Are dihydropyridine binding sites voltage sensitive calcium channels?

Life Sciences, Vol. 34, pp. 1205-1221 Printed in the U.S.A.

Pergamon Press

MINIREVIEW ARE DIHYDROPYRIDINE BINDING SITES VOLTAGE SENSITIVE CALCIUM CHANNELS? Richard J. Miller and Stephen B. Freedman Dept. of Pharmacological and Physiological Sciences, U n i v e r s i t y of Chicago, 947 E. 58th Street, Chicago, I l l i n o i s 60637

It is w i d e l y a c c e p t e d t h a t Ca +2 is an e x t r e m e l y importan~ i,== intracellular messenger. Normally, th~ intracellular .... Ca +L concentration is e x t r e m e l y l o w ( ~ i0-~ M). The e x t r a c e l l u l a r calcium concentration is a b o u t f o u r o r d e r s of m a g n i t u d e higher (reviewed in i). This large concentration difference is the r e s u l t of s e v e r a l processes. These include the a b i l i t y ~f several i n t r a c e l l u l a r organelles and proteins to sequester Ca +L , the p r e s e n c e of v a r i o u s p u m p s t h a t r e m o v e Ca +2 f r o m the cell interior against its e l e c t r o c h e m i c a l g r a d i e n t ~ and the l o w permeability of the cell p l a s m a m e m b r a n e to Ca +~. One w a y in which the intracellular Ca + L c o n c e n t r a t i o n can be r a p i d l y increased is by o p e n i n g selective permeability pathways for calcium in the cell m e m b r a n e . A m a j o r w a y of r e g u l a t i n g such calcium channels is by a l t e r i n g the m e m b r a n e potential (2,3). Thus, in many cells, m e m b r a n e d e p o l a r i z a t i o n causes the opening of calcium channels which allow the r~pid entry of Ca ÷~ dow n its electrochemical gradient. T h i s Ca + ~ can n o w i n i t i a t e v a r i o u s processes. I~ neurones, some endocrine cells, s m o o t h and cardiac muscle, Ca + L e n t r y can c o n t r i b u t e significantly to the cell action potential (2). Calcium entry in s u c h c e l l s is an essential link in the p r o c e s s e s of s t i m u l u s / s e c r e t i o n and stimulus/contraction coupling. Moreover, the c a l c i u m c u r r e n t itself and the various effects of Ca +L on the ionic p e r m e a b i l i t y of the cell m e m b r a n e is i m p o r t a n t in the r e g u l a t i o n of c e l l excitability (2-5). In the l a s t d e c a d e , a g r o u p of d r u g s of d i v e r s e c h e m i c a l s t r u c t u r e has b e e n d e v e l o p e d w h i c h h a v e the p r o p e r t y of b e i n g able to block voltage dependent c a l c i u m channels (VSCC), at least in s o m e t i s s u e s (1,6). These observations h a v e p r o v e d to h a v e great therapeutic i m p o r t a n c e and these drugs are already widely used for the treatment of angina and hypertension. Interestingly, the VSCC in different tissues do not necessarily have the same sensitivity to channel blockers (1,6). This again has t h e r a p e u t i c implications. For example, the drugs can be used to treat h y p e r t e n s i o n and coronary v a s o s p a s m w i t h o u t s u p p r e s s i n g neurotransmission or inhibiting cardiac function. The m o l e c u l a r implications of t h e s e o b s e r v a t i o n s w i l l be d i s c u s s e d in t h i s article.

0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.

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Calcium channel blockers not only v a r y considerably in structure, but a l s o in p o t e n c y . S o m e of the d i h y d r o p y r i d i n e s ( t y p i f i e d by n i f e d i p i ~ e ) act on s m o o % h m u s c l e at e x t r e m e l y low concentrations (< i0 - ~ M). Clearly, therefore, such drugs must form very high affinity c o m p l e x e s with VSCC or closely associated elements. It is therefore reasonable to suppose that they might be u s e d as p r o b e s for the i d e n t i f i c a t i o n and p u r i f i c a t i o n of VSCC. Indeed, the e n o r m o u s progress made recently in studies of voltage sensitive sodium channels is b a s i c a l l y due to the availability of drugs that form specific high affinity c o m p l e x e s with these structures (7,8). L a b e l l i n g of VSCC with radioactive calcium channel blockers has b e e n a t t e m p t e d previously (9). However, drugs with low affinity and s p e c i f i c i t y w e r e used. Recently, however, e x t r e m e l y potent d i h y d r o p y r i d i n e s (DHP's) such as nitrendipine, n i m o d i p i n e and nifedipine have been labelled to high specific activity. Theoretically, such drugs should make ideal m o l e c u l a r p r o b e s for VSCC. I n d e e d , m u c h d a t a has a l r e a d y b e e n o b t a i n e d on the i n t e r a c t i o n of t h e s e l a b e l l e d d r u g s w i t h "Calcium C h a n n e l s " in d i f f e r e n t tissues. The p u r p o s e of t h i s r e v i e w is to b r i e f l y d e s c r i b e s o m e of t h i s w o r k and to p r o v i d e some critical c o m m e n t s on it. General

