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Volume 309, numkr 3,253-257 FEBS llSl00 1992cdcrarion of Eurqxan
Biochemical Societies [email protected] September 1992
Ckimeric study of sudium channels from sat skeletal and cardiac
muscleL.-Q. Chcnb, M. Chnhinc, R.G. Kallct?, R.L, Barchicd and R.
MarnM
Received 20 July 1991Two soforms of voltngcsdcpcndcnt Na
chunnclr, cloned from rat skclctvI muulc, wcrc cxprcsscd in Xwopw
oocyks. The currcma of AkM t andrSkM2 differ functio nally in 4
propcrGcs: (i) tctrod otoxin (TTX) s cnriliv ity, (ii)ficono toxin
(ira) sensitivity , (iii) amplitude of sin& channelcurrcn ls,
and (iv) mtc of inactivnGon. rSkM I is scnsitivc to bolh TTX and
/LTX. rSkM2 h rcsiaant 10 borh torins, urrcms of AkMl hrv~P higher
single channel conductance and a slower rate of inactivation thnn
those of rSkM2. WC conaructcd (i) chimes by inrrrc hangingdon uin1
(DI) bctwccn tbc two iroforms, (ii) block muution r of 22 amino
acids in length Ihat intcrc bangcd parts of the loop kcclwccn
trmsmsrnbransscgmcnts SS and S6 in both DI and 04. and (iii) point
mudons in the SS? rcaion orthis loop n Dl. The IT% scnsitivi
tycould k switched klwccnrhc two isofonns by the cxcbangc of inglc
umino acid, tyrosinc -401 in rSkM I nnd cyslcinc.374 in rSkM2 in
S52 of DI. By conlnst most chimerasand point mutants had un
in&rmc dialc rcnsiliv ity topCTX when compared with the wild+@
chunncls . The point inumt rSkM1 (Y40lC) hadan inrcrmcd iatc ringlc
l yM) is prcscnt in both developing anddcncrvatcd skeletal muscle
as well PS n rat hsart [l&6].Adult Na channels from skclctal
muscle arc alsouniquely sensitive to y-sonotoxin @-CTX), a
propertynor shared either with the TTX-sensitive Nn channelsof
brain or with TTX-resistant Na channels of muscle[7,8]. Accordingly
the currents of rSkMI, but not ofrSkM2, arc blocked by nanomolar
concentrations of,u-CTX when the cRNA is expressed in Xcwpus
oocytcs[2,3]. At least two ocher functional diffcrcnscs are
ob-served between the Na currents of the wild-type chan-nels
txprcsscd ir: ,Ywroptrsoocytcs, namely the singlc-channel
conductencc is larger in rSkM1 than in rSkM2191, nd the rate of
inactivation is grcutcr in rSkM2 [2,3].
Corrcs~ondcncc adcfrcw R, Horn. Dcpartmcn t of Physiology ,
J&r-son Medical Collcgc, 1020 Locust Street, Phlludclphia, PA
19107,LJ5A. Fax: (215) YS5.2073.
WC have constructed chimeras and mutations thatexchange portions
of AkMl and rSkM2 to try to idcn=tify the regions of the protein
bat are rcsponsiblc fortheir sirnctionul differences. Most of the
mutations wereconstruct4 in the loop between the putative
transmcm-branc segments S5 and S6, either in the first (Dl) or
thefourth (D4) of the four homologous domains. Portionsof the SS-S6
segment from 011 our domains have beenshown to contribute to TIX
sensitivity irnd ainglc than=ncl conductance of rat brain Na
channels 1101.A resentreport dcmonstratcd that full m sensitivity
can bcrestored to rSkM2 by cxchangc of a single amino acidbctwccn
the two isoforms [111.This amino acid, tyrosincin rSkMl and
cystcinc in rSkM2, is located in the so-culled SS2 region of the
SS-S6 loop of Dl. An abstractof our data hus appeared [12].
2. MATERIALS AND METHODSChemicals and protoco ls wcrc obtained
from Fkthcxla R-archLabs, Sigma, VWR. Srratagcnc Ctoning Syslcms,
and Amcnham.
