density. Values are given as mean "S.E.M Ž y1 y1. Ž y1. 6 Ž y2 Conditions k mmol s k s i pa N =10 m. 01 10 Na 0 Amiloride-treated Non-depolarized Ž Ns6. Control 11.77" 0.56 8.03" 11.77 0.40" 0.11 106.8" 35.8 Cu 3.30" 0.17 9.91" 3.52 0.70" 0.16 83.6" 20.0 K-depolarized Ž Ns5. Control 10.63" 0.30 13.10" 6.29 0.36" 0.07 98.8" 23.3 Cu 3.28" 0.01 15.74" 0.32 0.35" 0.08 126.3" 33.0 CDPC-treated Ns7 Control 6.96" 0.29 295.3" 24.8 0.67" 0.11 28.03" 5.08 Cu 6.68" 0.20 351.2" 16.5 0.57" 0.06 40.45" 8.23
( ) M.L. Flonta et al.rbiohimia et Biophysia Ata 1370 1998 169 174 173 Fig. 5. Effet of 50 mmolrl Cu xm on the dependene of noise orner freueny on transepithelial voltage, in the ase of 5 frog skins. The onentration of amiloride in the muosal ompartment as 20 mmolrl. transepithelial potentials; this effet is redued by opper. 4. Disussion The large inreases in transepithelial Ž short-iruit. urrent and ondutane, produed by Cu, are assoiated ith an inreased entrane of Na through the apial membrane, as indiated by the reords in Fig. 1, hih sho that blokers of the Na hannel abolished the Cu-indued inrease. Thus, in the presene of the highest dose of amiloride Ž 30 mmolrl., I had virtually the same value ith or ithout Cu ŽFig. 1Ž A.. and even the less potent bloker CDPC ompletely eliminated the Cu-indued inrease above the ontrol value. The onentration-dependent inrease of the transepithelial urrent by Cu is refleted in the uasi-exponential fall of f of the noise indued by a fixed onentration of bloker Ž Fig. 2., as ell as in the inreased plateau values, illustrated in Fig. 3. A larger transepithelial ioni urrent, as produed by Cu ould, in priniple, be attributed to inreases in either the urrent passing through eah hannel Ž i., or the number of open hannels Ž N. Na 0, or both. The harateristis of the Na hannel, revealed by the analysis of amiloride-indued urrent flutuations Ž Table 1. shos that, in non-depolarized frog skin, Cu signifiantly inreased i Na, hile it tended to derease N 0. Hoever, hen the basolateral membrane as depolarized ith an exess of potassium, i as not influened by the addition of Cu Na, hile N0 tended to inrease, indiating the possible existene of a group of voltage dependent Na hannels. This ould be suggested also by the influene of transepithelial voltage on the opper indued derease of f values at higher positive or negative transepithelial potentials Ž Fig. 5.. The most onspiuous effet of Cu as the redution to one third of the assoiation onstant k 01 beteen amiloride and the apial Na hannel, in both non-depolarized and depolarized frog skins Ž Table 1.. Amiloride is thought to stabilize the losed onformation of the Na hannel via the interation of its positively harged guanidinum group ith a arboxyl group ritially plaed at the entrane of the hannel 15 x,possibly on the H hairpin struture16 x 2. The strong derease of k by Cu 01 ould arise from the eletrostati shield by this ation of the net negative harge present at the binding site for amiloride, on the Na hannel protein. Our data suggest that Cu binding, hih hampers the attahment of amiloride, does not prevent Ž and even favours. the passage of Na through the hannel, as shon by the inreased I. The idea of ompetition beteen Cu and amiloride for the same binding site is further supported by the fat that in the presene of high onentrations of amiloride, the stimulatory effet of Cu on I vanishes ŽFig. 1Ž A... In this ase the ondi- tions are very far from euilibrium beteen amiloride and opper. As one might expet ithin the ontext of suh a model, the basolateral depolarization did not signifiantly influene either the value of k01 in ontrol muosal solution, or the effet of Cu on it. On the other hand, the dissoiation rate onstant k 10, on hih Cu alone had virtually no influene, tended to be inreased in depolarized skins, the differene in k10 beteen non-depolarized and depolarized skins approahing signifiane in the presene of Cu. The basolateral depolarization, hih makes the intraellular ompartment less negative, might failitate the dissoiation of the harged bloker from its binding site, partiularly in the presene of the ompetitor Cu, as a result of redued transapial field. The same ombination of eletrostati onditions appears to promote the tendeny toards an inreased number of open hannels. The assoiation rate Ž k. 01 of the amiloride analogue CDPC ith apial Na hannels, i.e. the slope of the urves in Fig. 4Ž B., as only negligibly influened by Cu, strikingly different from amiloride
