Shaker shakes out potassium channels

he long wait is over. We have Shakershakesoutpotassiumchannels T met Shaker, and it is a channel. Actually, it is much more: K+

a 'nuclear family' of K + channels. In a recent series of papers of rare beauty TM, the Jan group has brought to a conclusion one phase of pursuit of the long-studied Shaker locus of Drosophila melanogaster and has simultaneously begun the next. By isolating both genomic and cDNA clones of Shaker, the Jans and their colleagues have achieved four results of fundamental importance. First, they showed that Shaker, originally selected 40 years ago as a behavioral mutant, is a structural gene for 'A-type' K ÷ channels, a conclusion whose validity was widely assumed but never established rigorously. Second, they provided the sequence of several channel clones, thu.sadding a second entry to the list of cloned voltage-dependent channels (or a third, if you count the dihydropyridine receptor as a Ca2+ channelS). Third, by expressing their cDNA clones in Xenopus oocytes, they provided a strong suggestion that the functional channel may be built as an oligomer of identical subunits. Finally - and this is a real humdinger - they demonstrated that Shaker codes for several structurally related but functionally distinct K + channels via alternate splicing of a primary transcript about 65 kilobases long. For years, electrophysiologists have realized that there are many different kinds of K ÷ channels carrying out a variety of repolarization tasks in excitable membranes. Though these varied K + currents are easily distinguished by their gating behaviors, many of their conduction characteristics - high selectivity for K +, block by Cs + and quaternary amines, occupancy of the pore by several K ÷ ions simultaneously - are so similar that the idea of a large molecular family of K ÷ channels has gained widespread appeal6'7. But purely functional studies, no matter how sophisticated (and single-channel methods provide functional informarion of unprecedented detail),

cannot address questions about the precise nature of the familial relationships among these channels. The cloning of Shaker now permits us to approach these kinds of questions, and many others. The Jan group, having located the region of genomic DNA contanfmg the Shaker locus by chromosomal walking, then proceeded to probe cDNA libraries with genomic fragments. They identified several clones large enough to predict the protein sequence, which conforms to a pattern now familiar for integral membrane channels. One of the clones codes for a 70kDa protein containing six clear strongly hydrophobic transmembrane stretches. Most significantly for a voltage-dependent channel, the protein displays a 22-residue hydrophobic stretch punctuated by arginine or lysine in every third position. This is a motif (named the '$4 region') seen in the voltagedependent Na + (Ref. 8) and Caz+ (Ref. 5) channels, but not in the voltage-independent channels gated by acetylcholine, GABA, and glycine. Though there is not yet any hard evidence showing that the $4 region really is the transmembrane voltage sensor in these channels, the sequence is so striking, so highly conserved, and so reminiscent of Armstrong's early prediction 9 of how a voltage sensor ought to be constructed, that this function for $4 is generally assumed. The appearance of an S4like stretch here in another voltage-dependent channel serves to reinforce this idea. Fig. 1 illustrates the impressive homology among the $4 regions of one of the Shaker clones, the Drosophila Na + channel, and the mammalian dihydropyridine receptor. According to precedents provided by other integral membrane channels, which are all about 200 kDa when fully assembled, a single copy of a 70 kI)a protein is too small to form a channel. It is thus a good guess that the A-type K +

channel is composed of multiple subunits, and this raises worries that the functional channel may require genes as yet uncloned. But these worries are deflected (though not put completely to sleep) by the finding that injection of only a single Shaker clone in frog oocytes is sufficient to produce typical A-currents in voltage-clamp experiments. This result strongly implies that the K + channel is composed of several identical subunits. The really novel piece of news emerges from a comparison of several apparently full-length cDNA clones. To approach the question of the splicing of the primary transcript, the authors asked where in the genomic DNA these different cDNAs hybridize. They then sequenced the genomic DNA to locate splicing points. They found that the different cDNAs originate from at least 12 exons scattered over 65 kilobases of genomic DNA. The surprising result is that there are four clearly different classes of clones, ShA, ShB, ShC, and ShD, which are built up from different combinations of exons, eight of which are shared in common. Fig. 2 shows how alternate splicing affects the composition of the mature protein of each class. All four classes of proteins share a common central region of about 380 amino acid residues, but differ from each other in the shorter regions on both N-and C-terminal sides (as well as in the untranslated regions presumably controlling expression). Four of the six putative transmembrane helices, as well as the $4 region, fall fully within the common region. The production of closely related proteins by alternate splicing of a primary transcript is hardly novel, but this is the first time it has been observed for an ion channel. It is intuitively satisfying that this first example has been found for a channel thought to be one of a large molecular family.

Putative voltage-sensor sequences for Shaker and other channels

ShA Na ÷ C a =+

~LyS- I i e-Leu4Ar~,Val-Leu-Agg'Val-Leu~g'Pro-Leu-A/'g-Ala- I i e-Asn-Arg-Ala-Lys-Gly-Leu~Lys-

TINS, VoL 11, NO. 5, 1988

© 1988, ElsevierPublications, Cambridge 0378 - 5912/88/$02.00

Christopher

Miller GraduateDepartment of Biochemistry, BrandeisUniversity, Waltham,MA 022549110, USA.

