TiPS -January

8 retention of the response developed under the olfactory stimulus, but not of that developed under the visual stimulus9,*o. The effect of amphetamine in each caudate region was shared by microinjections of the Dz receptor agonist quinpirole (1 Bg), but not by the D1 agonist SK&F38393 (Ref. 10). The results suggest that dopamine D2 receptors are invoked in consolidation processes, and that there is a regional specialization of the caudate nucleus according to the nature of the conditioned stimuli associated with each task. The findings are consistent with a previous observation that dopamine-specific lesions of nigrostriatal neurons (obtained by injecting 6-hydroxydopamine into the substantia nigra) block amphetamine-induced memory enhancementsll. Data obtained several years ago by Roberto Prado-Alcala and his co-workers in Mexico, using intracaudate microinjections of muscarinic acetylcholine receptor

agonists” and antagonists13, have suggested a role for muscarinic receptors in the caudate in memory processes. Brust-Carmona pointed to the possibility that the caudate nucleus could play a role in integrating sensory signals during conditioning14, which is consistent with White’s findings’,r’. It would now be interesting to study the interaction of Dr receptor mechanisms with muscarinic receptors and others (Nh4DA receptors, adrenoceptors) in the caudate during the process of consolidation”. It might also be worth studying the eventual anatomical and functional relation of the caudate mechanisms with those in the limbic system mentioned above2z4. IVAN IZQUIERDO Centro de Memoria. Depwtnmento de Bioquimicu, Insfituto de Biociencius, UFRGS Ccentroa),90049 Port0 Alegre, RS, Brazil.

References 1 McCaugh, J. L. (1966) Science 153, 1351-1358

A small K+ channellooms large In the past few years molecular biological techniques have produced unprecedented advances in our understanding of the structure of ion channels and receptors. What originally appeareu to be individual molecules have now invariably been found to be members of diverse families of proteins derived from multiple genes, splice variants and other crafty genetic tricks. Each individual member of a family has subtly different properties, and pharmacologists have realized that these may hold the key to understanding previously mysterious observations on drug specificity and action. These hopes are now being fulfilled in several instances, and recent observations on K+ channels serve as an example. Class III antiarrythmics typically block K+ channels. By comparing the effects of a drug on channels expressed in heterologous systems with drug effects on native cell processes, these agents should be useful tools in understanding the role of particular K+ channel genes in a variety @ 1992. ElsevierSciencePublishe:s Ltd (UK)

of cellular events. For example, in cardiac ventricular myocytes, block of K’ channels has been thought to be the mechanism by which some class III antiurythmic drugs retard repolarization and thereby prolong the action potential duration (see review in Ref. 1). However, it has been difficult to tell which K+ current is actually being blocked as many of the agents lack specificity, especially at high concentrations2. For example, clofilium is a quaternary ammonium compound that has been reported to increase action potential duration in isolated Purkinje fibers3. This compound was found to be more specific than its tertiary derivatives in its ability to block Ikv (delayed rectifier) but not IiR (inward rectifier) at l&lOOp~ in isolated guinea-pig ventricular myocytes4r5. By contrast, selective inhibition not of Ixv but of 1~ (transient outward) K+ currents by clofilium and its tertiary derivatives has recently been reported in rat ventricular myocyte&. However, as discussed further below, it has now been demonstrated that

2992 [Vol. 131

2 Izquierdo, I. and Medina, J. H. (1991) Trends Pharmacol. Sci. 12. 260-265 3 Squire, L. R. (1987) Memory and Brain, Oxford University Press 4 Izquierdo, I., _ Medina, J. H., Jerusalinsky, D. and DaCunha, C. Rev. Neurosci. (in press) 5 Cahill, L. and McGaugh. I. L. (1990) Behav. Neurosci. 104,53%~ 6 Wolfman, C. et al. (1991) Brain Res 548, 74-80 7 Zola-Morgan, S., Squire, L. R., AmaraJ, D. G. and Suzuki, W. (1989) J. Neurosci. 9,4355-4370 8 Hyman, B. T., Van Hoesen, G. W. and Damasio, A. R. (1990) NeuroZogy 40, 1721-1738 9 Viaud. M. D. and White. N. M. (19&g) Behav: Bruin Res. 32,3142 1 ’ 10 White, N. M. and Viaud, M. D. (1991) Behav. Neural BioJ. 55. 255-269 11 White, N. M. (1988) Life Sci. 43, 7-12 12 Prado-Alcala, R. A. and Cobos-Zapiain, G. G. (1979) Neurosci. Left. 14. 253-258 13 Prado:Al&a, R. A. and Cobos-Zapiain, G. G. (1977) Bruin Res. 138,190-196 I4 Brust-Carmona. H. (1981) in Learnine 2nd Memory - brugs as Reinforcer (Saiti, S. and Yanagita, T., eds), pp. 126-142, Excerpta Medica