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M o s t of the s t u d i e s r e p o r t % d so far h a v e u t i l i z e d one of the potent d i h y d r o p y r i d i n e s (DHP's) ~ H - n i t r e n d i p i n e or ~H-nimodipine. Binding s i t e s for these a g e n t s that are s a t u r a b l e , of h i g h affinity and s t e r e o s p e c i f i c have been described in many tissues. However, most detailed studies have e x a m i n e d binding in vascular and nonvascular smooth muscle (i0-22,71), cardiac muscle (i0,11,13,15-18,20-29), s k e l e t a l m u s c l e ( 1 1 , 1 3 , 3 0 - 3 7 ) and b r a i n (ii,15,18,23,38-55). The properties of t h e b i n d i n g sites ~dentified in e a c h c a s e a r e r a t h e r similar although some interesting d i f f e r e n c e s certainly exist. For example, sites in skeletal muscle are of lower affinity than those in other tissues e x a m i n e d (vide infra). Certain basic features are clear. Thus, although all DHP's interact with the labelled binding sites in a simple c o m p e t i t i v e fashion, other channel blockers with d i f f e r e n t structures, e.g. p h e n y l a l k y l a m i n e s , diphenylalkylamines and benzothiazepines ~o so in m o r e c o m p l e x ways. The s i t e on the VSCC labelled by a H - n i t r e r d i p i ~ e a p p e a r s to be a l l o s t e r i a l l y l i n k e d to o t h e r d r u g b i n d i n g s i t e s on the c h a n n e l . T h e r e ~s an obvious analogy here with the situation at the voltage sensitive sodium channel (7) or ~icotinic receptor linked channel (60). In the f o r m e r case, s e v e r a l d r u g and t o x i n b i n d i n g s i t e s h a v e been defined. A g e n t s a c t i n g at one site, e.g. s c o r p i o n v e n o m can alter binding of a drug to a second site, e.g. batrachotoxin. In the c a s e of the n i c o t i n i c r e c e p t o r c h a n n e l , d i f f e r e n t t y p e s of c h a n n e l b l o c k e r s a l s o i n t e r a c t w i t h the r e c e p t o r at d i f f e r e n t sites. It is p e r h a p s not surprising to f i n d t h a t s u c h a disparate group of structures as the calcium channel blockers do not all interact w i t h the channel at precisely the same locus.

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The best e v i d e n c e that DHP b i n d i n g sites and VSCC are identical has been obtained with smooth muscle. When examining this e v i d e n c e it is i m p o r t a n t to c o n s i d e r e x a c t l y how c l o s e l y b i n d i n g data o u g h t to c o r r e l a t e w i t h p h a r m a c o l o g i c a l and o t h e r data o b t a i n e d in f u n c t i o n i n g tissues. One m i g h t c o n s i d e r for example, the situation with the 6-adrenergic or opiate receptor (61). In these cases, the r e c e p t o r s can be l a b e l l e d with radioactive agonists or antagonists in isolated membranes from responsive cells. However, the precise affinity obtained for the drug receptor complex depends critically on the assay conditions employed. The p r e s e n c e of v a r i o u s ions or guanyl n u c l e o t i d e s w i l l a l t e r the a f f i n i t y of the d r u g r e c e p t o r interaction radically. These considerations together with others such as the presence of spare receptors and various desensitized states makes a precise comparison of in vitro binding data with responses in whole tissues rather difficult. Although some systems are rather s i m p l e and d i r e c t c o m p a r i s o n ~ can be made, the s i t u a t i o n w i t h VSCC may not be. Evidence suggesting the modifiability of VSCC in the heart (62) and the "run d o w n " of VSCC in s t u d i e s of isolated patches or perfused cells (63,64) implies that there may be factors in addition to membrane potential that can influence the state of the channel. M o r e o v e r , it is d i f f i c u l t to see how features such as the "use dependence" associated with the action of drugs such as verapamil (65) can be incorporated into binding studies in i s o l a t e d membranes. Clearly, factors that can i n f l u e n c e the state of the c h a n n e l in w h o l e cells may be a b s e n t in isolated membrane preparations. Several a g e n t s are k n o w n to c o n t r a c t smooth muscle in a d d i t i o n to d e p o l a r i z i n g s t i m u l i . D e p e n d i n g on the tissue in question, these might include norepinephrine (e-adrenergic effects), 5 - H T or v a r i o u s icosanoids. There is o f t e n a considerable difference in the a b i l i t y of c a l c i u m channel blockers to i n h i b i t depolarization and agonist induced contractions (6,66). One reason for this is that several agonist i n d u c e d r e s p o n s e s are t o t a l l y or p a r t i a l l y i n d e p e n d e n t of the influx of calcium from the cell exterior. Such responses appear to be m e d i a t e d by m o b i l i z a t i o n of c a l c i u m from i n t r a c e l l u l a r s t o r e s (1,6,67). However, c o n s i d e r i n g those a g o n i s t i n d u c e d responses that are wholly or partially dependent on the influx of c a l c i u m from the cell exterior, the q u e s t i o n a r i s e s as to the route by w h i c h this c a l c i u m enters. It has been s u g g e s t e d that s e p a r a t e p o p u l a t i o n s of c a l c i u m c h a n n e l s are r e s p o n s i b l e for depolarization and agonist induced calcium entry (i). Alternatively, agonists and depolarizing stimuli might recruit the s a m e pool of c h a n n e l molecules. If there are s e p a r a t e classes of c a l c i u m channels, they might exhibit different a f f i n i t i e s for c a l c i u m c h a n n e l b l o c k i n g drugs. Some evidence s u p p o r t i n g this c o n t e n t i o n has been o b t a i n e d (1,6,67). Thus, w h e n p e r f o r m i n g a b i n d i n g assay w i t h a l a b e l l e d blocker, it is possible that multiple binding sites would be expected. However, the e v i d e n c e to date w i t h DHP b i n d i n g u s u a l l y only s u g g e s t s the p r e s e n c e of a s i n g l e type of site. It is p r o b a b l y s i m p l e s t at

