Calcium Channels and Calcium Channel Drugs

From Institut für ßiochcmischc Pharmakologie der Universität Innsbruck (Austria)

Calcium Channels and Calcium Channel Drugs: Recent Biochemical and Biophysical Findings By H. Glossmann, 0 . R. Fcrry, A. Goll, J. Striessnig, and G. Zcmig

Summar)': Tllt' hi11ch1•111ic·ul und hluphysiral fi•a111res of thc 1y1/1111.:1•-d1•p1•11d1·111 l'uki11111 cha111wl.\ , us dist·uvt•rc•d in 1•itru h.1· 1111•t1m ofrudiulahc•lli•d drugs. are preJellled. The cw1c1•pt uftli\/illl't bttt reciprocall.1• a//usterically co11pled drug rer:epwr d11111uim· lii1kt'd tu ruld11111 binding sites is explained. Tlw 1•1·icl1•11n• Jfir tlw e.ri.11e11ce uf i.wcht1111rc•ls (and isorecep'""'' 1.1 r1•ric·11~·c/ Ul/U tlll' 1'u/IOJ.:l'·UC'fll'llllt•11fe 11[ /,4-dihydm· p1·ridi1w bi111/i111.: und aC'tiu11 is disc11.1·sed. Tlte struaure u/tlte c>1111111t'I is illl'l'St(gatcd b)' radia1iu11-l11uctiva1io11 and by pllotuaj/init)' /ube//i11g. Luw aßinit)• bi11di11g sites for calcium l'h1111111•/ tlmgs are sholl'n to reside 011 the 1111c/eoside carrier. Zusammenfassung: Kal:iumka11üle 1111d Kal:!i11111ka11al111r.k.1a1111• Pharmaka: Nt•uc• biu1:hcmi.1C'he 1111d biophysikaluche Befimde

Die Arb~Jt besclireiht die biochemischen 11nd biophysikaliJ'chen f..1g1•1ud1ajien d1•s J·pa11111111gsabltä11gigt•11 Kal:r:i11mka11als. die mit lli/fe radioaktiv markimer Pharmaka in vitro ellftirep•rat iun or membranc rra11mcn1s Irom different tlssues C:mli:lc musclc and smooth musclc organs: Organs (clcancd from conncctivc trssuc and fäl) arc lincly minced wilh scissors and bomogcnittd in icc-cold bufTcr A with a we1 wcight 10 volumc ratio of 1 : 3 by an ul1ra1urrax for 2 >< 1S s al 3/4 m:uimal specd and thcn have bulTcr A added 10 giv' a wel weighl 10 volumc ratio or 1 : 7. Thc dilu1ed homogcnatc is subjectcd 10 a 1S min l .SOO x g ccntrifugation. Thc s upcma1an1s arc ccntrifuged at 4S,000 x g for l .S min, thc pcllelS arc resuspcndcd in bufTcr B and washcd twicc as abovc. Bram: Wholc brain (minus mcdulla and cercbellum) is homogenizcd a1 a wc1 wcighl 10 volumc ratio as above and then subjected 10 a 4.S.000 x g ( l .S min) ccntrifugation. PcllelS arc than washcd 1wicc in bufTcr B a5 above. Skclc1al musclc: Guinca-pig hind limb skelcla1 muscles (15 g) is lincly min,cd in buffcr A and homogcn1ztd al a wc1 weighl t maximal spced. Disruptcd musclc is liltcrcd through two layers of chcese c:loth and 1hcn ccntrifugcd as dcscribed for cardiac muscle. This mcmbranc fraction is refcrrcd to as thc partially purilicd skclelal musclc transvcrsc 1ubulc mcmbranc. For preparalion of Hubulcs the 45,000 x g pellet is taken up in 0.15 mol/I sucrosc in buffer B (4.S ml for 1S g original WCI Wcight tissuc) and is homogenized With sevcn complcte strokes of a Douncc homOgenizcr (Bleassig Glass Company, Rochester, NY, USA) (light pcstle). 7 ml fractions are overlayed on a discontinuous sucrose gradicnt (8 ml of 40%, 35%, 30%, 7 ml 25% (w/w) sucrose in buffc r B). Oradienls arc spun in a Beckman SW 27 rotor (ßeckman lnslr., Munich, FR Gcrmany) for 1.SO mm at 2 '-4 ·c 3t 113,000 xg,,.., (25.000 rpm). Thc material collrcting a1 the 2Sl!b (w/ w) sucrose laycr/ovcrlay inlcnace is rcmoved with a syringe, dilutcd 20fold in a buffer B and centrifugcd for 120 min al 45,000 >< g. T bc membra.nes wcre kcpl in 2 ml aliquots in polypropylene vials and could bc storcd in liquid nitrogcn