2,2, Currxrruaion a/ chinwic and sits-qwcifk mulunUpSlSl and
pSIS2 wcr c connruc lcd from the full-length cDNAs ofrSkM1 (SI) and
rShM2 (52) by transfcrln g So/I-Sari and NlndlIl-Emiti iqpncnis
iitiiii $i#i+j iii5 fii%Gi crprcwhr WSiGi5 i.qinto pSclccl-I (PSI ;
Promcga), Chimeras were conswuckd from c%isbillg or newly crcutcd
cndonuclcasc rcstriclion sites or wilh new sites,
Putdished by EIsrvicr Scicncc Publl~hcrs E. V. 253
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Yolumc 09, numbsr 3 FEBS
Fig. I, Elfcctof TTX and JI.STX on currcms of rSkM I and
rSkM2.Current of (A) rSkMt und 05) r&M? nt -10 mV from il
holdingpolcnlhl of - 100 mY. Supcrimpwzd wscs indiulc the clTcrtr
of 100RM ITX rend 500 RM PCTX.diffcrcnsc in TTX sensitivity bctwccn
rkMl undrSkM2, has the following amino acid scqucncc:
rSkM 1: IULMTQDYW;NLFQLTRAAGKTYrSkM2:
lJLMTQD~WE~L~Q?jTLR~AGK~-l&h isoform contains ;I single
glutamate in SS2 (@).Tlziv rcriduc. when rcpleccd by glutaminc in
rut brainNtl chunncls. abolishes TTX sensitivity [ 181,All but
6amino acids (undcrlincd) arc idcnticcll in this region ofDl, There
is one difference in charge; a positivelycharged urgininc (R377) in
rSkM2 is rcplaccd by D ncu-tral srspuraginc (N404) in rSkM 1. These
residues, how-cvcr, arc not the primary dctcrminnnts for the
differentTM scnsitivitics of the wild-type isoforms, Satin ct al.[
1I] showed that the point mutant rSkM2(R377N) hasn lower, rather
than u higher, sensitivity to TX thanthe wild-type isoform, WC have
constructed the sumcmutation with equivalent results(data not
shown). WChave also constructed the complsmcntary mutation in
rSkM1. This mutant. rSkMl(N404R), hud little effecton cithcr TTX
sensitivity, with an &,of 5724 nM. or@TX sensitivity (I& =
55*7 nM).WC also cxchungcd the tyrosinc (Y4Ol) in rSkM 1 forthe
cystcinc (C374) of rSkM2 [1 11.Fig, 2 shows thut therxchangc of
this rcsiduc switched t>c TTX sensitivity ofthe two isaforms.
The I& for lTX block of rSkMl-(Y401C) was 1,5f0.3 PM, and for
rSkMZ(C374Y) was
TERS Scpwmtw 15%5.2&0,4 nM. rSkM2(C374Y) was cvcn more
scnsitivc tomX than wildqpc rSkM I. By contrast the sensitivityto
PCTX in rSkMI(Y401C) was reduced below thatfound in nutivc rSkM1
(I& = 19757 nM). but not tothe lcvcl found in native t5kM2.
Convcrscly the muta=tion rSkM2(C374Y) did not rccovcr the
scnsitivicy top-cTX found in native rSkM1.
It has been postulated that the ITX binding site hwnegatively
charged sites that could accumuhtc Ne ionelocally and thus increase
the condustancc of the Nnchrrnncl at physiological
ionicconccntrations[l5,19,20].In uccordancc with this idco rSkM1
has a larger singleshsrnncl conductansc than AkM2, cithcr in
dcvclopingmuscle [L6] or when cxprcsssd hctcrologously in
oocytcs[9,15]. This suggcstcd to us that TT%-scnsitirc mutantsmight
IIPVC tl higher single chnnncl conductance thunthose that urc
inscnsitivc to TTX. Fig. 3 shows the singlechannel current-voltugc
relationship for the two pointmutants tl1ilt intcrconvrrt TTX
sensitivity,rSkMl(Y40lC) and rkM2(C374Y). obtained fromoutsideout
patches. Linear regressions for thcsc dataarc shown us dashed
lines. For comparison, the bet-fitregression lines for the
wild-type channels arc shown assolid lines. bnscd on the duttl in
Gcllcns et ~11.92, Al-though the point mutation in rSkM2 had little
effect onthe amplitude and conductance of single channel cur-rents,
the complcmcntary mutation in rSkM1 caused tlsignificant dccrcasc
in the slop condusbncc, from 32pS in the wild-type to IS pS in rSkM
l(Y4olC), Our datathcrcforc show that the 7TX-scnsitivc mutant has
alower single chilnnd conductonsc than the lI%rc+timt mutant,
contrary to our cxpcctation.3.3. I~ructiwtion kirlcticsrSkM 1, but
not rSkM2. has abnormally slow kineticsof inactivation when
cxprcsscd in oocytcs, The timeconstunts of inactivetion arc
prcscntsd in Trtblc I for allthe chimeras and block mutations. The
wild-typeisoforms in our experiments had &fold diflcrcnccs
intime constants of inactivation. Block mutations in the
ITim COINIII~of intictivation r~ncl C, of7TX imd 1(.ffX
block
NumcWildatyp rSkM IWild-type
rSkM2CllitttcrupSlS1.1234pSlS2-1234Es&
ii;;:ti;rpSlSl.DSS2pSISbDISS1pS1Sl-D4552
