Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action?

TiPS - November 1989 [Vol. 10}

442

Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action? Antonio Laurenza, Elizabeth McHugh Sutkowski and Kenneth B. Seamon Forskolin, a naturally occurring diterpene, directly stimulates adenylyl cyclase and has been used extensively to increase cAMP and to elicit cAMP-dependent physiological responses. More recently, forskolin has been shown to inhibit a number of membrane transport proteins and channel proteins through a mechanism that does not involve the production of cAMP. Many of these channel proteins are predicted to have similar topographies in the membrane bilayer and it is tempting to speculate that forskolin may be binding at structurally homologous sites. Kenneth Seamon and colleagues discuss the cAMP-independent effects of forskolin and the structural similarity between forskolin and other physiologically important substances such as hexoses and steroids with respect to potential forskolin binding sites. Forskolin (Fig. 1) was first described in 1981 as a cardioactive drug that reversibly stimulated adenylyl cyclase in vitro and in vivo resulting in increased intracellular cAMP (see Ref. I for review). Since that time forskolin has been used extensively in physiological studies aimed at determining the consequences of increased cellular cAMP. Physiological consequences of forskolin administration have largely been consistent with the proposal that forskolin effects are due to increases in intracellular cAMP. However, several recent studies have demonstrated that forskolin has actions that are not mediated by increased cAMP and are apparently produced by forskolin acting at a site or sites distinct from adenylyl cyclase. Various criteria (Table I) have been used to demonstrate that forskolin produces cAMP-independent effects. All these cAMP-independent effects (Table II) are associated with the modulation of membrane transport proteins. A. Laurenza is a guest worker, E. McHugh Sutkowski is a National Research Council Associate and K. B. Seamon is Chief at the Laboratory of Molecular Pharmacology, Division of Biochemistry and Biophysics, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA. (~) 1989, Elsevier Science Publishers Ltd. (UK) 0165 -

Adenylyl cyclase The interaction of forskolin analogues and other agents with adenylyl cyclase can be determined by their ability to inhibit [3H]forskolin binding and by their ability to stimulate adenylyl cyclase. Direct binding studies with [3H]forskolin have demonstrated that there are high affinity binding sites for forskolin associated with the activated complex of the catalytic subunit and the Gs protein 1,2. There is a good correlation between the ICso of forskolin analogues to inhibit [3H]forskolin binding and the ECso for their stimulation of adenylyl cyclase. 1,9-Dideoxyforskolin, a naturally occurring analogue of forskolin, does not inhibit the high affinity binding of [3H]forskolin nor does it activate

adenylyl cyclase. Analogues of forskolin that are modified at the 1- and 9-hydroxyl groups or that have large lipophilic groups esterified at the 7-hydroxyl group are not potent at inhibiting [3H]forskolin binding (ICs0 > 10 ~M) or activating adenylyl cyclase (ECs0 5 0 ~ M ) 1"3. Water-soluble analogues of forskolin that have heterocyclic amino acids esterified at the 6- or 7-hydroxyl groups are very potent at activating adenylyl cyclase (EC50 < 10 ~M) and inhibiting [3H]forskolin binding (ICs0 < 40 ~M)3. Neither cytochalasin B nor D-glucose (both of which act at the glucose transporter) has any effect on high affinity [3H]forskolin binding or on the activity of adenylyl cyclase measured in the presence of forskolin 4.