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t h i s p o i n t to c o m p a r e binding d a t a w i t h that o b t a i n e d from depolarization, r a t h e r than a g o n i s t induced contractures in various smooth muscle preparations. T h i s w a y i s s u e s such as w h e t h e r agonists are u t i l i z i n g the same channels as d e p o l a r i z i n g stimuli or a separate population can be avoided. Certain features of the p h a r m a c o l o g y of c a l c i u m channel blockers are particularly useful when considering binding data. One of these is stereospecificity. Many channel blockers can be r e s o l v e d i n t o o p t i c a l i s o m e r s w h i c h have d i f f e r e n t potencies. For example with the DHP's nitrendipine and nimodipine (15,52,68), the (-)-isomers are more potent than the (+)-isomers w h e r e a s the reverse is true for nicardipine (69). For v e r a p a m i l and its m e t h o x y d e r i v a t i v e D - 6 0 0 the (-) i s o m e r s are a l s o m o r e p o t e n t than the (+) i s o m e r s (70). If DHP b i n d i n g r e f l e c t s the l a b e l l i n g of VSCC, it should also p r e s u m a b l y reflect the relevant stereospecificity. T h i s is i m p o r t a n t as s e v e r a l of the n o n specific effects associated with high concentrations of d r u g s such as v e r a p a m i l do not exhibit s t e r e o s p e c i f i c i t y (70). In m o s t s m o o t h m u s c l e p r e p a r a t i o n s , DHP's b i n d to a s i n g l e c l a s s of s i t e s w i t h v e r y h i g h a f f i n i t y . The K D r a n g e r e p o r t e d for n i t r e n d i p i n e is 0.1-2.1 nM (10-22). However, most studies are in one r a n g e 0.1-0.5 nM. The b i n d i n g site a p p e a r s to be a protein. Triggle and his colleagues have synthesized an i r r e v e r s i b l e VSCC blocker based on the DHP structure (22). Using this a f f i n i t y label, they h a v e d e m o n s t r a t e d that DHP's b i n d p r i m a r i l y to a protein of m o l e c u l a r weight 45,000 in the guineapig ileum. A b i n d i n g p r o t e i n of the s a m e m o l e c u l a r w e i g h t is a l s o p r e s e n t in the g u i n e a - p i g heart. T a r g e t size a n a l y s i s of guinea-pig ileal m e m b r a n e s h o w e v e r gives an apparent m o l e c u l a r w e i g h t of 2 7 8 , 0 0 0 for the D H P b i n d i n g site. Presumably as w i t h the s o d i u m c h a n n e l , the V S C C i ~ ~ i ~ are m a d e up of s e v e r a l subunits (8). Triggle and c o l l e a g u e s h a v e also p e r f o r m e d a detailed comparison of the e f f e c t of d r u g s or 3 H - n i t r e n d i p i n e binding in the g u i n e a - p i g ileum and their effects on VSCC in the s a m e t i s s u e (71). T h e r e is a v e r y g o o d c o r r e l a t i o n b e t w e e n the a f f i n i t y of DHP's for n i t r e n d i p i n e b i n d i n g s i t e s and the t o n i c portion of K + i n d u c e d muscle contraction. This correlation includes stereospecific inhibition of b o t h b i n d i n g and m u s c l e contraction by p a i r s of DHP i s o m e r s . Interestingly, ileal c o n t r a c t i o n s induced by m u s c a r i n i c receptor s t i m u l a t i o n are about an order of m a g n i t u d e less sensitive to DHP blockade than are K + induced contractions although t h e i r r a n k o r d e r of p o t e n c y is s i m i l a r in b o t h cases. It is p o s s i b l e that t h i s c o u l d i n d i c a t e the presence of a separate receptor linked channel as discussed above. However, other e x p l a n a t i o n s are also possible. Evidence with DHP's therefore clearly supports the n o t i o n that OHnitrendipine binding in the guinea-pig ileum is to some e l e m e n t that mediates the p o t a s s i u m induced contraction, i.e. the VSCC. Such a detailed comparison has not been c a r r i e d out in o t h e r tissues. Significantly, DHP's a l s o bind w i t h h i g h a f f i n i t y to membranes f r o m dog and rat m e s e n t e r i c arteries. However they inhibit K + induced contractions at 2 0 - 3 0 times higher concentrations (19). Thus, it r e m a i n s to be s e e n w h e t h e r the

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Biochemistry of Calcium Channels

correlaticn found in with other smooth muscle

the guinea-pig preparations.

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C a l c i u m channel blockers apart from DHP's also interact with the D H P b i n d i n g site, but in a m o r e c o m p l e x fashion. These include p h e n y ] a l k y l a m i n e s such as verapami], D-600 and tiapamil, diphenylalkylamines s u c h as c i n n a r i z i n e , flunarizine and l i d o f l a z J p e and b e n z o t h i a z e p i n e s such as diltiazem. For example, it has b e e n f o u n d in s m o o t h m u s c l e and in o t h e r t i s s u e s ( v i d e infra) that verapamil and D - 6 0 0 o n l y p a r t i a l l y inhibit DHP binding (11,18,24,71). This i n h i b i t / o n is s t e r e o s p e c i f i c , (-)D600 b e i n g m o r e p o t e n t t h a n ( + ) D - 6 0 0 (71). F u r t h e r a n a l y s i s has s h o w n that v e r a p a m J l and s i m i l a r a g e n t s f u n c t i o n as n e g a t i v e heterotropic regulatory agents reducing the a f f i n i t y of the DHP/VSCC interaction by a c t i n g at a s e p a r a t e site (11,24,71). D J p h e n y l a l k y ] a m i n e s can c o m p l e t e l y inhibit DHP binding, but also appear to do so by an a l l o s t e r i c m e c h a n i s m (ll,71). B i n d i n g data f r o m rat b r a i n to be d i s c u s s e d b e l o w s u g g e s t s t h a t all t h e s e agents may actually interact w i t h the same a l l o s t e r i c site (44). Some attempt~ in h e a r t and b r a i n h a v e b e e n m a d e to l a b e l t h i s site using ~H-verapami] (25,56), but these results are p r e l i m i n a r y as yet. A further c o m p l i c a t i o n concerns the effects of d i l t i a z e m . It is c l e a r f r o m its e f f e c t s in s m o o t h m u s c l e ( g u i n e a - p i g ileum, pig coronary artery, rabbit uterus) that this benzothiazepine actually stimulates DHP binding over its pharmacologically e f f e c t i v e dose range (11,12,21,71). Moreover, this effect is s t e r e o s p e c i f i c in that d - c i s - d i l t i a z e m produces it but its p h a r m a c o l o g i c a l l y less potent isomer l - c i s - d i l t i a z e m is m u c h less e f f e c t i v e (21). D i l t i a z e m also seems to influence DHP binding by i n t e r a c t i n g w i t h a separate site. Bolger et al. found that the effect of d i l t i a z e m in the g u i n e a - p i g ileum was mostly due to an increase in the n u m b e r 7~m~.x) of DHP sites rather than an e f f e c t on t h e i r affinity ( However, this is n o t n e c e s s a r i l y the mode of d i l t i a z e m action in every tissue as will be discussed. B o l g e r et al. have also d e m o n s t r a t e d that several other compounds not u s u a l l y thought of as c a l c i u m channel blockers could also enhance DHP b i n d i n g . These include yohimbine, c h l o r p h e n i r m i n e and tripolidine. It is i n t e r e s t i n g to a s k w h e t h e r in f a c t t h e s e a g e n t s do p o s s e s s s o m e c h a n n e l blocking activity. T h i s q u e s t i o n has b e e n a d d r e s s e d by S n y d e r and his c o l l e a g u e s (vide infra) (44,45). Apart from organic calcium channel blockers, inorganic channel blockers also i n t e r a c t w i t h D H P b i n d i n g sites. In the i l e u m as in m o s t o t h e r tissues, DHP binding is at l e a s t p a r t i a l l y dependent on the p r e s e n c e of d i v a l e n ~ c a t i o n s (ll). R e m o v a l of e n d o g e n o u s ions (probably mostly Ca +~) from ileal m e m b r a n e s with EDTA causes DHP b i n d i n g to fall to a b o u t 40% of c o n t r o l l e v e l s . Ca +2 a d d e d to i n c u b a t i o n s p e r f o r m e d under control c o n d i t i o n s slightly enhances binding (71). O t h e r ions s u c h as Co + L and La + 3 i n h i b i t D H P binding over approximately the s a m e c o n c e n t r a t i o n r a n g e as the b l o c k V S C C (11,71).

