Alamethicin channel permeation by Ca2+, Mn2+ and Ni2+ in bovine chromaffin cells

V o l u m e 283, n u m b e r I, $9=92 FEBS 09699 ~ 1991 Fcdc'ration or [h~ropean Bioclt~mtcal Societies O014:~79M~Jl/$3,~0 AI)ONIS 001457¢~39100412P

Alamethicin channel permeation by Ca +, Mn + and chromaffin cells

Ma~, 1991

• +

in bovine

R.,I. Fonteriz t, M , G . L6pe:ff.', J, O a r c i a - S a n c h o ~ and A , G . Garcia ~ ~Depa~'teJmento tie Fisialol~la y Bioq!dmlea. Faculrud de Afedictt~a, Univershhui tie Valladolid, 47005. Valhulnfid, Spah~ a n d =Dcpm'tctmento de Farmncolagla y Terup~utl¢¢~, Factdl¢~d ¢le Medlrttla, Uttlvcrsitlctd d~a~m~ma de Madrid, Ar:obi.~po, Mor¢lllo, 4, 2~029.Madrhl. Spct#~ Received 19 F e b r u a r y 1991 Alamethicin causes a concentration.dependent increase of [Ca ~"], m suspensions of bovine ~tdrcnal ehromaflln cells loaded with turn.2. The basal levels o1' Ca?" (234 + 37 nM: n ~,4) increased to a n~aximum of 2347 :t: 791 nM (t) ~, 3] with 1(10/qj/ml alamethicirl, In the pres©nCc of I mM Ca. = ) the increase reached a plale:m within about 2,-5 s. This increase wus due to Ca=* entry nto chromafin eel s. since in the absence of Ca, =" alam~thicin did not modify [Ca 2 *1,, This contrasts with ion~amycin (I tIM) which produced a Ca?" tr'.msient even in the absence of C a ~ ' . Mn 2 ' ions also entered chromaffin cells in ti~e presence of alulnethicin, as rneasuretl by the quenchin~l or fura,2 fluorescence following excitation a1360 nm, Resting chrornaMn cells had u measurable permeability to Mn =" which was drastically increased by cell depolarization by K* (50 raM) addition. This stJggest,~ tl~at Mn =° is able to pern~eate volt;ttle-dependcnt C=t=" channels, Ni =" uptake into either rcstin8 or K*.stimulated, chromaffin cells wets undetectable, but addition of alamethicin induced rapid uptake or this cation, The alamethicin,induced entry of Ni a" was decreased by 50 mM K °, Ovendl, the resuhs are compatible with the r'ornuttion by -'dam~thich~ ot'ion channels in ¢hromaflin cell plasma membranes, Ahtmethicin Calcium signal; Mang,anese: Nickel; Fura-2; Chromaffin cell

I. I N T R O D U C T I O N Alamethicin is a 20-amino acid peptide that forms voltage-dependent channels across membranes [1,2] and has been used as a model for voltage-gated channels. Recently, we demonstrated that this ionophore causes the release o f catecholamines from perfused cat adrenal glands in a temperature-, concentration- and Ca~" ÷ -dependent manner [3]. The time-course of secretion evoked by alamethicin (quick activation followed by a decline) considerably differs from the adrenal catecholamine release pattern seen with carrier-type ionophores such as A23187 [4] and ionomycin [51. Rather, the profile of secretion evoked by alamethicin resembles that o f nicotinic or high-K* stimulation o f cat adrenal medullary c h r o m a f f i n cells [6]. In the light of these results, we suggested that alamethicin might form Ca 2+ permeable artificial channels in chromaffin cell plasma membranes [3]. This possibility has been explored now in bovine adrenal chromaffin cell suspensions loaded with the Ca ~-÷ fluorescent indicator fura-2. T o study the cell permeabilities to Ca 2÷, Mn :+ and Ni z÷ we have taken advantage of the spectral characteristics Correspondence address: R,I. Fonteriz, Departamento de Fisiologia y Bioquimiea, Facultad de Medicina, Universidad de Valladolid, 47005-Valladolid, Spain

Published by Elsevier Science Publishers B. V.

o f fura-2 to estimate the entry o f b,ln 2÷ and Ni z÷ without interference with Ca 2+ signals when Ca~ + was present [7,81.