Glucose transporter Forskolin inhibits glucose transport in a number of different cells including adipocytes, erythrocytes, platelets, cardiomyocytes and bone cells 4-8. 1,9-Dideoxyforskolin inhibited glucose transport in rat adipocytes and did not increase cAMP (Ref. 4). Forskolin inhibited glucose transport in vesicle preparations from erythrocytes and adipocytes 5"6's. Because these vesicle preparations lack ATP, they do not produce cAMP. The inhibitory effects of forskolin cannot, therefore, be attributed to activation of adenylyl cyclase. Binding of forskolin to the glucose transporter has been demonstrated both b y direct binding of [3H]forskolin and b y the ability of forskolin to inhibit the binding of [3H]cytochalasin B, a fungal metabolite that inhibits the glucose transporter 4"6"s'9. [3H]Forskolin binds to the glucose transporter in human erythrocytes with a Ka of 2.6 ~M and the binding is inhibited by cytochalasins A and B and b y hexoses 9. Forskolin also inhibits cytochalasin B bind-

TABLE I. Criteria for defining cAMP-independent effects of forskolin • The inability of cholera toxin, analogues of cAMP, or hormonal stimulators of adenylyl

cyclase to reproduce an effect of forskolin • The inability of 2',5'-dideoxyadenosine (an inhibitor of adenylyl cyclase) to inhibit a forskolin-stimulated effect • The lack of correlation between the magnitude of the physiological response and the levels of intraceliular cAMP produced as a function of either concentration of forskolin or time of administration of forskolin eThe ability of 1,9-dideoxyforskolin (a naturally occurring analogue of forskolin that does not activate adenylyl cyclase) to reproduce a forskolin-like effect

6147/89/$02.00

TiPS - November 1989 [Vol. 10] ing to adipocyte membranes with a Ki of 200 nM which is identical to its Ki for inhibition of glucose transport in the same vesicles s. [3HlForskolinl° and an [125I]arylazido derivative of forskolin 11 are covalently incorporated into the erythrocyte glucose transporter when photolysed in the presence of erythrocyte membranes. Incorporation of both analogues is inhibited by cytochalasin B and by D-glucose, but not by n-glucose which does not bind to the glucose transporter. An 18 kDa fragment of the glucose transporter is photolabelled by [3H]forskolinl° and the [12SI]arylazido derivative of forskolin 1~. This fragment is also photolabelled b y [3H]cytochalasin B (Ref. 10) suggesting that forskolin and cytochalasin B may be binding at the same domain of the glucose transporter. There is good correlation between the potency of forskolin analogues to inhibit cytochalasin B binding and to inhibit glucose transport 4. Lipophilic analogues of forskolin inhibit the transport of glucose with a potency relative to forskolin that is much higher than their potency to activate adenylyl cyclase. The very lipophilic arylazido derivative of forskolin that is used to photolabel the glucose transporter is more potent than forskolin in inhibiting transport 11. Water-soluble analogues of forskolin that are equipotent with forskolin in activating adenylyl cyclase are less potent than forskolin in inhibiting glucose transport 4. 1,9-Dideoxyforskolin inhibits glucose transport and cytochalasin B binding although it is less potent than forskolin.4 The above results suggest that the forskolin binding site on the glucose transporter has a higher affinity for lipophilic analogues of forskolin than the forskolin binding site on adenylyl cyclase. Nicotinic acetylcholine receptor cAMP-dependent and -independent effects of forskolin have been observed on acetylcholineinduced desensitization of the nicotinic acetylcholine receptor. Forskolin increased the rate at which nicotinic receptors desensitized when exposed to agonist in rat muscle 12-1s and rat sympathetic ganglia 16. At a concentration of I~M, forskolin (but not 1,9-dideoxyforskolin) enhanced nicotinic

443

CH3 •

14

|

H3C 'CF OH OH I --

0

c.3 II

forskolin

~OH H0\\\~

OH OC-D-galactose

androsterone

Fig. 1. Structures of forskolin, o~-o-galactoseand androsterone. The parts of the A, B and C rings of forskolin that are structurally homologous to hexoses are encircled by the broken line.