SK&F38393: 2,3,4,5-tetrahydro-7,8dihydroxy-1-phenyl-lH-3-benzazepine hydrochloride

clofilium also blocks a unique Kf channel gene (expressed in Xenopus oocytes) at a concentration that prolongs the action potential duration in newborn mouse ventricular cardiac cells7,s. Clearly the time has come for direct study of clofilium and other class III agents (cf. Refs 9-15) on the actions of the host of cloned Kf channels that have been isolated, many of which have been cloned from, or detected in, cardiac tissue. New voltage-dependent K+ channels have been arriving at such a tremendous rate that it is difficult to assign precise functions to the growing number of genes that code for them. The Drosophila Shaker (Sh) locus was the first such K+ channel gene to be cloned and studied in heterologous systems (for review, see Ref. 16). In the past three years, multiple genes related to the Drosophila Sh gene, and its cousins the Shab, Shaw, and Shal families, have been isolated from mammalian cDNA and genomic libraries (for annotated bibliography, see Ref. 17). This year has alsc seen two additional Drosophila loci identified and sequenced, K-eag (ether-a-go-go) and

TiPS -January

2992 [Vol. 131

sZO’~,~~. Although more divergent from the previously identified families, the deduced proteins for these genes exhibit the precise transmembrane arrangements of the Shaker family and retain significant homology throughout the H4-H6 (helices 4 to 6) transmembrane domains, thought to determine the ion pore. While a definitive function has not yet been assigned to K-eag, do mutations s ecifically affect a Drosophila ($+-activated K+ channel, but this assignment is tentative pending the actual expression of the cDNA. Until recently it appeared that the structural requirements for building a voltage-sensitive ion channel were rather consistent. All the K+ channels discussed above are constructed according to the same basic principles as voltage-sensitive Naf and Cazf channels, with each K+ channel molecule being equivalent to a quarter of a Na+ or Ca2’ channel. However, a few years ago a report appeared claiming that a completely different type of structure could also produce a voltagesensitive K+ channel. One problem with this molecule was that it was ridiculously small. Its discovery was greeted with derision by most scientists. The channel, Ik,s, was called the mini K+ channel by some who were unimpressed with its size, or even ‘isK?’ by others who questioned its very existence. It was originally cloned by Takumi et aL2’ from rat kidney using expression-cloning methodology. A 700 bp mRNA was found by blot hybridization primarily in epithelial cells such as kidney, duodenum, stomach, pancreas, and submandibular gland, but not in brain or liver2’. While there seems to be a single gene enroding the protein, several mRNA splice variants have been found by RNA blotting7J1. HOWever, all the splicing seems to be outside of the coding region, creating messages that differ primarily in the 3’-untranslated region21. The protein has been localized to the apical membrane of epithelial cells by histochemical techniquesz2. The Ik,s channel has also been identified in uterine muscle, where it is hormonally sensitive to estrogen and diethylstilbestrol, and in eonatal, but not adult, rat heart’: 2

9 The rat 1k.s gene encodes a relatively small, 130 amino acid protein with a single predicted membrane-spanning domain20. A human gene encoding a similar functional channel protein of 129 amino acids has only 76% amino acid identity to the rat sequence”. The mouse sequence is also 129 amino acids, with 92% identity to the rat sequences. The 23 amino acid hydrophobic segment extends from residue 44 to residue 66, but there is 96% identity between the rodent and human sequences between residues 41 and 90. Just as in the case with %-like channels, the N-terminal and Cterminal portions are more divergent than the hydrophobic segment, but here the C-terminal is significantly more conserved. Two potential N-glycosylation sites and a cysteine residue are conserved in the N-terminal and Cterminal, respectively. The cysteine residue has been posited to participate in subunit interactions, but the nature of the functional organization of the active channel is unknown. Pragnell et aZ.= have pointed out the interesting relationship of this clone to the bacterial peptide toxins known as colicins. These bactericidal agents have a single membrane-spanhydrophobic ning domain, which may act as a monomer to form a slowly activating ion channel. Although there is minimal sequence homology, the IK,Sfamily may thus be distantly related to these bacterial channels. In their initial investigations Takumi et a1.” found that injection of Xenopus oocytes with in vitro transcribed RNA encoding the Ik,s channel gave rise to slo~~!y activating, voltage-dependent K+ currents, which did not undergo inactivation. The current was sensitive to tetraethylammonium (20 mM) and Ba2+ (10 mM)20.An initial concern was that this protein was not actually an ion channel but merely modified endogenous Xenopus oocyte channels. That concern has now been laid to rest thanks to further examination of the pharmacological properties and structure-function aspects of the channel in several more recent reports. Lazdunski’s group has now independent reported their cloning of Ik,s from neonatal