2. MATERIALS AND METHODS Bovine adre~al chromaffin celh were hotated as previously described [9], A cell layer from a pereoll gradient enriched in adrenaline cells was taken to perform these experiments, Cells were suspended in Dulbecco's modified Eagle's medium (DMEM; l0 n cells/2 ml), supplemented with 10% heat-inactivated fetal calf serum containing 50 iu/ml penicillin and 50l~g/ml streptomycin. Cells were kept in suspension under smooth continuous a~itation, in bottles with an MCS-140 tnicrocarrier stirrer, in an incubator (Hearaeus) under a n atmosphere of 5°7o CO2 in air at 37°C, for 2-3 days before the experiments. Cells were loaded with fura-2/AM (4/~M) for 45 rain at room temperature with continuous shaking in a standard medium (pH 7.4) containing (in raM): NaCI, 145; KCI, 5; MgCI2, 1; CaCI2, 1; SodiumHEPES, 10; glucose, 10, The loading incubation was terminated by dilution with 3 volumes of fresh standard medium followed by cert. trifugation at 80 x g for 10 mix, The cells were then suspended in standard medium at 1.5 × 10 6 cells/ml, Fluorescence measurements were performed at 37°C with magnetic stirring in a fluorescence spectrophotometer which allowed rapid alternation (30-300 Hz) of up to 6 different excitation wavelengths (Cairn Research, Kent, UK). Emit. ted fluorescence was measured at 530 rim. Fluorescent signals were integrated at 1 second periods, [GaZ+]i was estimated from the ratio of the fluorescence values excited at 340 tam and 380 nm [10], Uptake of Mn ~+ and Ni 2" was estimated from the quenching of fluorescence excited at 360 nm [7,8]. Alamethicin was obtained from Sigma, dissolved in ethanol and diluted in saline solution.

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+

suspension produced a sharp, quick r~se o f [Caa*]t to about tO00 n M (Fig. IB). The new [Ctt='h plateau was stable and did not suffer inactivation for at least a 5 rain period. This was a sitlnal specifically associated to alamethicin, since in the absence o f the i o n o p h o r e , Ca~* addition did not increase the basal levels of

medium

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100,

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~.a

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N.A 100

t...-..J t rain

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{Ca~÷h,

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Fill, I. Effects or alamethicln and ionomycin on lntraeellular level~of cytosolic Ca l', Cells (2.$x 10~/assay) Ioad~cl witl~ Fura.2 were suspended in a Cat'.freemedium containlntll m M EGTA (A) After the fluoresc©nce signal reflecting stabilized basal levels of Ca~*, alamethictn (ALA, 100/~g/ml) was added, One ~ltittlater, ionomydn ( I aM) was added to the same aliquot of cells, (B) A separate aliquot of cells suspend¢d in Caa'-free solution (I mM EGTA) was exposed to alamethlcin (ALA, 40.ag/ml). I minlater, Ca 2" (I raM) wasadded to the bathing solution. The recordings are copies of the orillinah obtained in a typical experiment.

3. R E S U L T S

AND

DISCUSSION

3.1, Effects o f alamethicin on intraceilular calcium levels in the absence and in the presettce o f external calcium T h e basal levels o f cytosolic Ca z÷, in the presence o f 2.5 m M Ca~÷~ were a r o u n d 234 ± 37 nM (n = 4). In the absence o f extracellular Ca 2÷ (with 1 mM E G T A present), alamethicin (100 ~ g / m l ) did not affect such levels. However, addition of ionomycin (1 ~M) still in the presence of alamethicin, increased [Ca2+]i from 60 to 200 nM (Fig. IA). T h e Ca 2+ signal decayed slowly with time. In a separate aliquot of cells, bathed in CaZ+-free m e d i u m , alamethicin (40 u g / m l ) did not alter basal [Ca2*]l; however, addition o f 1 m M Ca 2÷ to the cell

1 r'nM Co 2 +

ALA ( / ~ g / m l )

700

50 500, 400. 300. 2O

o m

200. 100.

o

~

~

3

"rime (rain) Fig,

2,

Effects

of

alamethicin

on

Ca~ + l e v e l s .