receptor desensitization after 30min in rat muscle, consistent with forskolin acting through cAMP (Ref. 12). By contrast, forskolin and 1,9-dideoxyforskolin, at concentrations of 20~M, both rapidly inhibited nicotinic receptors in rat muscle cultures; this is consistent with a cAMP-independent effect 13'1s. Forskolin and 1,9dideoxyforskolin directly blocked the open channel of Torpedo nicotinic receptors expressed in Xenopus oocytes 17 and forskolin also blocked the open channel of nicotinic receptors of rat sympathetic ganglia 16. Forskolin rapidly and reversibly inhibited the carbachol-stimulated influx of 86Rb+ through nicotinic receptors of PC12 cells and chick myotubes ls'19. In PC12 cells, inhibition by forskolin of carbachol-induced 86Rb+ uptake occurred more rapidly than the forskolinelicited increase in cellular cAMP levels. The inhibitory effect was also observed with 1,9-dideoxyforskolin TM. Carbachol-stimulated 86Rb+ uptake in chick myotubes was not inhibited by cholera toxin, isobutylmethylxanthine, isoprenaline, or 8-Br-cAMP, and inhibition by forskolin of S6Rb+ uptake was not inhibited by 2',5'-dideoxyadenosine 19. These data suggest that forskolin inhibits agonistinduced S6Rb+ influx through the nicotinic receptor by a mechanism that is independent of adenylyl cyclase and cAMP.

Carbachol and o¢-bungarotoxin binding to chick myotubes were not affected b y forskolin, indicating that forskolin has no effect at the agonist or antagonist binding sites of the myotube receptors 19. Forskolin quenched carbachol-induced ethidium fluorescence (ECso 20 ~M) and inhibited amantadine-sensitive high affinity binding (ICso 10 ~M) of [3H]phencyclidine in the presence of carbachol in Torpedo microsacs 19. Inhibition of [3H]phencyclidine binding by forskolin in the absence of carbachol was not as complete as in the presence of carbachol, suggesting that forskolin binds more efficiently to the channel. The high affinity ]phencyclidine binding site is a noncompetitive antagonist site which is postulated to be located in the ion pore 19. Since forskolin inhibited high affinity [3H]phencyclidine binding to nicotinic receptor, it was suggested that forskolin may act at this noncompetitive antagonist binding site or at another site that allosterically affects the phencyclidine binding site 19. Voltage-dependent K + channels Forskolin modulates voltagedependent K+ conductances through cAMP-dependent and cAMP-independent mechanisms 20-26. Two voltage-dependent K + currents (delayed outward currents) in neurons of Aplysia were

444

TiPS - N o v e m b e r 1989 [Vol. 10]

reduced by application of forskolin and the phosphodiesterase inhibitor, theophylline26. The inhibition by forskolin was reduced in the absence of theophylline suggesting an involvement of cAMP. Forskotin inhibited delayed rectifying K + channels in pancreatic ~-cells (EC50 13 ~M) 24 as well as voltage-dependent K + channels in Helix neurons (Ka 16 ~M)22, nudibranch neurons 21, h u m a n T cells 25 and PC12 cells (EC50 ~ 19 ~M) 23 i n a cAMP-independent manner. Forskolin greatly accelerated the inactivation kinetics of the K + curr e n t s 21"23"25. External application of phosphodiesterase inhibitors, analogues of cAMP, intracellular injection of cAMP, or intracellular injection of the catalytic subunit of cAMP-dependent protein kinase did not accelerate the inactivation kinetics. 1,9-Dideoxyforskolin was equipotent with forskolin in accelerating the kinetics of inactivation in h u m a n T cells and PC12 cells23"2s. Forskolin inhibition was

also observed on cell-free membrane patches from PC12 cells, pancreatic ~-cells and h u m a n T cells that are devoid of cytoplasmic components such as ATP (Refs 23-25). It has been suggested that the effects of forskolin on the kinetics of the K + currents are similar to those of hydrophobic quaternary a m m o n i u m ions and aminopyridines which reversibly block open voltage-dependent K+ channels 21,22. The interactions of forskolin with ligand- and voltage-gated channels have been assessed only by the modulation of channel function. Although there has not been a systematic study of the interaction of forskolin analogues with ion channels, limited information is available for some forskolin analogues. Forskolin and 1,9-dideoxyforskolin were equipotent in inhibiting voltage-dependent K + channels in PC12 cells and T cells23'2s. 1,9-Dideoxyforskolin was slightly more potent in inhibiting carbachol-stimulated S6Rb+ uptake

TABLE II. cAMP-independent effects of forskolin

Effect

Cell type

ECso (p,M)

Ref.

