Molecular mechanism of protein kinase C modulation of sodium channel α-subunits expressed in Xenopus oocytes

Volume 291, number 2, 441-344 Q 1991 Federation of European Biochemical Societies ADONIS 001457939300996E

W. Schreibmayer’,

FEBS 10283 Ofii&5793/91/$3 30

October 199I

I’d. Dasca12, I %otan2, M Wallner’ and I_.. We@’

‘Instlluk of Medtcal Physics and Btopl~ysm. KarWranzens

Wrwcrsrty Graz. A-8010 Graz, Aus~rra and 2Dcparrmcns of Physrology and Pharmacology, Sackim School of Medrcrtte, Tei AVIV Utwerslty, Ramal Aviv 69978, Israel Recetved

20 August 1991

The mechanism of modulation of sodmm channel cl-subunits (Type HA) by a protem kmdse C (PKC) activator was studled on smgle channel lcvcl It was found that (I) time constants for channel actlvatlon were peolonged, (II) mactlvatlon remained virtually unchanged. (III) peak sodium inward current was ecduced as evidenced by calculation ofaverage sodium currents, and (IV) time constants for current actlvatlon and decay were prolonged (I), (III) and (IV) were voltage dependent, being most promment at threshold potentials The data show that a voltage dependent action on the actlvatlon gate can account for the observed reduction of peak mward sodmm current and prolongation of current decay m macroscopic expcrimcnts Sodium channel modulation,

Protein kmase C (PKC), Phorbol ester, Xetlopus luevts, Patch clamp, RNA expression

1 IWTRODUCTION Part of the pivotal and diverse cellular regulation by protein kinase C (PKC) [ 11IS accomphshed via modulation of K+-$a’+- and Cl--channels of the plasma membrane ([2-53). The channel forrnmg a-subunit of the

voltage dependent sodium channel protein has been shown to be a target for PKC-medlated phosphorylatlon [6]. Regulation of functional sodium channels, expressed from vertebrate brain in Xenopus oocytes, 1s now well established [7-g]. Both an altered availablhty of the channels to open without affecting actlvatlon or mactlvatlon [7] and interference with the channel’s actlvation gate [S] have been suggested to be the mechanism of sodium channel modulation by PKC. Yn the present study this modulation was explored at the molecular level to gam detailed insight into the underlying mechanism 2 EXPERIMENT,

LL

2 I C/Iermu/~ ad solurron~ Phorbol 12.myrlstnte 13-&et&e (PMA) was purchdscd from Sigma (Germany) All other chemicals used were of reagent grade ND96 contained (III mM) NaCl 96, KCI 2, M&l2 2, HEPES/NaOH 5, pH 7 4 NAtOO contamed (m mM) NaCl 200, KCI 5 4, MgC12 2. Cat& 2, HEPES/NaOH IO, pH 7 4 PGl80 contained (In mM) K’/glutamate‘ 180, KCI 37 5, EGTA 2 5,MgCI, 0 5, cdq 0 25, glucose IO, HEPESIKOH 10, pH 7 4 2 2 Itt yllro Irmwtplfon otrd e\prebrrotl Cdpped cRNA was transcnbcd from C/u1 Imcarzed plasmid DNA

Corrcrporrdcfrcc c&/rcM W Schrcibtnaycr, Institute of Medlcal PhyUmvcrstty Grdz, A-8010 Grdz,