mouse cardiac RNA’. RNA blotting and in situ hybridization techniques were employed to demonstrate developmental and species-specific differences in gene expression. The message is highly expressed in the mouse neonatal heart and is greatly reduced, though detectable, in the adult’. However, mRNA levels in the neonatal rat heart were lower than the mouse and undetectable in the adult rat cardiac tissue, in agreement with Folander et aL8 Honore et al. also examined the regulation of IK,~ expressed in Xenopus oocytes by classical Kf channel blockers, the class III antiarrhythmics, Ca2+-cahnodulindependent protein kinase II (CAM-kinase II) and protein kinase C, and compared these results to those obtained in newborn mouse ventricular cardiac cells7. While the channel was insensitive to quinine, quinidine, dendrotoxin, apamin, fi-bungarotoxin, mast cell-degranulating peptide, charybdotoxin, amiodarone, bretylium tosilate and tedisamil, it was blocked by clofilium in a dose-dependent fashion7. Half-maximal inhibition was at 100 PM, accompanied by a shift in the activation threshold by 20 mV in the positive direction. These results confirm those of Folander et al.’ who found clofilium (100 PM) caused a 70% block of the Ix,s channel, also expressed in Xenopus oocytes. Honore et aL7 then compared these results to those obtained in electrophysiological studies on isolated ventricular cardiac cells from newborn mouse. Clofilium (100 PM) increased the action potential duration, decreased the beating rate and increased the force of contraction. A delayed rectifier outward current in the newborn mouse ventricular cells was similar in shape but faster than the Ix s expressed in Xenopus oocytes. This outward current was completely blocked at clofilium concentrations that blocked the Xenopus oocytes (i.e. 1K.S in that this 100 CL& suggesting endogenous IKS may Play an important role in mediating the effect of clofilium7. However, given the distribution of IK,Sin a variety of epithelial cells, the chances of unfavorable side-effects besides its antiarrythmic action would be high.

TiPS -Janua y 1992 [Vol. 131

10

fig. 1. Left: proposed membrane-spsnning orientation for the 1,s channel protein. Middle: pmpased membrane-looping orient&ion for ts. Right: current consensus model for Shaker family-related K’ channels. Consensus phosphorylation sites are shown for CAM-kinase II (Ce2+-&modulin-dependent protein idnz9.s //) and PKC (pmtein kinase CJ.

Honore et al. examined the effects of phosphorylation on 1K.S functior?. The rodent I&s channel contains a CAM-kinase II consensus phosphorylation site just upstream of the hydrophobic domain and a protein kinase C consensus phosphorylation site just downstream7*20. It is therefore quite interesting that injection of CAM-kinase II into oocytes expressing IK,s resulted in a twofold increase in current (reversible by the calmodulin antagonist W7) while phorbol myristate acetate (0.1 pi) inhibited it by abollt 70% (blockable with staurosporine). As Honore et al.’ point out, this might suggest that both the Nand C-termini are intracellular, and that the single hydrophobic domain of 23 amino acids folds back on itself (as shown in Fig. 1) or alternatively the manipulations of phosphorylation may not be affecting the Ix,s channel protein directly. It is pertinent to note that the human I&s gene is divergent in the region of the putative CAMkinase II consensus sequence (in single-letter code, the rat/mouse sequence at residues 3-l is SQLRDDSK; the human sequence is SPRSSDGK) and so might provide a test of the relevance of this site to channel regulation. Parallel studies on isolated newborn mouse ventricular cells found similar responses to clofilium and phorbol ester. Miller and colleagues have taken advantage of the small size of the gene to undertake sitedirected mutagenesis experiments employing a synthetic gene con-