Inhibition of glucose transport

rat and human adipocytes human erythrocytes human platelets rat cardiac myocytes rat adipose plasma membrane vesicles human bone cells

ND 7.5 2 ND, 0.3 0.2

5 6 a b,c 4",8

Inhibition of D-glucose-sensitive [3H]cytochalasin B binding to glucose transporter

human erythrocytes rat adipose plasma membrane vesicles human erythrocyte ghosts

ND 0.2, 0.27

6 4,8

ND, 3

9,10

Enhancement of nicotinic receptor desensitization

rat muscle cultures (myotubes) rat sympathetic ganglia rat muscle cultures (myoballs) Xenopus oocytes expressing Torpedonicotinic receptors

> 20 10-30 > 20 - 10

14,15 16 13* 17*

Inhibition of carbachol-mediated ion flux through nicotinic receptors

PC12 (rat pheochromocytoma) cells) chick muscle cultures (myotubes)

- 5

18*

20

19

Inhibition of amantadine-sensitive [3H]PCP binding to nicotinic receptors

Torpedoelectroplax microsacs

10

19

Inhibition of muscimol-stimulated CI- flux through GABAA receptors

rat brain synaptoneurosomes

14

Modulation of voltage-dependent K+ channels

neuron cell bodies of nudibranch mollusks helix nerve cells PC12 cells mouse pancreatic B-cells human T cells

ND

21

~16 19 13 50-65

22 23* 24 24*

ND

e*

Partial reversal of doxorubicin resistance

murine sarcoma $180 (multidrugresistant variants

1

d

35*

* Similar effects observed with 1,9-dideoxyforskolin. NB, not determined. "Kim, H. D. et aL (1986) J. Pharmacol. Exp. Ther. 236, 585-589; bShanahan, M. F. et aL (1986) Biochim. Biophys. Acta 887, 121-129; c Geisbuhler, T. P. et aL (1987) PflOgersArch. 409, 158-162; dvan Valen, F. and Keck, E. (1988) Bone 9, 89--92; "Wadler, S. and Wiernik, P. H. (1988) Cancer Res. 48, 539-543.

through the nicotinic receptor in PC12 cells is. The water-soluble analogue, MPB-forskolin, had no effect on the nicotinic receptors in rat muscle cultures 13. Forskolin has not yet been shown to interact directly with ligand-gated or voltage-gated ion channels. Definition of binding sites It is not possible to discriminate between cAMP-dependent and cAMP-independent effects of forskolin solely on the basis of concentration-response curves. Although forskolin can elicit physiological effects at concentrations that do not produce maximal increases in cAMP, the EC50 for forskolin elevation of cAMP in cells and tissues ranges between I~M and 20~M (Ref. 1). The ECs0 for forskolin to elicit cAMP-independent effects generally falls in the same range (Table II). It is therefore essential to define criteria to distinguish between the binding of forskolin at its different sites of action. • Effects of forskolin that are mediated through adenylyl cyclase and cAMP will not be reproduced by 1,9-dideoxyforskolin, nor will they be modified by D-glucose or cytochalasin B. Binding of forskolin to adenylyl cyclase will not be inhibited by 1,9-dideoxyforskolin, cytochalasin B, or Dglucose. • Binding of forskolin to the glucose transporter will be inhibited by D-glucose or cytochalasin B. 1,9-Dideoxyforskolin and water-soluble derivatives of forskolin will be less potent than forskolin and certain lipophilic derivatives of forskolin may be more potent than forskolin at inhibiting glucose transport. • Effects of forskolin that are mediated through inhibition of ligand- and voltage-gated ion channels may be reproduced by 1,9-dideoxyforskolin with equal or greater potency. These definitions are based on data derived from studies with only a few forskolin derivatives. It is clear that analogues of forskolin that are specific for one class of binding site would provide the best criteria for distinguishing different sites of action. In designing more specific forskolin analogues, it may also be helpful to consider the structural similarity between forskolin and endogenous corn-