sics and Blophyslcs, Karl-Fr,m/cns AUSIII~ Fax (43) 316 35566

contammg the IIA sodium channel Insert (pVA2580, Auld et al [IO]) with the ald of T7 polymeease as descnbcd [8] Adult female Xenopus fuevrs were purchased from H Kslhler, Germany, aad kept at 18°C Defolhculated stage V and VI oocytcs were prepared as described [8] After mJectlon of 3-20 ng RNA oocytes were Incubated at 21°C m ND96 plus I 8 mM CaCl,, 2 5 mM pyruvate/Na+, lOO@ml streptomycin and 100 units/ml pemcllhn Two to four days after mJectlon oocytes were tested with the double clcctrode voltage clamp techmque [8] m ND96 plus I 8 mM CaCI, and those with peak currents (1,,)>3 PA were taken for patch clamp recordmgs 2 3 Patch clamp recordtttgs Oocytes were placed for at least 5 mm m PGl80 and then the vltellme layer was removed Subsequently oocytes weee teansferred to plastic Petri dishes contammg 3 ml fresh PGl80 Patch clamp recordings were pcrformcd at ISOC accordmg to Hamill et al [I I] Sylgaed-coated plpets were pulled (List L/M-3P-A puller, Germany) from standard hdematocrlt cdplllartcs (Assrstcnl No 564, Germany) and filled with NA200 Patch current was amphfied with an Axopatch D (Axon Instr , USA) current amphfier and filtered at 5 kHz (4-pole Bessel filter) Sampling frequency was 33 kHz with the TL-I25 mlerface (Axon Instr , USA) connected to an IBM §X386-compatible computer (Steiner, Austrrd) Voltage pulses were supplied by the same device. Datd evaluation was pceforrned with the PClamp 5 5 I softwdrc (Axon Instr , USA) Holding potential was kept constant at -100 mV dnd every 2 s voltdge pulses rangmg from -90 mV to +5O mV dnd lastmg for 50 ms were dpphed (range and Increment were vandblc) Voltage Jump sequences were run repetltlvely to allow observation of channel behavlour m time 5 nM PMA was Introduced by the addIllon of 3 ~1 of a 5 ,uM PMA solution m PC180

3, RESULTS Sodium channel currents were recorded from cellattached membrane patches. Patches that did not dlsplay overlapping sodium channel opemng events at depolarlzatlons >-I-10 mV were consldercd single channel patches For calculation and evaluation of ensemble currents one membrane patch contammg many func-

FEES

Volume 291,number 2

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October

LETTERS

I

5 nM PMA

10 flrtt

20

latency

XI [rix]

5 RM PMA

e.

012345

012345 opentIme

2.5

pA

1

-10

opentIme

[me]

[ms]

ms

Fig I Effect of PMA dt -30 mV (d) Development of the PMA effect on first ldtencles (b) OrIginal smgle chdnnel traces, filtered dlgltally at 2 0 kHz, starts of voltngcJumps dre mxked by drrows (c-f) Nlstograms for first latencles and open times (g) Open probablhty vs tnnc as caicuiated by dvcrdgc current/open channel current DIgItal filtcrmg at I 5 kHz

tlonal sodium channels (‘macropatch’) was used m additlon In 4 cell-attached patches, between 5 and 25 mm after the addltlon of 5 nM PMA to the bathing solutlon (this time bemg variable from expenment to experiment). a ma1 ked effect on first latencles of sodium channel openmgs developed at -30 mV (Fig Id, Tdble I) TIUS effect could not be mlmlcked by control add&Ions (‘c’ m Fig Id) of bathing solution Selected single channel recordmgs before and after the dcvelopmcnt of this effect and corresponding histograms for first latencles are shown m Ftg I At -50 mV first latencies were so much prolonged dfter nddltlon of 5 nI’vl PMA, that calculation of their ttme constant (t,) became lmposslble for the given pulse length of 50 ms (Fig 2) In contrdst, at -10 mV rl)s were virtually not influenced by the phorbol ester At depol&z,ltlons > -10 mV the resolution of sodium channel openings was hnmpcrcd by single channel currents being small and fast, and rnlcloscop~c analysis has not been pcrformcd. Mean values for the measured time const‘mts are listed m Table I The patch-to-patch Wlatlon of I, (and also of open tlrnc constant, r,,) was consldcrablc (Table I), obscurmg the cffcct of PMA Thcrcforc, the % ctiangcs of time constants were calculated individudlly in each patch and the srgnific.mcc of the mean % change to bc ~0% was calculated according to [I21 (P in T,tblc I). In conttrtst to I/, the open time constrlnt I,, was not 342

markedly affected by the pholbol ester, albeit there was a tendency for a decrease at -50 mV (see Figs 1, 2 and Table I, respectively) In additional three single channel patches, PMA had no effect on the channel This emphasizes the stochastic

Tdble I Evdlitdtcd tlmc comtants E”‘,, WV)

Control

5 nM PMA

% chdnge

(mG

(ms)

(%I

Open times (I,,) -50 0 x-to 05 -30 0 3820 07 -10 0 5320 I3

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I-lrst Idtenues (I,) -30 2 49+1 07 -10 0 9540 39

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1 9SLO 54 081,035 0 98fO 59 5 lU:I 52 2 7x21 54 2 52kl.61

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3 45*l.l6 -IQ 2 34&l so +I0 2 231140 _p-___I____l_-“.