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Vol. 34, No. 13, 1984

Husele

B i n d i n g of l a b e l l e d DHP's in the h e a r t has c h a r a c t e r i s t i c s w h i c h are e x t r e m e l y s i m i l a r to those described in smooth muscle. H e r e a g a i n the b i n d i n g a p p e a r s s a t u r a b l e and of h i g h a f f i n i t y u s u a l l y to a s i n g l e set of s i t e s ( 1 0 , 1 1 , 1 3 , 1 5 - 1 8 , 2 0 - 2 9 ) . The r e p o r t e d K~ r a n g e for n i t r e n d i p i n e b i n d i n g is 0 . 0 7 8 - 6 nM, but m o s t of t ~ e s e v a l u e s fall i n t o the 0.1-0.5 n M range. Some controversy exists as to the exact locus of the DHP binding sites in heart. S o m e s t u d i e s h a v e s h o w n t h a t the m l t o c h o n d r i a l and light s a r c o p l a s m i c r e t i c u l u m fractions are relatively free of DHP binding, but p u r i f i e d sarcolemma is e n r i c h e d in t h e s e s i t e s (27,29). However, another report of subcellular f r a c t i o n a t i o n of canine myocardium demonstrated t h a t the r y a n o d i n e sensitive sarcoplasmic reticulum vesicle fraction had the highest concentration of b i n d i n g sites (28). As in s m o o t h muscle affinity labelling e x p e r i m e n t s have revealed that the DHP binding s i t e is l o c a t e d on a 4 5 , 0 0 0 d a l t o n p r o t e i n (22). Other aspects of D H P b i n d i n g in the h e a r t are a l s o s i m i l a r to s m o o t h m u s c l e . Binding is reduced by EDTA t r e a t m e n t although to a lesser extent than in smooth muscle or brain (ll). Binding can be reconstituted in EDTA treat~ed m e m b r a n e s by a d d i n g b a c k Ca +L or significantly, Ba +L or Sr +L w h i c h are a l s o a b l e to pass e a s i l y through VSCC. Verapami] and D - 6 0 0 inhibit DHP b i n d i n g but incompletely and by a negative heterotropic mechanism (11,17,24,26). Diltiazem enhances b i n d i n g (11,21). Inorganic channel blockers also block binding (Ii,18,24). One interesting study utilized 3 H - n i f e d i p i n e which has not been widely u s e d in DHP b i n d i n g studies (26). Among other things, this study showed that the interaction of both D-600 and verapamil w i t h the D H P b i n d i n g site w a s s t e r e o s p e c i f i c , (-) > (+). However, the d i f f e r e n c e between the p o t e n c y of the t w o isomers was considerably less than that found for their ability to i n h i b i t e l e c t r i c a l l y stimulated rabbit papillary muscles. Moreover, the data obtained in this study with nifedipine itself is i l l u s t r a t i v e of an i m p o r t a n t p r o b l e m in this field. T h a t is t h a t the p h a r m a c o l o g i c a l e f f e c t s of DHP's in the h e a r t o c c u r at 2-3 orders of m a g n i t u d e higher concentrations than their affinities for the D H P b i n d i n g s i t e s in t h i s tissue. T h i s is quite clear w h a t e v e r the type of assay used be it p h a r m a c o l o g i c a l or e l e c t r o p h y s i o l o g i c a l . For example, Lee and Tsien found that nitrendipine inhibited i n w a r d Ca + L c u r r e n t s in i s o l a t e d h e a r t cells with an EC50 of 154 nM (65). S i m i l a r l y high c o n c e n t r a t i o n s of n i s o l d i p i n e were found to be required by K a s s (72). Pharmacological essays reflecting VSCC activity in various parts of the h e a r t are s i m i l a r l y insensitive to DHP's (73). Such c o m p a r i s o n s are harder to make for verapamil and D-600 owing to the c o m p l e x i t y of their interaction w i t h the DHP binding site and the e x t r e m e use d e p e n d e n c e of t h e i r a c t i o n on the h e a r t (65). W h y t h e r e s h o u l d e x i s t such a l a r g e d i s c r e p a n c y between DHP binding and D H P e f f e c t s in the h e a r t is n o t c l e a r . One possibility is that DHP b i n d i n g in the h e a r t is not to VSCC. However, other less radical e x p l a n a t i o n s are also possible. The V S C C in the h e a r t are c l e a r l y " m o d i f i a b l e " in a n u m b e r of w a y s

