STRUCTURE Sato and
Electrotechnical Laboratory, SupermolecularScienceDivison, Tsukuba, Ibaraki 305, JAPAN
SUMMARY: On the basisof our recent resultsof the completeamino acid sequenceof the squidLoligo bleekeri sodiumchanneldeducedby cloning and sequenceanalysisof the complementary DNA (Sato, C. and Matsumoto, G. Biochem. Biophys. Res.Comm. 186, I), we have proposeda tertiary structuremodel of the sodiumchannelwhere the transmembrane segmentsare octagonally aligned and the four linkers of S5-6 betweensegmentsS5 and S6 play a crucial role in the activation gate, voltage sensorand ion selectivepore, which can slide, depending on membranepotentials,along inner walls consistingof segmentsS2 and S4 alternately. The proposedmodel is contrastedwith that of Noda et al. (Nature 320; 188-192, 1986). 0 1992Academl Press,1°C.
Squid giant axons have long beenstudiedin the attempt to elucidatemolecularmechanisms of nerve excitation and transmission(1, 2). Through thesestudies,it was first found that the voltage-gatedsodiumchannelis essentialfor the generationof action potentials(l-3). In particular, the gating mechanismsof both activation and inactivation of the squidsodiumchannel have beenintensively studied(4) pecausesquidgiant axonsare the only axonsamenableto good voltage-clampingexperiments. Since cloning and sequenceanalysisof cDNA for sodiumchannels of Eiectrophorus electricus (5) and rat brain (6, 7) were successfuflyperformed, however, studies on sodiumchannelshave mainly beenconcentratedon thoseof vertebrates. It hasusually been assumedthat they all have similar topology of structure (S-IO); four homologousdomains(I, II, III and IV) containing all six membrane-spanningstructures(SI, S2, S3, S4. S5 and S6) are repeatedin tandemwith 23 connectinglinkers betweenneighboringmembrane-spanning segments,C- and N-terminals. The S4 segmentcontaining basicresidues(arginine or lysine) at every third position in each domainis widely recognized to serve asa voltage sensor(5, 7, I I, 12). A cytoplasmic linker betweendomainsIII and IV is mainly responsiblefor inactivation (1 I, 13-15), in good agreementwith a plug-ball model (16). Furthermore,patch-clampexperimentson site-directedmutantsof the rat II sodiumchannelexpressedin Xeno>fpzls oocytes indicate that lysine at position 1,496and alanineat 1,791are critical in determiningthe ion selectivity of the sodium channel(17), and that glutamic acid at position403 and asparticacid at 400 interact directly with the pathway of the ionspermeatingthe openchanneland areclosely relatedasthe main determinantsof specific binding to the blockers,tetrodotoxin and saxitoxin (I 7- 19). These findings suggestthat comprehensiveunderstandingof sodiurnchannelsawaits to be solvedafter #To whom correspondenceshould be addressed. 0006-291X/92
186, No. 2, 1992
sophisticatedprogresson the sodiumchannelstructureconcept. Quite recently we succeededin determiningthe completeaminoacid sequenceof a sodiumchannelof the squidLoligo hleekeri deducedby cloning and sequenceanalysisof the complementaryDNA (20). The deduced sequenceof 1,522residues,approximately three-fourths of thoseof the rat sodiumchannelsI, II andIII, hasrevealedan organization virtually identical to that of the vertebrate sodiumchannel proteins(20). This particular simplicity of squidsodiumchannelsfacilitates the proposalof a tertiary structure modelof the sodiumchannel.