Each

ionophore

concentration was tested in separate aliquots of cells. Cells were first incubated in Ca2+.free solution (30 s), then exposed to 1 mM Ca~÷ (40 s) and finally treated with each concentration of alametlficin during the 2 rain recording period shown lathe abscissa. [Ca2*]i is expressed as the fluorescenceratio between excitation wavelengtlls of 340 and 380 am. 90

Fig. 2 shows the effects o f increasing concentrations of alamethicin on [Caa*lj Levels, Cells were first incubated in Ca2*-free medium for 30 s. T h e n i mM Ca 2. was added and, after ,tO s, alamcthicin (!0-100 t~g/ml) was added (each individual c o n c e n t r a t i o n was tested in separate aliquots o f cells). T h e Ca 2. signal was recorded for an addit tonal 2 rain period, The threshold concentration producing a cleat', measurable response was 20 #g/nil; from then onwards, a gradual concentration-dependent increase in [Caa*]t was observed. Note that the higher the i o n o p h o r e concen. tration, the faster the rise o f the [Caa*]s signal. Also note that the signal reached a plateau (especially at 50 and 100/~g/ml alamethicin) and stabilized f o r the 120 s period o f recording. Ionomycin is k n o w n to mobilize Ca 2÷ from intracellular stores, and more specifically f r o m smooth endoplasmic reticrtlum, therefore, the C a z÷ signal seen with this i o n o p h o r e in the absence o f Ca z÷ was likely to ,, ' be d u e to Ca 21" mobfllzaUon from some internal stole. T h e fact that alamethicin was unable to m o d i f y the [Ca '+ ]i in tlte absence o f external C a 2÷, and that Ca~* addition suddenly increased the levels o f Ca,~+, suggests that the c h r o m a f f i n cell plasma membrane, and not the e n d o m e m b r a n e , is the target for alamethicin action. In this respect, alamethicin behaves as nicotinic agonists or h i g h - K * - - t w o stimuli which produce Ca~ ÷ transients in bovine c h r o m a f f i n cells loaded with fura-2 only in the presence o f external Ca 2+ [11]. This analogy, and the fast rise o f [Ca2+]i are compatible with an ion channel formed by alamethicin in the c h r o m a f f i n cell plasma membrane.

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600. o 0o t~ "o,tl" er) o

May 1991

3.2. Permeation o f alamethicin-trectted cells by Mn 2÷ and Ni z+ In order to examine more directly, and on a more adequate time scale, the kinetics o f cell ion permeabilities activated by alamethicin, we tried the 'Mn z+ quenching' technique, which has been successfully used to demonstrate agonist-activated divalent cation entry into several cell types [7,8,12-16]. This technique detects Mn 2+ entry into cells by monitoring the MnZ+-induced quenching of intracellular fura-2 fluorescence excited at 360 nm (the isosbestic point for Ca2+). Under these conditions, the fluorescence signal is insensitive to changes o f [Ca2+]i. In the absence o f Ca~ +, addition o f 0.5 m M Mn 2+ p r o d u c e d a quenching o f fluorescence which developped slowly (Fig. 3A). This quenching presumably reflects gradual entry o f Mn 2+ into cells, suggesting that

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May 1991

un=+O.S

\--

ElM, 3, Quendtintl of the fluorescence ~iMnal by hln =" and Ni =- in Fura.2 leaded cl~romett'l'in eell~. CA} Mn:" (0,S raM) wa~ addc'd and then, sequentially, at Ih© points indicated by arrow~, alamelhi¢tn (]0 #illml) and K" (60 raM). (B) In tt dirl'erent ,;ell aliquot, Nt a" (,~ raM), K" ($0 raM) and alamethlcln {50#=troll were added as Indicated, The cell bathinli ~ohltion had no Ca a- tl mM EGTA), Arbhrary unh~ of fluorescence m 360 nm are expre~e¢l in the ordinate,