Volume 291,

FEBS LETTERS

number 2

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October

25

e:

50

first letency

[n?s]

8.0

opentIme Effect of PMA at -50 mV

(a) Orlgmal

traces as m

Fig

nature of the smgle channel behaviour and may be due to a high basal phosphorylatlon of those channels, or the absence of PKC molecules m these particular membrane patches Jn one particular patch (Fig 3) about 45 functtonal e-subumts were expressed (estnnated as I,/ P(,,), where Ip is the patch current, and P,,, the open probablhty) From the mspectlon of the macroscopic current traces and also from the 1, vs membrane potential (IZ) curve, It becomes apparent that I he PWIA effect IS voltage-dependent, being perceptable only at voltages between -50 and -20 mV (Fig. 3) Reduction of I,, was also accompanied by a prolongatron of both actlvatton and decay of the current (Fig 3a) as also seen for PtO) of single channels (Fig lg) Average patch currents for all 5 patches were calculated before and after the addition of 5 nM PMA. Ir was measured and normalized to Ip-30(contro,j (control Pp of a given experiment at -30 mV) Time constants were measured as the rlsetlme to 63% Ir (actlvatron time const‘mt) and decay to 37% (decay time constant) and are summarized m Table J The % effect was calculated for every potential and experiment, and was found to be strongly voltage-dependent (Fig. 4) ,

,PMA

PMA

/

la

(b-e)

[ITS]

I-hstograms

50

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1

2

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3

[fflS]

4

5

[rn+J

for first latencles and open times

4 DISCJJSSION Our smgle channel data show a marked decrease of the rate constant(s) for conformational transitions from the closed (c) to the active (a) channel state(s). This effect was most prominent at threshoid potentrais (-50 mV and -30 mV) and vanished at depolarlzatlons >-IO mV, as would be expected for a modulation affecting the actlvatron process. These findings are m agreement with those of Dascal and Eotan [g] who suggested, based on measurements of macroscoptc IdE relations, that the activation process of sodium channels 1s shifted to posltlve potentials by PMA As shown by the averaged patch current analysts ln combmatlon with tnlcroscopic assessment of channel actlvatton and mactrvatton (e g. Fig lc-g and Table I), the reduction of peak sodrum Inward current by PKC stlmulatron observed m this study (see also ([7,8,13]) can be explanted by slowmg down of the actlvatlon process. A parallel slowing of the macroscopic current decay was observed, as IS directly predicted from kmettc theory [14] In accord wrth our findings, a slowing of the sodium current decay has been prchmmarrly reported for

PMA

10

-80

1’

25

first

d.elzsbs

Fig 2

Q

1991

mV

pA

T

60

mV

25 pA , Em a. hg

3 M,wqwtctl

currcntF

~o~lrccut~~cswecpr, dIgItal

4.30 mv

10

ms

b.

-30

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(a) Ongmal tracts bcforc lmd after the dcvclopmcnt of PMA cffec~ at dG’crcnt voltapcs Average current from 10 filtcrmg dt 3 0 kI-I7 (l~)f,,!C rclatmn berorc (open cwh) .md after (filled clrclcr) ttrc dcvclupmsnt of PMA cfkct

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for mtracellular protem kmases), dlfferentlal modulation of sodium channel lsoforms might provide the evolutlonary clue to the existence of at least 5 tissue specific isoforms in one organism [IO, 17-211

20

dcknowledgemenrs We thank Prof R Dunn (Montreal, Canada) for provldmg the pVA2580 and pVA200 constructs and Dr D Platzer (Graz,Austna) for valuable dlscusslons about statlstlcal evaluation of the data SklllFul techmcal assistance and computer programs were provided by B Spreltzer and A Russ Fmanclal support by the Austrlan Resedrch, foundation (project S4505B). the Karl-Franzcns Universlty, Graz (fellowshlp to N D ) and the USA-[srael BSF IS gratcfully acknowledged

[mv]

Fig 4 Voltage dependence of effect on Ip before (open circles) and sftcr the development of PMA effect (filled circles) calculated from 5 different expenments % effect (diamonds) ISp!otted on separate scale Values are presented as mean&SEM