Figure 1 showsthe alignment of the amino acid sequenceof the squidsodiumchannel (20), together with thoseof rat I, II and eel (5, 6) sodiumchannels. Local hydropathy analysis hasrevealed (20) that the transmembranetopology of the squidsodiumchannelis quite analogous to the vertebrate sodiumchannel(6); that is, four homologousdomainscontaining all six membrane-spanning structuresare repeatedin tandemwith connectinglinkers between neighboring membrane-spanning segments,carboxyl and aminoterminals. Detailed comparisons of the amino acid sequenceof the squidsodiumchannelfor segmentsS2, S3 and S4 and for extracellular linkers which are hoFologously conservedbetweenSSand S6 for sodiumchannels (designatedas ~35-6regions)are illustrated in Fig. 2 with thoseof other sodium channels. For S2 segments,both positi*;ely chargedresidue, lysine or argininel75,se2.tW. 1697,at the 19th position (Fig. 2) and negatively chargedresidue,glutamic acid”‘, 8% IX92 ‘653,at the 15th position are completely conserved,while negatively chargedresidue,glutamic acid’61or asparticacidls’9, at the 5th position is conservedfor both I and III domains. From the viewpoint of charge distribution, I-S2 resemblesIII-S2, and II-S2, IV-S2. However, a notable difference in charged residuesof S2 segmentsis found in domainsI and III; at the 2nd position in I-S2, lysine residue’s8is completely conservedfor vertebratesbut not for squid. It is replacedby glutamic acid residuet5R.Similarly, glutamic acid’316and lysine1x2()at the 2nd and 6th positionsin III-S2 are conserved for vertebratesbut replacedby phenylalaninel3’hand isoleucine’3’” for squid. respectively. Thesefindings suggestthat the chargedresiduesessentialfor sodiumchannel functioning are lysinet9thand glutamic acidlsthfor all the domains,and glutamic or aapartic acidsthfor domainsI and III. For S3 segments,negatively chargedresidue,aspartic acidl97,ss& 1351,1674,at the 6th position is completely conserved. In domainsII and IV, negatively charged residues9081689, glutamic or aspartic acid, is conservedat the 16thand 21st positions,respectively, for both squid and vertebrates. A notabledifference in chargedresiduebetweensquid and vertebrate sodiumchannelsis found in domainsI and III; at the 17th position in I-%3, glutamic acidz()*is conservedin both rat and eel sodiumchannelsbut replacedby leucine20xin squid. At the I I th position in III-S_?.asparticacid1y-56 is conservedin rat but replacedby isoleucinein squid. For S4 segments,which have widely beenrecognized to serve as a voltage sensor(5, 7, I I, 12), squid S4scontain 4, 3, 5 and 8 basicresidues(arginine or lysine) at every third position in domainsI, II, 111and IV (Fig. 2), respectively, while thoseof vertebratescontain 4, 5. 5 and 8 basicresiduescorrespondingly (Fig. 2). The difference is only in II-S4 where two lysine 1159
SQUID I/ RAT I/ RAT II/ EEL I 11 10 30 I- @I
II- IIQPILvrPCPDSfIfflIAslAlIrASI~ I- YAISVLIPfCPDSflffIIIslAAIsQSI~~ I- IAIIf-SSAIPtlflRFlPOSLSP7iA-19
A00 nPI~1D-DOEnEPS?“SDLII6IXLPfI7~F~qIPPSSIsSPL~OtDP*V*“A IpI~~IDEDDF116P1PISDl6ACKSLpI~I~~l~tPPSNVSSPLSDlDPVlII, SCl~I----OPtSlPIIDLLICIPLp~:I~~~~PPSDLlSIPLEDLDPfVlIQ
220 III 231 232 225
253 943 ?15 322
:flt -..___.___--._------flflQAAAkASKHSASPSAA------------------~ItSDSS lAAA~~ASlLSRDfStA5-----------------E~(VfSISS IllA~)SQL,Q1QtA[I,DDGDDA,IEC161A,)~A,n,lLPS 101
AD __--____.__...______ v-S”,DDI __.____._______ _____ _____.-_ _._ ___-. BASILSSSSASEI~IItKl1~QSiQSC6ASSOO~DSFU~SSSSOSIlSIGfSfSIS6~S~~VKKAISSPHQSt~SI VASSLSSISEIILIItIKII~QISQACV~Il~DA--VllSlS~DS7ASICfQfSLSCSSLll~SffSSPSQSLtSI _ _____..__...__. ___- __.______..__.____._------.-----. I V.-I,,S ..___..__.___.___
3s 1 5S9 S62 491
720 JllI 679 673
09 ,9S 76 7 5,‘
554 Ill go3 il0
1_Comparisons of amino acid sequence of the squid sodium channel (1st row) with those of rat I (2nd row) and II (3rd row) and eel Efecrrophorus e/ec/ricm (4th row) sodium channels. The squid and other data are cited from Sato and Matsumoto (Biochem. Biophys. Res. Comm., 186,l) and Noda et&. (Nature 312: 121-127, 1984: Nature 320: 188-192, 1986). respectively. Identical and conservative am=acid residues are boxed with solid and broken lines. respectively. Spaces (-) are inserted into these amino acid residue alignments to achieve maximum amino acid sequence homology. Transmembrane segments S l-S6 are determined by the hydropathy profile analysis (Sato and Matsumoto: Biochem. Biophys. Res. Comm.. 186,l).