Vol. 34, No. 13, 1984

Biochemistry of Calcium Channels

1211

(62). cAMP mediated modification is the b e s t k n o w n of these. Consequently, it is l i k e l y t h a t the f u n c t i o n i n g heart VSCC may possess properties in situ which are not n e c e s s a r i l y reflected in isolated preparations in Xi~Z~. Some particular influence or modifying f a c t o r m i g h t be lost. H o w e v e r , it s h o u l d be p o i n t e d out t h a t D H P b i n d i n g to i s o l a t e d w h o l e rat m y o c y t e s is a l s o of e x t r e m e l y high affinity (29). ~

M

_

~

Calcium currents have been d e m o n s t r a t e d in skeletal muscle from various species. The locus of this c a l c i u m current appears to be the T - t u b u l a r system. Although there have been several s u g g e s t i o n s made as to the function of this calcium current, the matter is c l e a r l y u n r e s o l v e d at this t i m e (74). It has b e e n s u g g e s t e d t h a t the c a l c i u m p r o v i d e d by t h i s p a t h w a y c o u l d p l a y some role in the i n i t i a t i o n or c o n t r o l of s k e l e t a l muscle contraction. However, such m u s c l e s appear to be able to t w i t c h quite happily in the absence of e x t r a c e l l u l a r c a l c i u m or when the ~ a has been blocked p h a r m a c o l o g i c a l l y (75-81). For the purposes the p r e s e n t d i s c u s s i o n , it is c l e a r that V S C C in s k e l e t a l m u s c l e can be blocked by organic calcium blockers including DHP's (75-81). However, very little detailed pharmacological data on these effects has been o b t a i n e d . Usually rather high d r u g concentrations h a v e been u s e d (e.g. 0.3 pM n i f e d i p i n e , 30 pM D600). However, in one r e p o r t the Ica in s k e l e t @ l m u s c l e was blocked by d i l t i a z e m w i t h an IC50 of a b o u t i0 -~ M, w h i c h is a fairly modest concentration for t h i s a g e n t (76). DHP binding in skeletal muscle is c o n c e n t r a t e d in T-tubular m e m b r a n e s w h i c h is c o n s i s t e n t w i t h the e l e c t r o p h y s i o l o g i c a l d a t a (32,35,36). Indeed, the concentrations of D H P b i n d i n g sites in s u c h preparations are by far the h i g h e s t for any t i s s u e r e p o r t e d to date. P o s s i b l y for t h i s r e a s o n , D H P b i n d i n g in t h i s t i s s u e has been extensively characterized in s p i t e of the d i f f i c u l t y in making corre]ations with functional data. It should be pointed out t h a t the p r o p e r t i e s of DHP b i n d i n g s i t e s in s k e l e t a l m u s c l e d i f f e r in m a n y r e s p e c t s f r o m t h o s e in o t h e r t i s s u e s . DHP sites in skeletal m u s c l e are of l o w e r affinity than in other tissues by a b o u t an o r d e r of m a g n i t u d e . The r e p o r t e d r a n g e for KD'S for nitrendipine and n i m o d i p i n e are 0.5-3.6 nM but m o s t r a n g e f r o m 1.4-3.6 nM. As d i s c u s s e d a b o v e , t r e a t m e n t of c a r d i a c or s m o o t h muscle membranes w i t h E D T A r e d u c e s DHP b i n d i n g , however in skeletal muscle, 1 mM EDTA enhances DHF binding (ii). Some e x t r e m e l y curious o b s e r v a t i o n s have also been made concerning the pharmacological specificity of D H P b i n d i n g s i t e s in s k e l e t a l muscle. Thus, u n d e r n o r m a l a s s a y c o n d i t i o n s , ( - ) D - 6 0 0 d o e s not d i s p l a c e D H P b i n d i n g w h e r e a s ( + ) D - 6 0 0 d o e s (31). M o r e o v e r , the a f f i n i t y of n i f e d i p i n e for the D H P b i n d i n g s i t e s a p p e a r s to be e x t r e m e l y low (240 nM) (31). These same authors have shown that diltiazem e n h a n c e s DHP b i n d i n g in s k e l e t a l m u s c l e as it d o e s elsewhere (32). In t h i s t i s s u e , the e f f e c t is p r i m a r i l y to increase the number of high affinity DHP sites. Indeed, a survey of 4 r a d i o a c t i v e DHP ligands showed no c o n s i s t e n t e f f e c t of diltiazem on K D a l t h o u g h the B was increased in e a c h c a s e (32). Thus, the e f f e c t s of d i l t ~ e m are s i m i l a r t o t h o s e s e e n

1212

Biochemistry of Calcium Channels

Vol. 34, No. 13, 1984

in i l e u m (Bm~x) (71) r a t h e r than those s e e n in b r a i n (K D) (44,47,53). ~ n c o n t r a s t to a s s a y s p e r f o r m e d in the a b s e n c e of diltiezem, in its presence (-)D-600 does displace DHP binding and in fact is s l i g h t l y m o r e p o t e n t t h a n ( + ) D - 6 0 0 (31). Under both conditions, stereoisomers of DHP's show the correct specificity. Surprisingly, F o s s e t et al., (35,36) in t w o p u b l i c a t i o n s have c l a i m e d that d i l t i a z e m has no effect on DHP binding in skeletal muscle. Moreover, this group also reports an a f f i n i t y for nifedipine of 4 nM, q u i t e d i f f e r e n t to the r e s u l t s reported above. This discrepancy could be related to the fact that Fosset et al., p e r f o r m their incubations at r a t h e r l o w t e m p e r a t u r e s (lO°C). DHP binding in skeletal muscle can be regulated by other c h a n n e l b l o c k e r s in a n e g a t i v e h e t e r o t r o p i c m a n n e r as in o t h e r tissues (Ii). It h a s been found in t w o reports that diphenylalkylamines are consistently more potent blockers of DHP binding in skeletal muscle than in heart, ileum or brain (ii,30). Inorganic blockers will block binding as they do e l s e w h e r e (ll). S o m e data is a l s o a v a i l a b l e on the m o l e c u l a r n a t u r e of the DHP binding site in skeletal muscle. Target size analysis gives an apparent m o l e c u l a r weight of 210,000 daltons w h i c h is close to that f o u n d in i l e u m and h e a r t (37). Glossmann and F e r r y h a v e succeeded in s o l u b i l i z i n g skeletal muscle DHP binding sites using either digitonin or C H A P S (33). It is i n t e r e s t i n g to note that this solubilized preparation could still be r e g u l a t e d by d i l t i a z e m in a s i m i l a r manner to the particulate state (Increase in B - a x , d e c r e a s e i d i s s o c i a t i rate). M o r e o v e r , ( - ) D - 6 0 0 is stJl~ c o n s i d e r a b l y ~ e s s p o t e n t °inn the s o l u b i l i z e d preparation than (+)D-600, although in the p r e s e n c e of d i l t i a z e m , the t w o i s o m e r s are a b o u t equal. In c o n t r a s t to r e s u l t s r e p o r t e d for particulate preparations, however (ll), c h e l a t o r s of d i v a l e n t m e t a l c a t i o n s s u c h as E D T A or E G T A r e d u c e DHP b i n d i n g in the soluble preparation. These effects could be c o m p l e t e l y reversed by addition of Ca +~ or even Mg +~. The solubilized binding sites could be retained on a variety of lectin c o l u m n s suggesting their glycoprotein nature. This would not be s u r p r i s i n g for an integral m e m b r a n e protein. DHP binding sites can be purified up to 4 0 - f o l d on l e c t i n columns or 1 0 - f o l d by s u c r o s e density centrifugation. Neuronal