cultured embryonic rat bram neurons [13]. Dlfferenccs to our findings (i.e. lack of voltage dependence, removal of mactlvatlon) might be due to differences m a-subunit and/or PKC lsoforms, higher phor bolester- and/or Na’concentrations in [ 131 The mactlvatlon process was httle or not at all mfluenccd by PI+&4 Indeed, if the prolongation of the macroscopic I\Ja’ current were due to slower mactlvatlon, then modulation affectmg the a-S process (e-subunit mactivatlon) should be most promment at higher membrane potentials and become of minor relevance near threshold potentials, were C-XI and a-% teansltlons dominate the time course of the macroscopic current. However, microscopic inactivation (tranations to the inactivated channel state (I)), as assessed by measuring the dwell times m the open state (&,,Table I), was little 01 not influenced m our experiments Especially at higher potentials (-30 mV and -10 mV), where the inactlvatlon process dominates I, [ 141, 1,‘s were practlsally identical Our findings demonstrate that the effects of PKC on sodium channels expressed from mRNA (total rat brain) and of cRWA (VA200 and IIA ar-subunits, respectively) are clearly different from those by the CAMP-dependent protein kmase, that do not affect the activation process [I 51, suggestmg differential regulation by different sequence-specific kmases. Detailed kinetic analysis of macropatch currents m combination with site-directed mutations will be aecessary to distinguish whether (i) the channel’s gating charge is reduced, (ii) the movement of gating pdrtlcles, i c S4 segments [16] or (iii) conformational changes of the entire a-subunit, accompanying channel dctivatlon, are hmdyred allosterlcally by subunit phosphoryl,ttion As different ar-subunit isoforms differ mostly in their intracellular regions (and hence arc potentially targets

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REFERENCES Ill Nlshlzuka, Y (1984) Nature 308, 693-698 I21Strong, J A , Fox, A P , Taen, R W and Kaczmarek L K (1987) Nature 325, 714-717 [3] Tohse, N , Kamcyama, M , SeBlguchl, K , Shearman, M S and Kanno. M (1990) J Mol Cell Cardlol 22(6), 725-734 [41 McCann. J D and Welsh, M J (1990) Annu Rev Physlol 52, 115-135 151LI, M , McCann, J D , Anderson, M P, Clancy, J P , Lledtke, C M , Nalrn, A C , Greengard, P and Welsh, M J (I 989) Science 244(4910), 1343-1356 161Costa, M R C and Catterall, W A (1984) Ccl1 Mol Neuroblol 9.29 l-297 L71Slgel, E and Baur R (1988) Proc Nat1 Acad SCI USA 85, 6192-6196 [8] Dascal, N and Lotan, 1 (1991) Neuron 6, 165-175 [9] Lotan, I , Dascal, N , Naor, 2 and Boton, R (1990) FEBS Lett 267, 25-20 [IO] Auld. V J. Goldm, A L , Krafte. D 5. Catterall, WA, Lester, H A .and Davidson, N (1990) Proc t&l Acad SCI USA $7, 323-327 III1 HamIll, 0 P , Marty, A , Neher, E , Sakman, B and Slgworth, F J (1981) Pflugers Arch 391, 85-100 1123Kreyszlg, E (1979) Statlstlsche Methoden und lhre Anwendungen, Vandenhoeck & Ruprecht, Gottmgen 1131 Numann, R , Scheuer, T and Catterall, W A (1991) Biophys J 59,262a [I41 Yue. D T , Lawrence. J 1-i and Mdrbdn, E (1989) Sclcnce 244. 349-352 [l51 Gershon, E , Wc~gl, L , Lotan, I , Schrcibmayer, W and Dascal. N (submltted) [l61 Stuchmcr, W , Contl, F , Suzuki, H , Wang, X , Nod& M , Yahag, N , Kubo, I-I dnd Numa, S (1989) Ndture 339, 597-603 [I71 Auld, V J , Goldm, A L. Kraftc, D S , Marshall, J , Dunn, J M , Catterall. WA, Lester, H A, Ddvldson. N and Dunn, R J (1988) Neuron I. 449-461 1181Nodd, M q Ikeda, T , Suznkl. H , Tdkcshlma, H , Takdhdsh!, T , Kuno, M and Numd, S (1986) Ndturc 322.826-828 iI91 Kaydno. T ,Noda. M , Flockerzl, V . Tdkahashr. H dnd Numd, S (1988) FEBS Lett 228, 187-194 [20] Trlmmcr, J S , Coopcrmann, S S , Tomlko, S A , Zhou, J , Crcdn, S M , Boyle. M B , Kdtten, R G , Shcng Z,, Barcht, R L , SIP worth, F J , Goodmann. R H , Agnew, W S and Mandcl, G, Neuron 3, 33-49 [21] Bogart, R B , Cnbbq, L L , Mugl~d, L K, Ksph.lrth, D D md Kaisct , M W (1989) Proc N&l Acnd, SCI USA 86,B 170-8174