TiPS - November 1989 [Vol. 10]

445

pounds that can affect membraneassociated proteins. Structural similarities between forskolin, hexoses and steroids Forskolin shares a number of structural features with some endogenous bioactive compounds (Fig. 1). The B ring of forskolin, part of the A ring and the ether oxygen of the C ring are structurally homologous to hexoses. Forskolin has four hydroxyl groups that are superimposable with those of 0C-D-galactose. The 1hydroxyl group of forskolin on the A ring is homologous to the 6hydroxyl group of 0C-D-galactose and the 9-hydroxyl and 6-hydroxyl groups of the B ring of forskolin are homologous to the 4-hydroxyl and 1-hydroxyl, respectively, of a-Dgalactose. The 7-acetoxy oxygen and the 8-ether oxygen of forskolin are homologous with the 2hydroxyl and 3-hydroxyl oxygens, respectively, of a-D-galactose. The structural similarity between forskolin and hexoses may be the basis for forskolin binding to the glucose transporter in a similar manner as transported hexoses 4. This structural homology does not apply to the forskolin binding site on adenylyl cyclase. Hexoses have no effect on the interaction of forskolin with adenylyl cyclase (A. Laurenza and K. B. Seamon, unpublished) and there is no evidence that hexoses interact with ligand-gated or voltage-gated channels. Forskolin is also structurally homologous with steroids. The A, B and C rings of forskolin are homologous with the same rings in steroids. Both forskolin and steroids have axial methyl groups on positions 10 and 13. The major differences between forskolin and steroid structures is the presence of hydroxyl groups at the 1, 6 and 9 positions of forskolin and the methyl groups at positions 4 and 8. Forskolin also differs from steroids by the presence of the ketone at position 11, the ether oxygen in the C ring, the double bond at position 14 instead of the D ring of the steroid, and the acetate group at position 7. Steroids have been shown to interact with the glucose transporter and the GABAA receptor 27"2s both of which are inhibited by forskolin (Table II). However, a number of different hydroxylated derivatives of andro-

glucose transporter

channels nACh GABAA

++4-

+-!''1"

+++

+'i"

+

++++

+++

ND

ND

+

ND

ND

+

ND

ND

ND

ND

+

+

ND

ND

o..-i~,,,' \\

H0

= /

adenylyl cyclase

00

"H

"

OH

forskolin

~CH3 1,9-dideoxyforskolin

HOO~r~,,,\\ ~ 1

~

0 1 6HI

0 0 II IIH '~ N OCCH2CH2CNCH2CH2~ 3

+

+++++

IAPS-forskolin .o

o...1~,,,\\

= ['~°11

Oo

~_~

++

MPB-forskolin OH

OH l',,nT~,' ,,'OH

O~I~OH OH

oc-o-glucose

=0

pregnenolone sulfate

- - O 3 S O ~ O II

o

ndrostenedione

Fig. 2. Potential ligands at forskolin binding sites. Relative potencies at adenylyl cyclase, glucose transporter, nicotinic acetylcholine receptor (nACh) and the GABAA receptor are indicated by the number of + signs. - , No effect; ND, effect of agent is not well documented.