struct, showing directly that the Ix,s protein in fact determines the channel pore25*26. In a clever application of synthetic methods, the entire I&s gene was synthesized via seven overlapping oligonucleotide duplexes of about 60 bases each. These sequences took advantage of the degeneracy of the genetic code to incorporate 40 new restriction enzyme sites. The 5’-untranslated region was optimized for translation and the entire gene then placed in an easily movable cassette26. The new restriction sites also created a cassette by which the entire coding region for the 23 amino acid hydrophobic domain could be removed and replaced by synthetic duplexes in which specific mutations had been introduced. The mutant channels could then be conveniently studied after injection of cRNA into Xenopus oocytes. The first conclusion was that a variety of point mutations in the hydrophobic domain did not dramatically alter expression of the voltage-gated currents. Ion selectivity was altered by specific mutations at phenylalanine 55 (F55); the threonine mutants were about threefold more permeable to NH4+ and Cs+. Other mutations at this and nearby sites give similar changes, and double mutants could restore wild-type behavior. The mutant channels at F55 were several-fold more sensitive to open channel block by tetraethylammonium and Cs+. Goldstein and Milleti conclude that, although the Ix,s channel protein has not yet been purified

and functionally reconstituted, these studies strongly suggest that the gene does in fact code for a channel. Significant questions remain. Does the action of clofilium on adult heart also work through the IK,S channel, or do the significantly reduced levels of I&s mRNA in the adult mouse heart and absence of the message in adult rat heart imply that other K+ channels are involved? No systematic study of the class III antiarrythmic agents has yet appeared on the other cloned K+ channels expressed in heart, Does the presence of the Ix,s channel in multiple epithelial cell types suggest that potent and unavoidable ‘side-effects’ will forever limit the utility of this class of drug? What is the meaning of sexhormone induction of the IK,s message, and might modulators of this channel be useful agents in the pharmacological manipulation of labor? Are these hormones inducers (or capable of being inducers) of this channel in pathological states (e.g. congestive heart failure, liver disease with elevated estrogen levels)? It will be very interesting to see the effect of this channel in transfected mammalian cells and transgenic animal models. We shall not be surprised if multiple new members of this family are uncovered. LOUIS H. PHILIPSON RICHARD

AND

J. MILLER*

Departmen& of Medicine and *Pharmacological and Physiological Sciences, University of Chicago, Chicago, ii. 60637, USA.

References 1 Arena, J. P., Walsh, K. 6. and Kass, R. S.

(1990) Prog. Clin. Biol. Res. 334, 43-63 2 Sanguinetti, M. C. and Jurkiewicz, N. K. (1990) j. Gen. Physiol. 96,195-215 3 Steinberg, M. I. et at. (1984) in New Drugs Annual: Cardiovascular Drugs (Scriabine, A., ed.), pp. 103-121, Raven Press 4 Arena, J. P. and Kass, R. S. (1988) Mol. Pharmacol. 34. 60-66 5 Sanguinetti, M. C., Siegel, P. K. S. and Zip;aro, G. S. (1989) I. Mol. Cell. Cardiol. 21:s21 6 Castle, N. A. (1991) J. Dharmacol. Exp. Ther. 257, 342-350 7 Honore, E. ef al. (1991) EMBCJ 1. 10, 2805-2811 8 Folander, K. et al. (1990) Proc. Natf Acad. Sci. USA.87, 297512979’ 9 Lynch, 1. 1. et al. (1984) 1. Cardiovasc. Pharmacot. 6,1132-1141 10 Bacaner, M. 8. et al, (1986) Proc. NaN Acad. Sci. USA 83,2223-2227 11 Bkaily, G. et al. (1988) Eur. 1, Pharmacol. 151,389-397

TiPS -January

1992 [Vol. 231

12 Schohysik, G. (1987) Naunyn-Schmied. Arch.

Pharmacol.

335,692-696

13 Scholtysik, G. et al. (1938) /. Pharmacol. Exu. Ther. 245. 773-780

14 Guilt, M. et al: (19SSj Circuiaiion 78,150 15 Guilt, M. et al. (1989) I. Mol. Cell. Cnrdiol. 21, Sll ’ 16 Jan, L. Y. and Jan, Y. N. (1990) Trends Neurosci. 13, 415-419 17 Pemey, T. M. and Kaczmarek, L. K. (1991) Curr. Opin. CeN Biol. 3, 663-670 18 Atkinson, N. S., Robertson, G. A. and