174 ( ) M.L. Flonta et al.rbiohimia et Biophysia Ata 1370 1998 169 174 ŽFig. 4Ž A... This indiates that the CDPC moleule, hih is neutral at physiologial ph, bloks the hannel upon binding at a different site from amiloride. The dissoiation rate onstant of the CDPC Na hannel omplex, k 10, as more than ten times higher than for the amiloride Na hannel omplex Ž Table 1.. This is in agreement ith Helman and Baxendale 17x and ith previous results from this laboratory, hih indiated a lifetime of the blokerhannel omplex muh shorter for CDPC Ž 4 ms., than x for amiloride 75 ms 18. Cu still inreased k 10, in agreement ith its tendeny Ž above diussed. to promote the open onfiguration of the hannel. In summary, this study indiates a strong ompetition beteen Cu and the harged pyrazine derivative amiloride, for binding on the apial Na hannel of frog skin, and the lak of suh an antagonism beteen Cu and the neutral pyrazine derivative CDPC, thus pointing to the to blokers having different binding sites on the Na hannel. Moleular tehniues have revealed additional epithelial Na hannel family members. If the d-enac is oexpressed ith the b- and g-subunits, instead of the a-subunit, the hannel is 30-fold less sensitive to amiloride and has a unit ondutane tie as high 19 x. This suggests that the amiloride binding site ould be loated on the a-subunits, and it ould be interesting to determine the CDPC sensitivity of this novel hannel. Aknoledgements M.L.F. aknoledges support from the European Community ŽS & T Cooperation ith Central and Eastern European Countries -PECO Grant 1993.. She also expresses her gratitude to Dr. Gordon Reid for improving the English. Referenes x 1 C.M. Canessa, L. Shild, G. Buell, B. Thoreus, I. Ganthl, J.-D. Horisberger, B.C. Rossier, Amiloride-sensitive epithelial Na hannel is made of three homologous subunits, Nature 367 Ž 1994. 463 467. x 2 D.J. Benos, C.M. Fuller, V.G. Shlyonsky, B.K. Berdiev, I.I. Ismailov, Amiloride-sensitive Na hannels: Insights and Outlooks, Ne Physiol.Si. 12 Ž 1997. 55 61. x 3 I.I. Ismailov, M.S. Aayda, B.K. Berdiev, J.K. Bubien, J.E. Luas, C.M. Fuller, D.J. Benos, Triple-barrel organisation of ENaC, a lonned epithelial Na hannel, J. Biol. Chem. 271 Ž 1996. 807 816. x 4 W. Mertz, The essential trae elements, Siene 213 Ž 1981. 1332 1338. x 5 D.D. Perrin, R.P. Agaral, Multimetal multiligand euilibria: a model for biologial systems, in: H. Siegel Ž Ed.., Metal Ions in Biologial Systems, vol. 2, Mixed-ligand omplexes, Marel Dekker, NY, 1973, pp. 167 206. x 6 K.T.G. Ferreira, The effet of Cu on isolated frog skin, Biohim. Biophys. Ata 203 Ž 1970. 555 567. x 7 K.T.G. Ferreira, The effet of ooper on frog skin. The role of sulfhydryl groups, Biohim. Biophys. Ata 510 Ž 1978. 298 304. x 8 K.T.G. Ferreira, The mehanism of ation of Cu on the frog skin, Biohim. Biophys. Ata 552 Ž 1979. 341 345. x 9 W. Van Drieshe, W. Zeiske, Ioni hannels in epithelial ell membranes, Physiol. Rev. 65 Ž 1985. 833 903. x 10 I. De Wolf, W. Van Drieshe, Voltage dependent Ba blok of K hannels in apial membrane of frog skin, Am. J. Physiol. 251 Ž 1986. C696 C706. 11x W. Van Drieshe, B. Lindemann, Voltage dependene of the bloking rate onstants of amiloride at apial Na hannels, Rev. Si. Instrum. 49 Ž 1978. 52 57. 12x S.I. Helman, N.I. Kizer, Apial sodium ion hannels of tight epithelia as vieed from the perspetive of noise analysis, Current Topis in Membranes and Transport, vol. 37, Aademi Press. In., 1990, pp. 117 155. 13x W. Van Drieshe, W. Zeiske, Spontaneous flutuations of potassium hannels in the apial membrane of frog skin, J. Physiol. 299 Ž 1980. 101 116. 14x W. Van Drieshe, Noise and Impedane Analysis, in: J.A. Shafer, G. Giebih, P. Kristensen, H.H. Ussing Ž Eds.., Methods in Membrane and Transporter Researh, R.G. Landes Company, 1994, pp. 27 80. 15x J. Warnke, B. Lindeman, Voltage dependene of the bloking rate onstants of amiloride at apial Na hannels, Pflugers Arh. 405 Ž 1985. 89 94. 16x C.M. Canessa, What is ne about the struture of the epithelial Na hannel?, Ne Physiol. Si. 11 Ž 1996. 195 201. 17x S.I. Helman, L.M. Baxendale, Bloker-related hanges of hannel density. Analysis of a three-state model for apial Na hannels of frog skin, J. Gen. Physiol. 95 Ž 1990. 647 678. 18x D.G. Margineanu, W. Van Drieshe, Comparison of amiloride and CDPC effets on the frog skin eletrophysiology, Pflugers Arh. 415 S1 Ž 1990. R33. 19x R. Waldman, G. Champigny, F. Bassilana, N. Voilley, M. Lazdunski, Moleular loning and funtional expression of a novel amiloride-sensitive Na hannel, J. Biol. Chem. 270 Ž 1995. 27411 27414.