stane do not inhibit high affinity [3H]forskolin binding to bovine brain membranes (A. Laurenza and K. B. Seamon, unpublished) and there is no well established direct interaction of steroids with adenylyl cyclase. It will be interesting to determine if forskolin and steroids interact with the GABAA receptor at identical sites. Interestingly, forskolin, cytochalasin B and androstenedione can photolabel the same 18 kDa component of the glucose transporter 1°,11,28. Many forskolin analogues and structurally similar compounds

affect the function of different membrane proteins (Fig. 2). It is anticipated that an understanding of the structural similarities between forskolin and endogenous compounds will allow endogenous regulators that act at forskolin binding sites to be postulated. Further investigations should also determine whether the effects of forskolin can be differentiated on the basis of similarities to a steroidal structure or a hexoserelated structure. []

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[]

TiPS - November

446 It is clear that forskolin can elicit b o t h c A M P - d e p e n d e n t a n d cAMPi n d e p e n d e n t effects. These effects a p p e a r to involve different sites of action since t h e y can b e disting u i s h e d b y a n u m b e r of criteria (see above). H o w e v e r , a n u m b e r of i n t e r e s t i n g q u e s t i o n s arise f r o m the o b s e r v a t i o n that forskolin can modulate membrane transport p r o t e i n s i n d e p e n d e n t l y of cAMP. Does forskolin act t h r o u g h a direct interaction w i t h m e m b r a n e p r o t e i n s or b y n o n s p e c i f i c p e r t u r b a t i o n of a m e m b r a n e - p r o t e i n interface? A direct i n t e r a c t i o n of forskolin w i t h adenylyl cyclase has b e e n d e m o n s t r a t e d b y affinity c h r o m a t o g r a p h y of the e n z y m e o n i m m o b i l i z e d forskolin 29. B i n d i n g of forskolin to the glucose transporter has b e e n d e m o n s t r a t e d directly b y p h o t o a f f i n i t y labellingl°,~L There is indirect e v i d e n c e to suggest that forskolin d o e s n o t affect ion channels in a n o n s p e c i f i c m a n n e r . The i n t e r a c t i o n of forskolin w i t h the nicotinic r e c e p t o r in T o r p e d o m i c r o s a c s w a s m o d u lated b y carbachol s u g g e s t i n g that forskolin p r e f e r r e d to b i n d to the o p e n - s t a t e c o n f o r m a t i o n or to the d e s e n s i t i z e d r e c e p t o r 19. It w a s also s u g g e s t e d that forskolin b o u n d tighter to the o p e n c o n f o r m a t i o n of the K + channel in PC12 cells 23. Forskolin a p p e a r s to b i n d to a site on ion channels w h o s e accessibility a n d affinity are d e p e n d e n t on the c o n f o r m a t i o n of the protein. H o w e v e r , a direct i n t e r a c t i o n of forskolin w i t h the nicotinic receptor or a n y o t h e r ion c h a n n e l p r o t e i n has yet to b e d e m o n strated. There h a v e b e e n n o reports of direct effects of forskolin on G p r o t e i n - c o u p l e d r e c e p t o r s such as the o~-adrenoceptor n o r d i d forskolin affect C a a + - d e p e n d e n t K + channels 22'24. Therefore, it s e e m s unlikely that the effects of forskolin are d u e to a general p e r t u r b a t i o n of the lipid bilayer. It is i n t r i g u i n g that forskolin is able to i n h i b i t different t y p e s of ion channels. Is there a family of ion channels that are (1) structurally h o m o l o g o u s , (2) distinct f r o m the family of G p r o t e i n - c o u p l e d receptors a n d (3) contain an allosteric r e g u l a t o r y site for forskolin? The nicotinic r e c e p t o r is a p e n t a m e r i c t r a n s m e m b r a n e p r o t e i n ass e m b l y c o m p o s e d of four t y p e s of homologous glycoprotein subunit each of w h i c h contain five membrane-spanning r e g i o n s 3°.