fnuctivation his) 71% wnrilivity j&TX sensitivity10.65 f:
0.8 34.9 f 9 nM 54 t6 nM1.2 t 0.3 3 *l PM > IO PM2.1 + 0.3 tl 22
nM 1.3 r 0.1 PM2.1 f 0.8 1 fO.lpM 0.5 z 0.3pM
11.9 & 1.2 1 2O.tpM 0.1 + 0.4pM1369 + 1 20 t Q nM 96,4 f 17
nM10.9 & 0.8 34.3 ?: 4 nM 287 f U9 nM
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Volume 30% number 3 FEES LE-lTERS September 1992
0%Fiy. 2. Point mutunts (A) rkMI (Y4olC) and (BI tSkM2
(C374Y),The Jatn wcrc obtained under the sumc conditions PI in FiQ.
1. Therime constant for inactivation of rSkM1 04OIC) was 11 I tZ
ms.andfor rSkM2 (C374Y) WYO .97fO.l ms.
SS2 region of either Dl or D4 had no significant cffcctson the
kinetics of innctivation of tSkM 1. The same wastrue for all the
point mutations WC xominsd, however,the two chimeras in which Di
was exchanged bctwccnthe isoforms had intermediate rates of
inactivation,each tipproximately 2-times slower than that of
rSkM2(Table I). Also, a block mutation in the 8Sl region ofDl in
&Ml caused a slower rate of inactivation thanobserved in the
wild-type isoform. These dota suggesta role of Dl in the rate of
Na-channel inactivation inoocytcs.4. DISCUSSION
We cxamincd four functional properties of NP cur-rents that
differ in the isoforms rSkMl and rSkM2,when expressed in oosytcs.
Thcsc properties arc TTXsensitivity, yCTX sensitivity, single
channel conduct-nncc, and inactivation kinetics. Our rcsuks show
that 3single residue accounts for most of the difference in theTTX
sensitivity between rSkM1 and rSkM2. The otherthree propcrtics,
however. cannot be explained so sim-ply, suggesting that more
extensive differences bctwccnthe proteins, or difference at
multiple sites, must us-count for the diffcrcnccs in function.
Fig. 3. Sin& chenncl amplitudco, Data were obthcd from
outsidc-OYI patches of the indicated mutations. and arc plotted as
mean 2S.E.M., along with best-fit linear regression linca. Solid
rcgrcrvionlines wcrc for the wild-type isoforrns, 85 found in
Gcllcn5 ct al. [9!,
The diffcrcnsc in TTX sensitivity between these twomuscle
isoforms is primarily explained by u single rcsi-due in the W-S6
loop of Dl (Fig. 2). Our datu do notshow whether a cystcinc at this
position inhibits TTXblock, or whcthcr the tyrosinc is ncccssary
for block byTTX, however. other TTX-sensitive Na channels of
ratbrain or clcctroplnx have either a phcnylaloninc or atyrosinc at
this position [2l-231, suggesting that an are-matic group here may
be ncccssary for the action ofTTX. The mutation of the cystcinc
(C374) of rSkM2 tophcnylolnninc also confers lTX sensitivity to
thisisoform [ll]. The aromatic group may contribute di-rcctly to
the TTX binding site. as suggested by the roleof aromatic rcsiducs
in the block by tctrecthylammo-nium in K channels [24] or I\&
he binding of asc-tylcholinc to usctylcholincstcrasc [25]. The
nature ofintcruction between the cationir toxin and the
aromaticgroup is unknown, but clsctrostatic interactions arc
bc-licvcd to occur between cutionic amino groups and aro-matic side
chains [26]. The SS2 regions of the four do-mains of Na chunncls
each cantain from one to threearomatic residues, any of which may
contribute to toxinbinding. Tyrosinc-401 in rSkM 1 is the only SS2
residuewhich does not huvc a corresponding aromatic residueat the
equivalent position in rSkM2. Since RkM2 hasull of the
ubovcmcntioncd ncgucivc rcsiducs. as well PSall-but-one of the
trromutic residues in SS2 regions, itnpparcntly bus all of the
structural requirements forTTX binding, with the cxccption of the
one aromaticresidue in Dl. Further mutagenic cxpcrimcnto arc
re-quired to understand the roles of the ocher aromaticresidues of
SS2 regions in sensitivity to TTX block.Although TTX andp.CTX show
competitive binding[271. the mutations thut restore TTX sensitivity
torSkM2 do not do the sums for @TX. This is notsurprising, since
all isoforms of TTX-scnsitivc Na chan-nels, cxccpt those of
skclctal muscle. arc insensitive top-CTX. Also y-CTX is a much
larger molecule thanTTX [28], and experiments using analogs of
y-CTXsuggest that widely spaced rcsions of the molecule con.tribute
to its ability to block Nu channels [29,301,TableI shows, in fact.