residues91sv 9’s are replaced positions
(Fig. 2), respectively,
which repeat at every third position, 21st position
in I-S4 is conserved,
and 24th positions
and glutaminegt* It should
(Fig. I ) at the 12th and 15th that
All these findings, topology
I and III; that is, lysine243 at the
and the basic residues (arginine
are both conserved
analysis (20), suggest that the transmembrane analogous
or Iysine)tT9”, together
1397 at the 19th
with local hydropathy
of the squid sodium channel is quite
to that of vertebrate sodium channels (5-10).
In considering transmembrane
the tertiary structure of the squid sodium channel, we assume that all the
the charged side chains are clustered
but not a-helices.
In the 3to-helix
one side of the helix, the opposite side being 1160
652 lb32 1623 326
1299 1732 1722 I515 1 I20
2!li ,522 5009 1Ob5 ,120
1 - Continued
occupied mostly by nonpolar sidechains(5,20). Further, the chargedresidueconfiguration in S2 and S3 segmentsfor the respectivedomains(Fig. 2), together with the peculiar positive charge configuration in S4 segments,leadsto the conclusionthat the octagonalstructureillustrated in Fig. 3a-b would most probably be realized where the U-6 regions, composedof 31, 30, 32 and 39 residueslocated betweenS5 and S6 in domainsI, II, III and IV respectively, are stabilized to interact with the inner surfaceof the core pore formed by the S4 segmentsand the neighboring S2 segmentsaligned with S4s. It is noted that the U-6 region possesses a strongly negatively chargedmoiety; that is, the total effective chargenumberin the SS-6 regionsis 9 for nominalnegative charge(in other words, the difference betweennegatively and positively chargedresiduesin S5-6sasa whole) in squid, and IO-1 1 in rat brain I-III (6, 7). The core pore of S2 and S4 1161
LKVANYVFTTVFVL LTVGNLVFTCI FTA
4; it IA
RATSCPIIA RATSCiII FSBSCPEEL 4th domain
RATSCPIIA RATSCIII FSBSCPEEL
LYWINLVFIVLFT LSRINLVFIVLFT LSQINVIFVIIFT
TVIKISYITLLLQTI TVITFAYVTEFVDLC TVITFAYFTEFVNLC SVIVHAYVTEFVDLG SVVTITPITEFIDLR
CWNI CWNI GWNI FE: FWTI AWCW AWCW AWCW AWCW
TIVIITVISLAASGL LIVDVSLVSLTANAL LIVDVSLVSLTANAL LIVDVSLVSLVANAL VIVGASIMGITSSLL
LWNi ;g GWNI GWNV
VVVILSIVGYFL VVVILSIVCYFL AVVVISIIGLLL
s3 AWNC PWNW PWNW
~ATSCPI IA RATSCIII FSBSCPEEL 3rd domain ;;WJ;;pS;p 1
1st domain SQUIDSCPI RATSCPI RATSCPIIA RATSCIII FSBSCPEEL 2nd domain
YTN YTS YTS YTS YTN
RATSCPIIA RATSCIII FSBSCPEEL 2nd domain
RATSCPIIA RATSCIII FSBSCPEEL 3rd domain
RATSCPIIA RATSCI II FSBSCPEEL 4th domain
Ki KVN KVN
QVNF TFGKTFLLLV&ATSACWN YFNF FGNSYICLFQITTSAGWYFNF FGNSYICLFQITTSAGWMFRF FCISYICLFQITTSACIIFNF c FGKSYICLFEITTSAGW-
RATSCPIIA RATSCI I I FSBSCPEEL
LLAPILNSKPPD LLAPILNSGPPD LLAPILNSAPPD LLLPTLNTGPPD
Detailed comparisons of aminoacidsequence for S2, S3andS4segments andthe S5-6 regionsin domainsI, II, 111 andIV of squid(SQUIDSCPI).rat I (RATSCPI),rat II (RATSCPIIA). rat III (RATSCIII) andeelElectrophorous electricus (FSBSCPEEL)sodium channels. The dataarecitedfrom SatoandMatsumoto(Biochem.Biophys.Res.CommJ86, 1).