19 18

for up to 3 months. Thc samc mc1hods wcrc applicd to isolale 1hc mcmbrancs from frog, rabbit and chicken. Hippocampus: The hippocampal formation is dissccled out of thc guinea-pig brain on icc. Afier gcntlc homogcnisation (10 compl.eie s1rokcs) in a Dounce homogenizer, cquipped with 1he 1ight pcstlc, in ice-cold bufTer A (at a wct-wcight 10 volumc ratio of 1 : 10) tbe bomocenalc is spun at 45.000 x g ( 15 min) al 2 ·c. Tbc resulling pelle· tis resuspcndctl in icc-cold bulfer B by 4 strokes of 1be homogcnizcr and lhc ccnlrifull'ttion rcpcalcd twicc as bcforc. The final pellet is Oash-froz.cn in liquid nitrogen and was stortc.I ror up 4 wcck.s w11hou1 loss ofbinding. Red blood ccll ghosts: T hcy wcrc preparcd from human erythrocytcs as dcscribcd (9). 3.2. Solubilil:ltlun or t'21clum channels Thc skclellll musclc panially purilicd transvcrse tubulc mcmbranc

(6 mg of protcm) is suspended in S ml ice'in>I 1hcm. Thc casstlles arc s1orcd, a1 room 1emper11turc. Expo'ur~ um•.,, rJni;• frorn 3 to 8 wccks for thc 11-ligand-lubcllcd tissucs or 1- 2 J.1)> for l"'l)lodipinc. Films arc dcvcloped in D-19 for S min a1 20 ·c, rin:.cJ In watcr and lixcd .S min ul 20 ·c in Ek1ano Fixer tKoJa~. Mun11:h. FR Gcrmany) (1 : 4 wilh W'Jter). ARcr 20 min of nn:.1ng in running wa1er thc films are dried in air. Scc1ions can bc rc-cxposcd or coun1ed ror radiouctivity. labclling Guinca·ptg .~clctal musclc 1ran5vcl'Sc 1ubulus mcmbrancs (prolcin: 1-2 mglml) in bufl'cr B arc incuba1cd wi1h 2.S nmolll of('H)aiido· pinc (101al bindrng), or w11h 2..S nmolll ('MJaz.idopinc in 1hc pres· cncc of 100 nmol/I of 1he (+)c:nan1iomcr or PN 200- 110 (blank b1nd1ngJ or 01hcr drug5 in a volurnc of 4 ml. Ancr 60 min inc:ubauon a1 25 ·c 1hc a>say mu1urc is ccntnfui:cd al 35 000" g for 10 min at 2 ·c. Thc lirst supcrnatanl is disc-.mlcd and thc pcllcts arc rc>U>pcndcd In 4 mJ o( iCC·COld buJTcr ß, )Upplemenlcd with 0.25 mg/ml bov1nc scrum albumin {fat frc:e: Sisma). Thc ccntrifugation is n:pc:alcd, thc supcm:uant 1s discarded and 1bc pcllcts are rcsuspcnd· cd in 4 ml of 1ce-cold bufTcr ß. Th~ suspeiuions arc irradiated with a Ph1hr» TL 40 W108 black-li~lu lamp in 311 8 cm di3mc1er Pc1ri dish JI a Ji)IJncc of 10 cm for (usually) J mm on i~c. Thc srrndia1ed susn

Ko •3'2 • 40pM

ö

(/)

0

-• _...:;...-=----.