Fig, 1. Sequencesof 54 regionsareshown for three voltagesensitivechannels: done ShA of Shaker, the DrosophilaNa + channel1°(54-4),and the rabbitmuscle dihydropyridine receptorS(putative Ca2÷channel;54-///). Conservedamino acidsare indicatedby shading 185

s.A

I\ \ \ \ \ I-c°°e

s.8

IHHHj-cooe

s,c

.,,%1

I\\\',,\

s.o H -tqh.3 I

IVB'B'D'Fcooe I

2 0 0 Residues

t-cooe

I

HI

I

I

I

I

n l

H2 H3 $4 H4 H5 H6

Fig. 2. The figure shows only the translated regions of the four classes of Shaker clones. Regions shaded identically have identical amino acid sequences and arise from identical exons. The common central region originates in eight exons. The locations of the putative transmembrane helices H1-H6, and the $4 region are indicated.

Do these alternative splicings mean anything? Apparently they do, since the A-type currents observed after expression of two of these clones, ShA and ShB, differ significantly. Each clone, when injected into oocytes, produced an A-current, but the detailed characteristics of these currents were not identical. Specifically, ShA inactivated at about half the rate of ShB, and ShB, in contrast to ShA, did not inactivate completely after a long depolarizing pulse. These results leave open the question of the actual situation in the fly neuron or mus-

de. By the arguments above, each of the A-type channels expressed in the oocytes must be composed of identical subunits, but in Drosophila neurons, where numerous types of Shaker messages may be simultaneously present, perhaps channels can be fine-tuned in their function by assembling heterologous subunits together. Questions like this have for many years been fascinating to contemplate, but now they can be approached directly via mixing experiments and detailed single-channel analysis. The general strategy of isolating

Potassiumchannels- whato n the protein chemistrycontribute? J. Oliver Dolly

E xcitable cells display a variety

Departmentof of K + conductances that may Biochemistry,Imperiai be governed by membrane potentCollege,London~/ir7 ial (e.g. delayed and anomalous 2AK UK. rectifiers, M-current), receptor

ligands, intracellular Ca 2+, ATP or other mediators (reviewed in Ref. 1). Amongst these are K + currents displaying a characteristic pattern of voltage activation2 and sensitivity to 4-aminopyridine a. The responsible channels activate rapidly in response to membrane depolarization, and control cell excitability and synaptic transmission, by regulating the frequency of nerve firing and affecting action potential duration. The numerous modulatory roles served in most excitable cells~ by such fastactivating, repolarizing outward K + currents, together with heterogeneity and cell specificity of the 186

channels, make them ideal targets for a new generation of therapeutic agents. Thus, it is very helpful that elegant molecular genetic studies 4-7 have recently provided amino acid sequences for one set of homologous proteins constituting the 'Atype' K + channel 2 in Drosophila. Moreover, characterization of a family of related K + channels present in mammalian neurons s'9 is being aided by the use of dendrotoxin zo, a convulsant protein from snake venom that inhibits selectively these K + conductances 9' n-15 and labels a putative K + channel protein l~-ts. Properties established for this macromolecule in vertebrate brain, taken in conjunction with the fundamental data derived from the Drosophila studies, ought to yield insight into the complexity of K + channel structures.

© 1988, ElsevierPublications.Cambridge 0378- 5912/88/$02.00

genes by chromosomal walking from an appropriate marker is a familiar one, but this case provides the first example of an ion channel hunted down in the absence of detailed biochemical information at the protein level. Now, with Shaker providing a wedge in the gate, a band of happy warriors armed with both biochemical and molecular weapons will be able to storm the palace housing the K + channel family. Selected references 1 Papazian, D., Schwarz, T. [., Tempel, B.L., Jan, Y. N. and Jan, L.Y. (1987) Science 237, 749-753 2 Tempel, B. L., Papazian, D. M., Schwarz, T.L., Jan, Y.N. and Jan, L. Y. (1987) Science 237, 770-775 3 Schwarz, T. L, Tempel, B.L., Papazian, D. M., Jan, Y. N. and Jan, L.Y. (1988) Nature 331, 137-142 4 Timpe, L. C. etal. (1988) Nature 331, 143-145 5 Kamb, A., Iverson, L. E. and Tanouye, M. A. (1987) Cell 50, 405-413 6 Hille, B. (1984) Ionic Channels of Excitable Membranes, Sinauer Associates 7 Yellen, G. (1987)Annu. Rev. Biophys. Chem. 16, 227-246 8 Noda, M. et al. (1986) Nature 320, 188-192 9 Armstrong, C. M. (1982) Physiol. Rev.

61,644-683 10 Salkoff, L et al. (1987) Science 237, 744-749

H e t e r o g e n e i t y of fasta c t i v a t i n g K + c o n d u c t a n c e s in mammalian neurons

Recently, dendrotoxin from the Eastern green mamha (Denavvas~ angusticeps) 1° has been employed successfully to dissect these rapidly activating, aminopyridine-sensitive currents in various neurons 9'II-15. At least three can be discerned (Table I) based on differential inactivation rates and susceptibilities to the specific blockers; for simplicity, these will be referred to as Acurrent variants. The slowly inactivating variety in sensory A cells is most sensitive to dendrotoxin or 4-aminopyridine (which, however, is orders of magnitude less potent), whereas much higher concentrations are required to inhibit the transient K + current in hippocampal neurons. On the other hand, the rapidly inactivating conductance seen in the sensory C cells or neurons of superior cervical ganglion is unaltered by the toxin and only partially blocked by 5 mM 4-aminopyridine. Note that with TINS, Vol. 1 I, No. 5, 1988