t, nstimulated chromaffin cells are quite permeable to Mn a*. The rate of decline of fluorescence was further accelerated by alamethicin (30 #g/ml), suggesting that Mn 2. was also entering the cells through alam~thicin pores. Finally, K* addition (50 raM) caused an additional drop in fluorescence suggesting that voltage. sensitive Ca 2÷ channels are highly permeable to Mn2*. In the light of these results, it seemed that Mn 2. would not be an adequate Ca 2÷ surrogate to study the kinetics of alamethicin channels in chromaffin cells. Therefore, Ni 2* was tried as an alternative divalent cation. Voltage-gated Ca 2* cl~annels are not permeable to Ni ~+ which, i n addition, is a potent blocker of such channels as well as of K+-evoked catecholamine release [17]. Ni z* did not affect the basal fura-2 fluorescence in unstimulated cells. Neither did K + addition alter this fluorescence (Fig, 3B) confirming that Ni 2÷ was not permeating voltage-dependent Ca z+ channels. Alamethicin (50/~g/ml) caused a gradual fluorescence quenching, suggesting that Ni 2. was permeating chromaffin cells only in the presence of the ionophore. This double component (blockade of Ca 2÷ channels and permeation of alamethicin channels) makes Ni 2÷ an ideal cation to study the kinetics of the latter channels, as demonstrated by the following experiments. Ni 2+ permeation of alamethicin channels was a slow process, as deduced f r o m the experiment shown in Fig. 4 . Upon addition of alamethicin (100/zg/ml) to cells incubated with 3 mM Ni z+, the dissipation of the fluorescence declined gradually with an approximate t~/2 of 40 s. This contrasts with the quick rise of CaP + levels evoked by this same concentration of alamethicin (Fig. 2), which exhibited a t~/2 of about I s. The Ni 2+ uptake process did not apparently exhibit inactivation, since fluorescence decayed almost linearly. Aiamethicin-evoked Ni 2+ entry was insensitive to the i,4,dihydropyridine derivative nisotdipine (I #M), an L-type Ca 2+ channel blocker [8]. Thus, dissipation of the fluorescence signal at 360 nrn followed the same rate either in the presence or the absence of nisoldipine (not shown). In a different experiment, 1 /~M nitren-

S' t~

iv3

Id,,, V

80

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K + 15=

60"

Z

K ÷ 30s

" 40

Control

~--=-~-~ 15s

Fill, 4. Efrect~ o f d d a y e d addition or K* on the rate of disdpalion by Ni~" of fluorescence of fura,2 lo:~decl ehromafrin ¢ell~ treated with alamethlein. C'elI~ were excited at 360 nm and their fluorescence e~timated (arbitrary, unil~ in the ordinate), Alametlli¢in (100 ~+8/ml) was added at time 0 and the rate of fluorescence decay followed for the next 60 s {abscissa), In 2 different alkluots of cells, K" (50 raM) was added 15 and ~10 s ~fter alatncthicin (arrow~).

dipine (another dihydrowridine) did not affect Ni 2. entry into alamethicin-treated cells (not shown). Thus, Ca a*, Mn 2÷ and Ni 2+ permeated alamethicin channels. The Ca 2. permeability was very fast, follow. ed by the intermediate Mn 2. permeability and the slowest permeability to Ni 2.. These permeabilities dif. fer only slightly from those for Ca z÷ channels. For instance, in high-K* solutions, which recruit voltagedependent Ca 2. channels in chromaffin cells [19], fura-2 loaded cells were highly permeable to Ca2 +. less so to Mn 2. and not at all permeable to Ni 2÷, We took advantage of the differential permeabilities of alamethicin channels and Ca 2+ channels to Ni 2+ in order to study, in isolation, the effects of the membrane potential on the kinetics of ionophore channels. 3.3. Modulation (by voltage) o f the t:inetics o f ala. methicin channels Fig. 4 shows the fluorescence decay evoked by alamethicin (100 /~g/ml) in the presence of Ni 2+ (3 mM). When K ~ (50 mM) was added 15 or 30 s after alamethicin, the rate of decay was almost immediately slowed down. This indicates that cell depolarization may be closing the alamethicin channels thereby decreasing Ni 2+ uptake through them. These results are consistent with previous reports in lipid bilayers, where depolarization decreased the conductance of alamethicin channels [2]. This behaviour is consistent with the model inferred from the crystal structure of this ionophore where gating of the channels is facilitated by burial of the channel in the membrane driven by a transmembrane potential difference [20].

3.4. Functional correlates The concentration-dependence of [Ca2+]~ increase 91

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(this paper) and of adrenal catecholamine release [31 evoked by alamethicin are closely correlated. Thus at 10 ,.g/rnl. where [CaZ*h was only slightly modified, alamethicin caused only a mild increase in secretion. But 20-100 /~glml caused parallel increases in both parameters, indicating that Ca ;=* entry through alamethicin channels exhibited a functional role in activating the secretory machinery underneath th e plasma membrane, The time-courses of the increase in [Ca ~']l and secretion cannot be comparedappropriatety. In our previous experiments [31 secretory rates were measured at 2 rain intervals but here, Ca~ transients caused by alamethicin are very rapid, specially at 50-100 /~g/ml (Fig. 2). Nevertheless, we can infer that the secretory effects also develop quickly since peak secretion is always reached during the first 2 min collection period. The rapidity of the effects o f alamethicin contrasts with those o f tonomycin and A23187 which, although causing a rapid rise of [CaZ*h in bovine chromaffin cells [21,22], nevertheless initiate a slowly.developing secretory response