Tissue

The a b o v e d i s c u s s i o n illustrates t h a t in s p i t e of c e r t a i n discrepancies, the p r o p e r t i e s of DHP b i n d i n g s i t e s in s m o o t h , cardiac and skeletal muscle are mostly consistent with the notion that these sites represent VSCC. Clearly, VSCC do exist in these t i s s u e s and it is c l e a r that they can be i n h i b i t e d by o r g a n i c calcium channel blockers. A fundamentally different situation exists in neuronal tissue. Thu~ there i ~ no evidence ~ h a t s o e v e r

organic c a l c i u m channel blockers. This is not to say that drugs s u c h as v e r a p a m i l w i l l not b l o c k c a l c i u m c u r r e n t s in n e r v e s at gigantic concentrations (Table 1). However, at t h e s e e l e v a t e d concentrations, p h e n y l and d i p h e n y l a l k y l a m i n e s are e x t r e m e l y

Vol. 34, No. 13, 1984

Biochemistry of Calcium Channels

1213

nonspecific in t h e i r a c t i o n s and it is m o s t u n l i k e l y t h a t the e f f e c t s o b s e r v e d are m e d i a t e d by the n o r m a l c h a n n e l b l o c k i n g m e c h a n i s m s of these agents. More specific VSCC blockers that act at l o w e r c o n c e n t r a t i o n s s u c h as DHP's s h o w no a b i l i t y at all to b l o c k V S C C in n e r v e e v e n at e l e v a t e d c o n c e n t r a t i o n s ( T a b l e i). In spite of this, the brain is one of the richest sources of high a f f i n i t y DHP binding sites (11,15,18,23,38-55). This i n t e r e s t i n g d i s c r e p a n c y d e s e r v e s some f u r t h e r discussion. Calcium channels e x i s t in all p o r t i o n s of the n e u r o n e (2). However, their concentration varies considerably with neuronal type, time of d e v e l o p m e n t and region. In general, VSCC are highly c o n c e , t r a t e d in nerve endings (synaptic regions) and are f o u n d in l o w c o n c e n t r a t i o n s in axons. The cell s o m a m a y contain VSCC d e p e n d i n g on neuronal type and d e v e l o p m e n t a l state. Indeed, ICa'S are often recorded from the cell soma as a "model" for VSCC in nerve t e r m i n a l s w h i c h are i n a c c e s s a b l e to r e c o r d i n g e l e c t r o d e s (e.g. 112). VSCC also occur in d e n d r i t e s (2,89). The best charactefized f u n c t i o n for V S C C in n e r v e is to p r o v i d e an i n f l u x of Ca + ~ for the r e l e a s e of n e u r o t r a n s m i t t e r s from nerve e n d i n g s and p o s s i b l y d e n d r i t e s (stimulus/secretion coupling). The e x a c t " f u n c t i o n s " of V S C C in o t h e r p a r t s of the n e r v e cell are not k n o w n e x c e p t of c o u r s e that these calcium currents c o n t r i b u t e both directly and i n d i r e c t l y to neuronal e x c i t a b i l i t y (2,4). V S C C can be m o n i t o r e d in n e r v e s in v a r i o u s w a y s . The most direct method is by m e a s u r i n g calcium currents Ica~ Car electrophysiologically. A second method is to m o n i t o r uptake via VSCC usSng radioactive Ca + ~ or c h a n g e s in t h e fluorescence of Ca +z s e n s i t i v e dyes. A third method is to m e a s u r e the release of n e u r o t r a n s m i t t e r w h i c h under the correct c i r c u m s t a n c e s b e c o m e s an assay for nerve t e r m i n a l VSCC. Table 1 illustrates the e f f e c t s of o r g a n i c c a l c i u m c h a n n e l b l o c k e r s on these various parameters. E l e c t r o p h y s i o l o g i c a l studies show that D - 6 0 0 and v e r a p a m i l b l o c k ICa'S in v a r i o u s p r e p a r a t i o n s very poorly. Even at c o n c e n t r a t i o n s greater than lO - ~ M the block is usually incomplete. D a t a on the e f f e c t s of DHP's is as yet unavailable. It should be stressed that at c o n c e n t r a t i o n s above a b o u t i0 - ~ M, v e r a p a m i l and D - 6 0 0 w i l l b l o c k v i r t u a l l y every b i o l o g i c a l process k n o w n to man. This includes the i n t e r a c t i o n of most neurotransmitters with their receptors (6,70), prostaglandin synthesis (113), transmitter uptake (114) and sodium channels (97,115). Most of t h e s e effects are not stereospecific (6,70). Consequently, e v e n the m e a g r e e f f e c t s o b s e r v e d w i t h t h e s e d r u g s on n e r v e V S C C m u s t be r e g a r d e d w i t h suspicion. T h i s v i e w is a m p l y c o n f i r m e d by d a t a on Ca +L f l u x e s and n e u r o t r a n s m i t t e r release w h e r e the effects of DHP's have also been analyzed ( T a b l e i). In s u c h cases, verapamil and D - 6 0 0 again produce some effects at high c o n c e n t r a t i o n s w h e r e a s drugs s u c h as n i f e d i p i n e are u s u a l l y completely inactive e v e n at concentrations a b o v e 10 -5 M. T h i s is at l e a s t 4 o r d e r s of m a g n i t u d e higher than those c o n c e n t r a t i o n s at w h i c h DHP's produce e f f e c t s on s m o o t h m u s c l e and 2 o r d e r s of m a g n i t u d e h i g h e r t h a n those at w h i c h they produce their effects on the heart.