Nodaetal. (Nature=: 121-127. 1984: Naturem: 188-192, 1986) and Kayano et (FEBS Lett. 228: I87- 194, 1988). Identical and conservative amino acid residues are boxed with solid and broken lines, respectively, and aligned vertically to be in the same positions by inserting spaces (-). a/.
possesses strongpositive charge in lines facing its inner surface,with its pore diameterestimated to be 0.6 nm, i.e., sufficiently wide to accept the 55-6 regions inside it (Fig. 3). The S5-6 regionscan be partly integratedinto the pore in the resting stateof sodiumchannels,presumably up to the positionswhere the negative chargeresiduesof the S2 segments(designatedas the stoppingresidues)are located, which resultsin the stabilization of the octagonalstructureof the channel(Fig. 3c-d). More concretely, the tips of the S5-6 regionsremained,in the resting state, at the positively chargedsitesof their respectiveS4 locatednearestto the stoppingresidues. In our model of the sodiumchannel,the S5-6 region plays a principal role in activation gating, voltage sensingand ion-penneableselectivity. First, (1) the respectiveS5-6 region makes a loop from the outsideto the insideand back again,forming the central pore. The S5-6 regions 1162
Schematic illustrations of a proposed tertiary structure model of sodium channel. Octagonal topology of transmembrane segments Sl-S6 and the P-6 regions for the domains I-IV is shown, as viewed firom the extracellular side (a) and from the transmembrane side (b). respectively. Resting configurations of the S5-6 regions, and C- and N- terminals are also illustrated along line a(c) and line p(d). where cross sections of the segments (I and II) and (III and IV) are viewed, respectively.
are all negatively tips
charged as a whole, and it may be hypothesized that they are halved, making their charged
is quite contradictory to the conventional view that S4s are voltage sensors (5, 7, I I, 12). Instead, the S4 segment in our model may act as a rail for the S5-6 train. In our present model, S4s, together with S2s, form a guiding pore through which the S5-6 regions can slide. (2) The negatively charged residues can select ions through the pore formed by the four SS-6 regions, together with its pore size (3). with the result that the SS-6 can determine the ion selectivity. (3) In the resting state, the tips of half-turned S5-6 regions partially enter into the S4-S2 pore up to a position which forms a balance with the attractive force due to positively charged residues of S4, and at the same
of S2 (Fig.
d). The repulsive force partly comes from both the resting membrane potential and the carboxyl terminal segment which possesses strong negative charge as a whole (24 negatively and 18 positively charged residues in squid sodium channels) since the C-terminal segment is most likely to be located at the mouth of the S4-S2 pore on the intracellular side in the resting state (Figs. 3 and 4). This configuration is reasonable because both resting membrane potential and the positive charges on S4 cylinders plug the negatively charged C-tail inlo the S4-S2 pore, and are further stabilized by the III-IV linker which is highly positive (I3 positively and 4 negatively charged residues in the III-IV linker of the squid sodium channel), as illustrated in Figs. 3 and 4. 1163
Schematic illustrations of a proposed tertiary structure model of the sodium channel corresponding to the resting (a), activated (b) and inactivated (c) states. The lqper- and lower pictures for each drawing represent the transmembrane and intracellular side views of the channel, respectively.