The s u b u n i t s s h o w significant a m i n o acid s e q u e n c e a n d structural h o m o l o g y w i t h the s u b u n i t s of the GABAA r e c e p t o r 31, the glycine receptor 32 a n d the 5-HT3 r e c e p t o r 33. S o m e n o n c o m p e t i t i v e i n h i b i t o r s of the nicotinic r e c e p t o r also i n h i b i t the GABAA r e c e p t o r g a t e d C1- c h a n n e l 34. Interestingly, forskolin a n d 1,9-dideoxyforskolin i n h i b i t GABAA r e c e p t o r - m e d i a t e d C1- fluxes in b r a i n s y n a p t o n e u r o s o m e s 3s. The effects of forskolin a n d 1,9-dideoxyforskolin s h o u l d be d e t e r m i n e d o n o t h e r ligandg a t e d ion channels, such as the glycine receptor a n d the 5-HT3 receptor. The glucose t r a n s p o r t e r , the Pglycoprotein multidrug transp o r t e r a n d s u b u n i t s of different v o l t a g e - d e p e n d e n t ion c h a n n e l s contain six m e m b r a n e - s p a n n i n g helices 36-3s. Bovine b r a i n a d e n y l y l cyclase has recently b e e n cloned a n d is p r e d i c t e d to consist of t w o regions, each w i t h six m e m b r a n e s p a n n i n g helices t o p o g r a p h i c a l l y h o m o l o g o u s to the t r a n s p o r t e r s a n d the v o l t a g e - d e p e n d e n t ion c h a n n e l s 39. It will b e i n t e r e s t i n g to d e t e r m i n e w h e t h e r forskolin can interact w i t h t h e s e functionally d i v e r s e p r o t e i n s at a structurally or topologically h o m o l o g o u s site. The d i v e r s i t y of the actions of forskolin at a d e n y l y l cyclase, glucose t r a n s p o r t e r a n d liganda n d v o l t a g e - g a t e d ion c h a n n e l s s u g g e s t s that there are different sites of forskolin action; this is a n a l o g o u s to the actions of o t h e r neurotransmitters and hormones. H o w e v e r , one m u s t b e cautious in d e f i n i n g sites of action for forskolin until there is a good physicochemical b a s i s to indicate a specific i n t e r a c t i o n of forskolin w i t h the u n i q u e site. A direct i n t e r a c t i o n of forskolin w i t h p r o teins has only b e e n d e m o n s t r a t e d for adenylyl cyclase a n d the glucose transporter. Forskolin h a s structural h o m o l o g y w i t h transp o r t e d hexoses 4 a n d this p r o v i d e s a rationale for d e s i g n i n g a n a l o g u e s of forskolin that m a y h a v e h i g h e r affinity at the glucose 'transporter. The i n t e r a c t i o n of o t h e r forskolin a n a l o g u e s w i t h ion c h a n n e l s s h o u l d b e d e t e r m i n e d to investigate w h e t h e r these sites are structurally similar to each other. It s h o u l d be p o s s i b l e to use a l i m i t e d n u m b e r of a n a l o g u e s to define the t y p e of site r e s p o n s i b l e for an o b s e r v e d effect of forskolin.

1 9 8 9 [ V o l . 10]

Therapeutic indications for forskolin h a v e , until n o w , b e e n b a s e d on the p r e m i s e that its effects are c o m m e n s u r a t e w i t h its ability to increase intracellular cAMP. H o w e v e r , it s h o u l d n o w be p o s s i b l e to look for p h a r m a c o logical actions of forskolin that are distinct f r o m its effects on c A M P metabolism. References

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Molecular sites of anesthetic action in postsynaptic nicotinic membranes Stuart A. Forman and Keith W. Miller Theories of general anesthesia have traditionally been based on correlations between potency and the properties of simple models such as apolar solvents, lipid bilayers and soluble proteins. However, mechanisms can now be determined directly by studying excitable proteins in their membrane environment. Stuart Forman and Keith Miller describe the physiological, biophysical and molecular biological evidence pointing to the location of a discrete allosteric site on the nicotinic acetylcholine receptor at which local anesthetics act. General anesthetics, while superficially resembling local anesthetics in their actions on the receptor, do not appear to act upon such a site.