5 x 10 -4 M 10 -4 M

V D D 10-5-10 -4 M V D i0-4-2xi0 -3 M

Limnea Stagnalis neurones

Euhadra Quaestia neurones

10 -4 M 10 -4 M 10 -5 M 10 -5 M 10 -4 M 10 -4 M

D D DZ V V D D Nit

Toad rods

Guinea-pig cerebella Purkinje cells

C-cells, rabbit nodose ganglion

NIE-II5, neuroblastoma (Nb)

58

96 97

V bl0ck~d IC50 = 10 -4 M 3 x 10 -4 M Nif ineffective 10 -5 M of all drugs ineffective under normal conditions blocked Ca signal (IC50 = 3 x 10 -4 M) but also blocked sodium channels

Nit Nimodipine Nisoldipine V D

Rat brain synaptosomes

Squid axon

Rat brain synaptosomes

blocked,

V Nif

V

Guinea-pig brain synaptosomes Synaptosomes

94

V & D partially blocked both fast and slow phases of Ca uptake

95

10 -4 M 10-4 M

V D

Rat brain synaptosomes

93

92 & Personal Cormn.

91

90

89

88

87

V & D blocked flux about 60% Nif was ineffective

D blocked Ca spike with IC50 = 10 -4 M, Nit was ineffective

No effect on Ica

10 -5 M blocked Ca spike

blocked ICa in dendrites and soma

blocked Ca spike

"some reduction" in Ica

blocked ICa and other parameters

86

85

blocked Ca spike,

IC50 = 10 -4 M

84

83

82

Reference(s)

blocked ICa,INa and ICI

40% block of ICa

80% block of ICa

Effect

IC50 = 2 x 10 -5 M

10 -4 M 10 -4 M 10 -5 M

V D Nif

Depolarization induced Ca fluxes in neurones

Rat brain synaptosomes

B.

NGI08-15 Nb x glioma hybrid

10 -2 M

D

H. Pomatia & H. Levantina pacemaker neurones

Aplysia neurones

3 x 10 -5 M

V

Drug

Soma of Helix neurones

Physiological measurements

Perfused neurones from Helix & Limnea Stagnalis

A.

TABLE i

Effects of Organic Calcium Channel Blockers on VSCC in Nerve

Oo

Lo

o

Lo

o

Q

o o

Flunirazine DZ Nicardipine V

Isolated cat heart

Rabbit ear artery

ileum

cortical

ileum

Guinea-pig

Guinea-pig

Guinea-pig

10 -5 M did not block evoked Ach release No effect on m.e.p.p.'s

V

blocked NE release, IC50 = 20 ~M, DZ = 25 ~M, 50 uM Nif h~d no effect

Did not block evoked Ach release at 10 -6 M

10 -4 M no block of NE release

D Nicardipine DZ

V = verapamil, D = D-600, DZ = diltiazem, Nif = nifedipine, NE = norepinephrine and Ach = acetylcholine.

98

30

iii

ii0

93

109

108

107

106

ICa = calcium current,

Chick retinal PCI2 cells

Nit = nitrendipine,

blocked transmitter release with IC50 = 10 -8 M in both cases

Nicardipine

cells and

2 x 10 -5 M no effect on evoked NE release

No effect on evoked DA release at 10 -5 M

Various Nit

Small effect on ejp's at 10 -5 M

V Bepridil DHP's

5 x 10 -5 M

104

103

102

No effect on evoked NE release below 10 -5 M 105

Did not block evoked NE outflow at 3 x 10 -6 M

Nif no effect up to 2 x 10 -5 M

evoked NE outflow at > 10-6 M

i01

I00

54,55,98,99

Rat phrenic nerve

neurones

arteries

Cultured nigro-striatal

Guinea-pig mesenteric

junction

V

Rabbit basilar and facial arteries

Frog neuromuscular

Nif

Rabbit ear artery

D DZ Nif

Nif V Nif

Rabbit heart Reduces

cGMP formation

No effect below 3 x 10 -4 M

coupling

blocked Ca dependent IC50 = 2 x 10-5 M

Drugs block at low concentrations

Crayfish neuromuscular transmisV sion and frog, myoneural junction Pr enylamine

Stimulus/secretion in neurones

D

NIE-II5

C.

Various

PCI2 NCB-20 NGI08-15 N4TGI NBrI0-A

~"

==

m"

C)