Will the real NMDA receptor please standup? A long-awaited moment in molecular neurobiology arrived on 7 November 1991 with the publication by S. Nakanishi and coworkers (Kyoto University) of a protein sequence (NMDA-Rl) from a functional NMDA receptor’. Rumours of such an advance had been current in the neuroscience community for some months, but for most of this period these derived from conference presentations made by another group, E. K. Michaelis and co-workers (University of Kansas), as reported in TiPS in September*. That laboratory has now reported an entirely different NMDA receptor sequence, in the same issue of Nature3. Non-NMDA ionotropic receptor cloning, initiated by S. Heinemann et al. to yield the GluRl subunit sequence4, had already identified seven channel-forming related subunits (GluRl-GluR7 or equivalents) nlus KA-1, a high-affinity kainate-binding subunit5 (see Ref. 6). Simultaneously, recombinant NMDA receptor subunits have been widely sought by academic and pharmaceutical laboratories. Three approaches were in using use: cross-hybridization, GluR cDNAs or PCR primers; cross-hybridization at low stringency from another cation channel

receptor, the nicotinic type; and oocyte expression cloning. A general lack of success with the first two had suggested that the NMDA receptor - which has such

11 Ganetzky, B. (1991) Science 253,551555 19 Warmke. J., Drysdale, R. and Canetzky, B. (1991) Science 252, 1560-1564 20 Takumi, T., Ohkubo, H. and Nakanishi, S. (1988) Science 242,1042-1045 21 Iwai, M. et al. (1990) J, Biochem. (Tokyo) 108,200-206 22 Sugimoto, T. et nl. (1990) J. Membr. Biol. 113,3ti7 23 Pragnell, M. et al. (1990) Neuron 4, 807-812 24 Murai, T., Kakizuka, A., Takumi, T.,

Ohkubo, H. and Nakanishi, S. (1989) Biochem. Biophys. Res. Commun. 161, 176181 25 Hausdorff, S. F.. Goldstein. S. A.. Rushin, E. E. and Miller, c. (1991)

a different and characteristic pharmacology and physiology to that of the non-NMDA receptors is only very distantly related structurally to the latter. In expression cloning the general experience that the NMDA-induced oocyte membrane current was decreased, rather than increased, by the fractionation of cRNA, suggested that more than one type of subunit was obligatory for function. In fact, with hindsight, one can point to several short regions of good nucleotide homology between NMDA-Rl and GluR sequences where the first approach could work. The second approach, one can now see, would never work at any stringency, although there is slight but significant localized homology with neuronal nicotinic receptor subunits. It was the perseverance of the Nakanishi laboratory, pursuing fractionation in the face of the declining oocyte signal, that led to their success with expression cloning. They found’ that the final cRNA still gave a much smaller response to NMDA/glycine than did the crude poly(A+)RNA, indeed suggesting that a complementary subunit is missing. cDNAs encoding such related subunits are now expected immediately. What of the totally unrelated sequence (GBP) claimed by the Michaelis group as an NMDA receptor subunit? This has (including the signal peptide) a molecular mass of 57 kDa, contrasting with 105 kDa for NMDA-Rl. A staff writer in Science, reviewing’ those two Nature papers on November 8 (itself significant, since neither of those august journals normally deigns to write about a report in press in the other) states in her heading ‘only one of them can be right’ and concludes by saying ‘the final determination of which group

reaily has the receptor can be worked out only by performing more functional studies on protein chemistry’. Both of these statements are questionable, even at this stage. Firstly, in principle both couId be right. Why should it be excluded a priori that some NMDA receptors contain two or more unrelated subunit types within one oligomerit protein? While that situation has not been found so far with the heterooligomeric ionotropic receptors, only a few classes of these are known, so it is rash to generalize; it is not in fact uncommon for enzymes to contain several subunits of different sizes and completely different sequences that cooperate to give a single function. Mammalian examples include many protein kinases. Even some receptors in other series follow this pattern, e.g. the IL-2 receptors or the CD3 complex of the T-cell antigen receptor’. While it may be thought less probable that any NMDA receptor has this type of structure, at present this can only be a guess. Secondly, while expert opinions supporting the Nakanishi conclusion are quoted’, the question is left open as to ‘which group really has the receptor’. But these are not two equal cases to be weighed similarly. The evidence that the Nakanishi subunit is truly from a native NMDA receptor is overwhelming. It is, indeed, remarkable that so many functions characteristic of or specific to the NMDA receptor are encoded in a single polypeptide: the agonist pharmacology, the NMDA competitive antagonist site, the glycine enhancement, the specific blockade of the glycine site, the Ca*+ permeability, the MK-801 (dizocilpine) sensitivity, the Zn*+ inhibition and the voltage-dependent block by Mgz+. (Only the poly-

Biochemistry

30,3341-3346

26 Goldstein, S. A. N. and Miller, C. (1991) Neuron 7,4Ow8 W7: N-(6-aminohe+)-5-chloro-lnaphthalenesulfonamide hydrochloride

~1992.ElsevierklencePublishenLtd

(UK)