that mutations in borh Dl and D4affect @ZTX sensitivity. It seems
likely, therefore, thatthe y-CTX docking site is comprised of more
extensiveparts of the N;I channel thun the TTX binding site.
Thecompetitive interaction between the binding of the twocompounds
indicates that their binding sites may ovcr-lap, but nor
necessarily thut the site arc identical. Ourdata do suggest.
however, that the two sites share spc.cific amino acid residues,
because the point mutationthat renders rSkM I TTX resistant also
causes u3.6.foldincrease in the fC& for POX,4.2. Sing/c chrtle:
c0nc/l6cru1zceModels in which TlX biocks Nu channels by cntcr-ing
and plugging the pore require that the TTX binding
256
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Yolumc 309, number 3 FH3S UTTERS Scptcmkr 1992rite is intimately
associated with the rxtraccllul~r mouthof the channel. Our data
show that the point mutution,rSkMl(Y40iC). that reduces TTX
sensitivity also dc-crca~scs in& chunncl conductance Also.
mutcltions inhomologous regions of D3 and D4 of bruin No
chmmslsaffect the monovulrnt/divalcnt sclctivity of the
channel[31]. Finully, the point mutant rSkM2(C374Y) chungcsthe
I&, for Cd: block [l I], which is believed to occurwithin the
pcrmcation pathway 1321.The accumulateddntsr. thcrcforc. provide
strong support thut SS2 regionsfrom part of the pore. and that TTX
binds to P sitewithin it,
The rtrtc of inactivation of rSkM1 Na current is ab-normnlty
slow when cxprcssed in oocytcs, by compnri-son both with the
situation in native tissue nnd with hctc-rologous expression in
mammiiliim cells [2.14.33.34].WC huvc found thut the two isoforms
have compnr;lblcrUtcs of inoctivtltion when expressed in TSA-201
cells,LL uman kidney ccl1 lint (unpublished obticrviition). Itis
not clctlr why the intrctivntion in oocytcs is abnormalfor rSkM I.
It may dcpcnd on the itbscncc in oocytcs ofu necessary coftlctor,
such POP subunit of the Nn chan-ncl found in mtrmm;rlitln cels
[35]. Altcrnatiucly. theoocytc may not correctly proccvv the rSkM 1
protein. Itiu also not clcor why rSkM2, by contrtrst, hers
rapidinactivation kinetics in oocytcs. Ncrvcrthclcss our
datasuggest that Dl htls LImodcrtrtc hflucncc on the inucti-vtltion
rate, since chimrrns in which Dl is swappedbctwccn the isoforms
hnvc intcrmcdiutc rates of inrrcti-vution. This could be due to
intcrdomttin 5~swell usincrtldomain inccructions. The
intcrprctotion of thesedata will dcpcnd on bcttcr understanding of
the influ-cncc of the oocytc cxprcssion system on the processingof
Nn channels.Ar~rrcl~\./c~~~*~t~~~/~~C hank David Meyer and C;ltcc
Da Kntlcn forIheir nrrist;mcc, Bob French und Let Chilb;ll;l for
prcpubticution man-uscripls. und Chris Miller for insighlful
discurrionr, The work wiIXsupportat by Amcricirn Hcurr
Ar&x\ian, MulkuInr Dyr;trtiphyFoundation. Research Foundaion
CI~I~CUniversity of Pcnnsytviulir.S~crling Rcrcnrch Lubontory. and
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