600

1000

*>O

FREE [:!ti}AZIDOPINE lpM l FIR. 6 : ('ll]Azidopinc usoc:ia1ion wi1h Hubul~ c:ilclum channcls from guinca·pfg skcleul muselc (upper) and dissoclation by unlabetlcd nimodipinc (ln• Kb. In othcr words, the allinity of the DHP for the ca1ion frce channcl must bc extrcmcly low. Having converted the channel into [Ch) wc can follow the format ion of the high affinity [Ch . Cal• . DI IPI complcx by "relilling". lnstead with Cah we can also rcfill with othcr cations (c.g. Sr2', ßal•) and measure thc rcconstitution as a function of ion concentration. The results arc shown in Fig. 14. Clcarly thcsc rcfilling isotherms arc indicativc: of morc than onc meta! binuing silc intcracting wilh urug rcccptor sitc "onc'', as thc apparenl Hill slopcs arc significantly smallcr than unily (ßal•. Srh) or may approach cvcn lwo. Ions which block the channel havc biphasic recons1i1ution curves. Wc. can also form the complex [Ch · Ca?• . DHPj and pcrturb thc equilibrium by rcmoving free Ca?• (and olher divalent cations) by adding a largc exccss (5 mmol/I) EDTA. Acconling 10 cquation 3 we thcrcbr form 1hc low aflinity (Ch· DHP) complex which mosl likely dissociates (aceording 10 cquation 4) into (Ch) and frec DHP. In a normal chase experiment we block the forward reaction (cquation 2) with a !arge cxcess of unlabcllcd nimodipine (1 µmol/ I} and measure the dissociation rale-constant of thc highaffinily complex. This dissociation is monophasic anJ is cxemplilied in Fig. 15 at 25 'C. Thc dissociation of [Ca2• ·Ch · DHP] by EDTA is also monophasic and has (not shown) a different tcmpcrature depcndencc compareu 10 thc dissociation induccd by cxccss unlabcllcd ligand. lf thc highaffinity complex is pcrturbcd simu ltancously by EDT A. and unlabcllcd nimodipinc the decay is monophasic and thc overall rate constanl is approximately thc sum of thc individual rate conslants. Wc mcnlioncd above that K. > Kh. lt follows thal K,. > K0 • Thus, thc tightncss of Ca1+ binding incrcascs (mo$1 likcly morc lhan IOOfold) ifthe DHP is bound to thc channcl. Thus, we conclude thal the high affinity complex of the channcl with a DHP antagonis1 holds Cal• so tightly that it will not pass. Fig. 16 shows an ex.perimcnl whcrc: the decay of 1hc Ann>OCiauon was induccd by addilion of unlahcllcd hgand (lo 1 µmul: ll, , „ „er: As af>ovc. bul Jossoda11on Wa> in 100) ties for R and 1. lhe RL complex (whcre L is an antagonist) may not be dis- The abovc model postulates that the binding of thc covercd in ligand binding eitpcriments, as the dissociation 1,4-dihydropyridines is vollagc-dependenl. In the most si mconstanl is mainly a function or the dissociation rate con- ple casc, if nearly all channcls are in the R conformation stant. lf the former is high, a very fast dissocialion rate of (c.g. k, < 1) the Kapp for blockadc in pharmacological ex[RL] will only allow the collection of [IL) compleitcs e.g. by the dilution-lillration tcchnique. Let us now consider a si- periments will be identica.I to K„ conversely if J_> 1 R tuation whcre chemically related 1,4-dihydropyridinc antagonists have dilferent allinities (reflected by lheir different the K.pp will bc closc to K, (sec Fig. 21 ). Thc modcJ prcdicts lhat for channels in the resting slatc thc mcasured dissociaratios or ~: ) for the resting (R) and inactivated (1) slalc tion constant or a 1,4-dihydropyridine blocker must be orders of magnitude grcatcr than for channcls in the inactivatcd of lhe channel. Assume that in ligand binding expcri- state. ßean [43) and Sanguineui and Kass (48) havc shown ments we can only isolale lhe [ILJ complex and furthermorc that thc high-affinity statc of the channel for nisoldipine or that the distribution of channels in 1 and R statc in isolated nitrendipine is thc inactivated state and thc low-allinity state membranes is govemed by tcmpcraturc. (In the intact, polar- the resting channel. The ratio of the rcspective dissociation ized cell the distribution is govemed by voltage.) In Fig. 22 constants (assuming block with 1 : 1 binding) was 1340 (niwc have assumed that thrce different 1,4-dihydropyridines soldipine) or 1944 (nitrendipinc). The Ko values (for thc 1 (A, ß, C) with c= 10.000, c= 1000 and c= 100, respectively, statc) were 1 nmol/l (nisoldipine) or 0.36 nmol/ I (nitrendiare used to label the channel in vitro. lf l/R is 0.001 the pinc) in excellent agrecmcnt with binding data. In Fig. 23 wc 1,4-dihydropyridine A (al saturating concentrations) will la- show the voltage depcndcnce of calc.ium channel availability bel 90% of all channels, B 50% and C less than 10%. lf we (distribution of l/ R states) in Purkinjc liber bundles from

.!. "