Upon depolarization, the SS-6 region further slides into the S4-S2 pore on the cytoplasmic side along the “linear rail” of S4 cylinders, depending on the membrane potential (Fig. 4-a-b). The activation energy required for the respective S5-6 region to pass through the pore is determined basically by the electrostatic interaction between a SS-6 region and its associated S4 segment wall which has a complete rail of IV S4 but some incomplete rails of other S4s (Fig. 3c, d). The activation energy also depends on the distance between the tip of the S5-6 region and the negatively charged C-tail surface facing the core pore. The IV S5-6 region can pass through the core pore with the least energy since the IV S4 segment possesses 8 positively charged residues, the maximum number among the other S4 segments. The S5-6 regions for domains III and I may follow the IV S5-6, and lastly the II SS-6 may come. At the motnent when the S5-6 regions 1164
approach the cytoplasmic side, the C-tail which has covered the S4-S2 pore from the cytoplasmic side is removed by both of the repulsive electrostatic interactions between negative charges of the C-tail and those of the S5-6 regions, and between negatively charged C-tails and the membrane potential (Fig. 4-b). The C-tail thus removed from the pore is most likely to be attracted to the Nterminal region because the squid N-terminus is positive-charge rich (14 positively and 4 negatively charged residues; see Fig. I ). The other possible sites to which the C-tail is attracted are the II-III linker, I S2-3 and I S4-5 regions, which are all positive-charge
rich (nominally 4
positive charges among the last 23 bases for the II-III linker, one positive residuelgO and one positive residueZs4 for S2-3 and S4-5, respectively).
When the four SS-6 regions pass through
the S4-S2 pore to form an ion-selective channel and the C-tail is completely removed so that cations can pass through (Fig. 4-b). the activation is completed. Accompanied by the activation in which the SS-6 regions pass through the S4-S2 pore to the cytoplasmic side and the C-tail is repelled from the pore. the III-IV linker, which has well been understood to play a crucial role in inactivation (I I, 1% IS), moves away from the channel together with the C-tails (Fig. 4-b). In the configuration of the activated state (Fig. 4-b). the III-IV linker interacts more with positively charged sites of the C-tail than when it is in the resting state (Fig.4a). The III-IV linker in this configuration is, both electrically and elastically, unstable, somewhat attracted by the negative charges at the tip of the S5-6 pore (Fig. 4-b-c), and eventually reaches the inactivated state (Fig. 4-c). On repolarization to the resting membrane potential, the C-tail is more attracted to the membrane side, and at the same time, the III-IV linker is more attracted to the cytoplasmic side. As a result, the C-tail interacts more with the III-IV linker, leading to the original resting configuration (Fig. 4-a). At the same time, the C-tail which will be restored to the original resting site, togelher under the action of the membrane potential, repels the P-6 regions to the extracellular side. The present paper has described our proposed tertiary structure model of the squid sodium channel, based 011 the results (20) of its complete amino acid sequence deduced by cloning and sequence analysis of the complementary DNA (Fig. I). In this model, the S5-6 regions play an essential role in (1) sensing membrane potentials, (2) forming the ion-permeable pore, specifically and selectively, for sodium ions, and (3) functioning as the main part of the activation gate. The C-tail works with the SS-6 regions as activation gating. The III-IV linker plays a crucial role in inactivation, as has been well established by others (1 I, 1% IS, 2 1). Our proposal that the S5-6 regions form the ion-permeable and selective pore is supported by other experiments employing site-directed mutagenesis; substitution of a single residue in the S5-6 region renders rat sodium channel I1 insensitive to tetrodotoxin (TTX), markedly reduces its single channel conductance (18, 19, 22), and alters the ion selectivity (I 7). The dynamic characteristics of the S5-6 regions, voltage sensing and activation gating, are consistent with the experiments that the ion selectivity is markedly voltage dependent, reduced and enhanced upon hyperpolarization and depolarization. respectively(23), and that TTX not only blocks sodium current but also reduces a component of the sodium gating current (24, 25). Our model that the SS-6 regions are, as well, activation gates, is supported by the linding that L~iur-KY scorpion toxin binds or unbinds to some localized sites in the I SS-6 region (26) in a voltage dependent manner as a function of the steady state of activation (27). Our model has further described a molecular mechanism of the coupling between activation 1165
and inactivation, which has been in question since Armstrong and Bezanilfa (16, 28) discovered the coupling from their measurements OPsodium gating currents using squid giant axons. Furthermore, the question raised by McClatchey et al .(29) as to why substitution of a single residue in the II S5 segment caused hyperkafemic periodic paralysis (HPP), a disease caused by inactivation deficiency, may be answered only by our octagonal configuration model of the sodium channel where the II SS segment directly neighbors the IV Sl segment; that is, in this configuration it may be possible that a single point mutation in the II S5 segment affects the IV Sl segment, resulting in changes in the inactivation. Quantitative understanding of the sodium channel remains to be established in future.
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