O

O

r~

O O

%0 Oo

O~

Z O

%0

o

1216

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In s p i t e of t h i s , D H P b i n d i n g sites in b r a i n exhibit properties e x t r e m e l y s i m i l a r to those seen in smooth and cardiac muscle. One possibility, that the binding sites are localized on c e r e b r a l b l o o d v e s s e l s has b e e n d i s m i s s e d by a u t o r a d i o g r a p h i c studies (42,50). D H P b i n d i n g s e e m s to be c o n c e n t r a t e d in the synaptosomal fraction. Neuronal DHP binding sites are regulated in a s i m i l a r fashion to those in other tissues. V e r a p a m i l and D600 partially inhibit binding by a n e g a t i v e heterotropie mechanism (11,44,46,53) and diltiazem enhances b i n d i n g by increasing the affinity of the binding interaction (11,44,47,53). There is no effect on Bma x in this case. DHP binding in brain is d e p e n d e n t on the p r e s e n c e of d i v a l e n t c a t i o n s and is r e d u c e d by t r e a t m e n t of m e m b r a n e s w i t h EDTA. Binding can be r e c o n s t i t u t e d by Ca +~, Ba +2 or Sr + 2 (ii). Binding is a l s o i n h i b i t e d by inorganic blockers, such as C o + L at slightly higher concentrations t h a n in o t h e r t i s s u e s (11,43)4 Moreover, such blocking ions w i l l b l o c k the a b i l i t y of Ca +L to r e c o n s t i t u t e binding in E G T A t r e a t e d m e m b r a n e s (43). The p h a r m a c o l o g y of b r a i n D H P b i n d i n g s i t e s has b e e n s t u d i e d in d e t a i l . It has the correct s t e r e o s p e c i f i c i t y for the isomers of DHP's and v e r a p a m i l (40). Curiously in the presence of diltiazem, the stereospecificity for v e r a p a m i l is no l o n g e r retained (40). Overall there is a good c o r r e l a t i o n b e t w e e n the affinity of drugs for b r a i n D H P s i t e s and t h e i r e f f e c t s on p o t a s s i u m induced contractions of r a b b i t a o r t i c strips (52). T h i s is f u r t h e r evidence for the s i m i l a r i t y b e t w e e n brain DHP sites and those in smooth muscle. A c a r e f u l a n a l y s i s of the w a y in w h i c h v a r i o u s d r u g s i n t e r a c t w i t h DHP b i n d i n g has l e a d M u r p h y et al., (44) to propose that inhibitors such as the phenyl and diphenylalkylamines and enhancers such as d i l t i a z e m all act at a single site. This may also be the case in other tissues although other authors have suggested that separate allosteric sites may exist (116). As d i s c u s s e d above, it w a s f o u n d that in the guinea-pig ileum, certain compounds that are not usually classified as c a l c i u m c h a n n e l b l o c k e r s are a b l e to e n h a n c e D H P b i n d i n g in a s i m i l a r f a s h i o n to d i l t i a z e m . This implies that t h e y m i g h t be a b l e to b l o c k V S C C in a d d i t i o n to the p r o p e r t i e s usually attributed to them. Murphy et al., (44) h a v e also o b s e r v e d t h a t a v a r i e t y of a g e n t s c o u l d i n t e r a c t w i t h b r a i n D H P binding sites in a d i l t i a z e m like fashion. These drugs included the n e u r o l e p t i c thioridazine. T h e s e a u t h o r s then d e m o n s t r a t e d t h a t t h e s e a g e n t s c o u l d i n d e e d b l o c k V S C C in the g u i n e a - p i g ileum. Most recently this s a m e group have shown t h a t the neuroleptic p i m o z i d e and c e r t a i n r e l a t e d s t r u c t u r e s c o u l d a l s o act at this allosteric site (45). These drugs inhibited VSCC in the rat vas d e f e r e n s at r a t h e r l o w c o n c e n t r a t i o n s . The D H P b i n d i n g site f r o m b r a i n has b e e n s o l u b i l i z e d using digitonin (53). This solubilized site can still be r e g u l a t e d by b o t h verapamil and diltiazem. Binding activity can be retained on a wheat germ lectin column. A 30-fold p u r i f i c a t i o n can be achieved by this method. Target size analysis has provided an e s t i m a t e of 185,000 for the m o l e c u l a r w e i g h t of the brain VSCC complex (39). A l t h o u g h D H P s e n s i t i v e V S C C h a v e not b e e n f o u n d to d a t e in authentic neurones, several neuronal clonal cell lines have been

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shown to p o s s e s s voltage sensitive calcium fluxes t h a t are b l o c k e d by DHP's at n a n o m o l a r concentrations. These cells also possess high affinity DHP binding sites (54,55,98,99). It is p o s s i b l e t h a t t h e s e V S C C are the c o r r e l a t e s of the D H P b i n d i n g sites detected in brain. Thus, it is p o s s i b l e t h a t on f u r t h e r careful analysis, such p a t h w a y s will also be found in a u t h e n t i c neurones. At any rate, D H P b i n d i n g s i t e s in b r a i n are so l i k e those in o t h e r tissues that it s e e m s likely that t h e y do represent VSCC. Actually determining the e x i s t e n c e of s u c h c h a n n e l s is a p r e s s i n g t a s k if D H P b i n d i n g in b r a i n is u l t i m a t e l y to prove meaningful.

Conclusions Although a good c a s e can be m a d e t h a t D H P b i n d i n g l a b e l s V S C C in s o m e cases, s e v e r a l c h a l l e n g i n g questions remain. For example, low affinity DHP binding sites have been occasionally reported (15,59) and it is possible that these represent receptor linked channels. This important p o i n t is yet to be c l a r i f i e d . The f u n c t i o n a l n a t u r e of D H P b i n d i n g s i t e s on n e r v e is a l s o an intriguing p u z z l e s t i l l to be s o l v e d . C l e a r l y not all V S C C are sensitive to organic c a l c i u m blockers to the same extent. Those in smooth m u s c l e are clearly more sensitive than those in heart and at l e a s t s o m e of t h o s e in b r a i n are not b l o c k e d at all. Elucidating the m o l e c u l a r b a s i s for t h e s e d i f f e r e n c e s w i l l be most interesting. In a d d i t i o n , V S C C in o t h e r t i s s u e s are a l s o important. What for e x a m p l e is the n a t u r e of the V S C C in e x c i t a b l e endocrine cells? Most evidence suggests that these may be r a t h e r DHP i n s e n s i t i v e l i k e t h o s e f o u n d in n e u r o n e s (e.g. 117). A final i n t e r e s t i n g point relates to t h e newly d i s c o v e r e d DHP's that actually enhance the flow of Ca +L through VSCC rather than blocking these channels (118). The m a r i n e toxin m a i t o t o x i n appears to have a s i m i l a r action (119,120). Such substances will provide new tools for probing the structure and function of VSCC.

Acknowledgements NATO

Supported by PHS g r a n t s (U.K.) Fellow.

DA 0 2 1 2 1

and

DA 02575.

S.B.F.

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