=

1928

Annc.om.-Fonch./ ON& Res. JS (II), Nr. 12a (19151 Glos.tmonn tr. 10, 276 ( 1984)- (24) Gan:ia, M. L, Truhh:. M. J.• Rrnbcn, J . P., Kacwrowski. G. J., J . Diol. Chcm. 259, 15103 ( 1984) - (25) G lossmann, H., Linn, T ., Rombusch, M ., Fcrry, D. R., FEßS Leu. 160. 226 (1983)- )26) Lnngcr. S. Z.. Schocmnkcr, H.• Rrit. J. Pha rmarol., in press (1985) - 127] Rcngasamy, A„ Pta· ~icn,~ i. J .. Ho~y. M. M .. lliochcm. Bioph)'!i. Res. Comm. 126, 1 { 19115)- (2!1) (ilossmann, H., Fcrry, D. R.. Goll, A., Rombusch, M„ J . Card10,'asc. Pharmacol. 6 ,5608 (1984)-(29) Fcrry, D. R., Glossmann. H .. Naunyn-Schmiedcbcrg's Arch. Pharmacol. 321, 80 ( 1 98~)- (30) Glossmann, H., Ferry, D. R., Lilbbccke, F., Mcwcs, R., Hofmann. F., Trends Pharm. Sei. 3, 431 ( 1982) - (31) Glossmann, H .• Fcrry, D. R .• Drug Develop. Eval. 9 , 63 j(l 983) - [32) Glossmann. II.. Fcrry, D. R., Goll, A„ Stricssnig, J„ Schober, M .. J. CarJ10\-:t.SC. Phurmacol., in press ( 1985)- [33) G lossmann, H., Goll. A., Fcrr)'o D. R., G lossmnnn. H., Transact. ßiochcm. Soc. 12, 94 1 11984) - f34) Spcdding, M., Naunyn-Schmiedeberg's Arch. Pharmacol. 328. 464 (1985) - [35) Renaud, J. F., Meaux, J, P., Romey, G., Schmid. A., Lnzdunski, M., Biochcm. Diophys. Res. Commun. 125, 405 (19841 -136] Albus. U .. Hu bcrmann, E., Fcrry, D. R., G lossmann. ll.. J. Ncu rochcm. 42, 1186 (1 984)-(37] Schramm. M., Towan. R„ J. Ph)·sioL (Lond.) 349, 53P (1984) - [38] Schramm. M., T homas. G .. Towart, R .• Franckowiak, G„ Nature 309, 535 (1983a) - [39) Schramm, M.. Thomas, G., Towart, R., Franckowiak, _G., Arzneim.-Forsch.lDrug Res. 33 (II ), 1268 ( 1983b)- (40] Spedding, M .. Berg. C„ Naunyn-Schmicdcbcrg's Arch. Pharmacol. ~28, 69 ( 1984) - (41 ) Kokubun, S., Reuter. H., Proc. Natl. Acad. Sei. (USA) 8 1, 4824 (1984) - 142) Middlcmiss, D. N.• Spedding, M., Nature 3 14, 94 ( 19851 - (43) Bcan, B. P., Proc. Natl. Acad. Sei. (USA) 81, 6388 ( 1984)-(44] Boles, R. G~ Yamamura, H. I.. Schocmakcr, H., Rocske. W. R .• J. Pharmacol. E..p. Thcr. 229, 333 (!984)- (~5) Fcrry. D. R.. Goll, A .. Glossmann, H., Naunyn Schm1cdcbcrg s Arch. Ph:irmacol. 323, 267 (1983) - (46) De Povcr, A., Grupp, 1. L., Grupp, G., Schwartz.. A., Biochcm. Biophys. Res. Comm. 114 , 92~ (1983)- (47) Bolgcr, G . T ., Gengo, P., Klockowski, R., Luchowsk1, E. Siegel H„ Jan is, R. A., Triggle, A. M„ Trigglc, A. J .. J. Phannacoi. Exp.'Ther. 2S5, 291 ( 1984) - (48) Sanguinctti, M. C., Kass. R. S., Circ. Res. 55, 336 (1984) - (49) Murphy, K. M . M„ Gould, R. J., Snydcr. S. H .. Eur. J. Pharmacol. 81, 517 (19821 -150) Cones, R•. Supavilai. P., Karobath, M .. Palacios, J . M .• Neurosci. Leu. 4;, 249 ( 1983) - [5 1) Concx, R., Supavilai, P., Karobath, M., Palac1os, J.

An;ne1m.-Fonch.I Druf Rcs. lS ( II), Nr. 111(l98S)

Glo.>m.nn Cl al. -