[4,5,231. In conclusion, alamethicin seems to form ionic channels in chromaffin cells which are permeable to Ca 2., Mn 2÷ and Ni 2+. As previously shown in artificial membranes [21, these channels exhibit voltage,dependence: they undergo inactivation in depolarized cells and reactivate under polarizing conditions. Because alamethicin mimicks the functional and kinetic aspects o f the natural voltage-dependent Ca z+ channels, it may become a useful tool to investigate the access and role of Ca 2÷ in activating secretory processes. Conversely, chromaffin cells are a good model alongside the widely used artificial membranes or membrane vesicles, to study the kinetics of ion permeation of alamethicin channels. They offer the advantage of a functional correlation between these ion permeabilities and a fast cellular event such as the exocytotic release of catecholamines. Acknowledgements: Supported in part by grants from DGICYT (No PB89-0359 and PB87-0092.C03.01), FISS and Fundaci6n Ram6n areces, Spain.

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REFERENCES Ill ~Isenberjl.M,, H~II, J.l~,and Mead, C.A, t19~3) J, M~mbr. 11101, 14. 143-176, 121 Latorre, R, ~nd Alvare~, f3, (19#I) Physiol. Ray, 61, ?'/=,I$0. [3] Arud~jo. A., Montld. C,, Sdnehex.G~rd~, P., Ueeda, G., Gunnies, LM. anti G~r¢|a, A.,G, (1990) Biodlem, Biophy~, Res. Commun. 169. 1~[04~121a. [4] Gar¢itt.A.G.. KirPek:tr,S.M. and Ptltl,J.C, (197~)J. Physiol. 244. 2~-262, [~] Carv~lho, M.H,. Prat, J,C,, Gar¢ta. A.G. and Klrpekar, S,M. (1982l Am, J, Physiol. 242, IEI37-EI45, 16] Sehlavone, M,T, and Klrpekar, S,M. (1982)J. Pharma¢ol, I~xp. Ther, 223,743-'/49, [7) Alonso, M,T.. Stlneh¢~. A, and Garc[a.Sancl~o, J, (1989) Bio. chen~, Biophys. Res. C o m m u n 162.24-29, [81 Garci~t.St~ncho. J,, Alonso. M,T..n~.l Sfinch~, A, (1~J89) Biochem. Sac. Transact, 17. 980-982. [9] Moro, M.A., Ldpex, M,G., Gandia, L,, Michclena. P. and Gore[a, A,G, (1990) Analyt. Biochem. 185,243-248. (10] ¢]rynkiewicz, G., Poenle, M, and Tsien, R,Y, (1981] J Biol, Chem. 260, 3440=3450. 11I1 Kim, K-T, and Westhead, IE,W. (1989)Proc. Natl. Acad. Sci, USA 86, 9881-9888. [12] Hallam, T,J, and Rink. T,J. (1985) FF.BS Left, 186, 175-t79. [13] Mcrrlth, J.E. and Hallam, T.J; 0988) J, Biol, Chem. 2~3, 6161-6164, [14] Sage, S.D,, Merrit. J.E,, 14allam, T,J, and Rink, T.E (1989) Biochem. J. 2S8. 923-926. [15] Mcrrith, jr=., Jacob, R, and Hallam T,J. (1989) J. Biol, Chem, 264, 1522-1527. [16] Jacob, R. (1990) J, Physiol. 421, 55-77, [17] Gandfa. L,. LOpcz. M.G., Fonteriz, R.i.. Artalejo, C,R, and Garcia, A.G (1987) N~urosci. Left, 77, 333-338, [18] Tsien, R.W., Lipscombe, D,, Madison, D.V., Bley, K.R. and Fox, A.P. (1988) TINS 11, 431-438, {19] Artalejo, C.R., Garcia, A.G, and Aunis, D. (1987) J, Biol. CItem, 262, 915-926, [20] Fox, R.O, and Richards, F.M. 0982)Nature 300, 325-330. [21] Burgoyne. R,D. (1984) Biosci, Rap, 4. 605-611, [22] Stauderman, K.A. and Pruss, R,M. (1989) J. Biol, Chain, 264, 18349-18355. [23] Artalejo, C,R. and Garcia, A,G, (1986) Br. J, Pharrnacol, 88, 757-765,