Theories of general anesthetic action are mired in controversy. Work on both lipid bilayers and soluble proteins suggest that each might be the primary site of action. H o w did this state of affairs come about? It is necessary to appreciate the pharmacological features of general anesthetics that make definition of a specific molecular site of action difficult. General anesthetics differ from site-specific ligands since they lack specific antagonists and range in structure from simple gases to complex steroids. Moreover, they exhibit little stereoselectivity and are effective only at high concentrations. All theories of general anesthetic action must account for: (1) the strong correlation between the potency of an anesthetic and its ability to partition into a hydrophobic phase, first noted by Meyer and Overton 90 years ago; (2) the reversal of S. A. Forman is a Research Fellow at Harvard Medical School, and K. W. Miller is Edward Mallinckrodt Jr Professor of Pharmacology at the Department of Anesthesia, Massachusetts General Hospital, Boston, M A 02114 and the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, M A 02115, USA.

general anesthesia by elevated hydrostatic pressure; and (3) the so-called cut-off effect which arises in homologous series of simple anesthetic alkanes and alkanols, where there is a regular increase in anesthetic potency with lengthening of the alkyl chain until potency is suddenly lost with the addition of a further methylene group (reviewed in Refs 1 and 2). According to the lipid solubility hypothesis, during anesthesia all general anesthetics have the same concentration in cell membranes. Most hypotheses assume that a structural change is required in the membrane, but there is no consensus - membrane expansion, lipid 'fluidization' and lateral phase separation of lipids have been suggested (reviewed in Ref. 1). Lipid theories also specify that these membrane perturbations ultimately alter intramembrane protein functions 3. However, at concentrations that produce anesthesia in vivo, structural perturbations in the membrane are often small and can also be induced by small elevations in temperature that do not cause anesthesia 2.

38 Catterall, W. A. (1988) Science 242, 50-61 39 Krupinski, J. et al. (1989) Science 244, 1558-1564

IAPS-forskolin: 7-iodoazidophenethylamine succinimidylforskolin MPB-forskolin: 7~- [y-(N'-methylpiperazino)butyryloxy]-7-desacetylforskolin

A priori it would seem unlikely that a single protein cleft could account for the diverse structural pharmacology of general anesthesia. Indeed, proteins such as myoglobin and hemoglobin form complexes with general anesthetics which have been studied by X-ray crystallography. The hydrophobic pockets that the anesthetics occupy are relatively rigid and exclude many agents (reviewed in Ref. 1). However, the possibility that direct general anesthetic-protein interactions might be important is established by a lipid-free soluble protein, firefly luciferase, which can be inhibited b y a variety of inhalational general anesthetics with potencies that correlate with lipid solubility 1"2. Although general anesthetics appear to inhibit b y competing with the enzyme's substrate luciferin, the prediction that general anesthetics should compete among themselves for occupation of a 'hydrophobic patch' has not been tested. Recent studies have shifted the focus from these simplistic models to excitable proteins in their membrane environment. One of the more promising experimental models for the study of general anesthetic mechanisms at the molecular level is the nicotinic acetylcholine receptor from Torpedo the most thoroughly characterized neuronal membrane protein 4. The Torpedo nicotinic acetylcholine receptor is closely related to that in mammalian muscle, so that comparison with a substantial body of data can be made s. The receptor consists of four homologous subunits with stoichiometry o~2~y6and a total molecular weight of approximately 290 kDa. Each o~-subunit has one acetylcholine (agonist) binding site, and the transmembrane cation channel probably involves all subunits. The structural genes for the four subunits have been cloned, enabling site-directed mutagenesis

1989,ElsevierSciencePublishersLtd. (UK) 0165-6147/89/$02.00