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Neuroscience 148 (2007) 164–174

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ENERALIZED EPILEPSY WITH FEBRILE SEIZURESLUS–ASSOCIATED SODIUM CHANNEL �1 SUBUNIT MUTATIONSEVERELY REDUCE BETA SUBUNIT–MEDIATED MODULATION OF

ODIUM CHANNEL FUNCTION

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. XU,a E. A. THOMAS,a E. V. GAZINA,a

. L. RICHARDS,a M. QUICK,a R. H. WALLACE,b,c

. A. HARKIN,b,c,h S. E. HERON,b,h S. F. BERKOVIC,d,e

. E. SCHEFFER,d,e,f J. C. MULLEYb,g,h

ND S. PETROUa,h*

Howard Florey Institute, The University of Melbourne, Parkville, Mel-ourne, Victoria 3010, Australia

Department of Genetic Medicine, Women’s and Children’s Hospital,orth Adelaide, South Australia, Australia

Department of Paediatrics, University of Adelaide, South Australia,ustralia

Department of Medicine (Neurology), The University of Melbourne,ustin Health, Heidelberg, Melbourne, Australia

Department of Paediatrics, The University of Melbourne, Royal Chil-ren’s Hospital, Parkville, Melbourne, Australia

Neurosciences, Monash Medical Centre, Melbourne, Australia

School of Molecular and Biomedical Sciences, University of Adelaide,outh Australia, Australia

Bionomics Ltd., Thebarton, Adelaide, South Australia, Australia

bstract—Two novel mutations (R85C and R85H) on the ex-racellular immunoglobulin-like domain of the sodium chan-el �1 subunit have been identified in individuals from twoamilies with generalized epilepsy with febrile seizures plusGEFS�). The functional consequences of these two muta-ions were determined by co-expression of the human brainaV1.2 � subunit with wild type or mutant �1 subunits inuman embryonic kidney (HEK)-293T cells. Patch clamptudies confirmed the regulatory role of �1 in that relative toaV1.2 alone the NaV1.2��1 currents had right-shifted volt-ge dependence of activation, fast and slow inactivation andeduced use dependence. In addition, the NaV1.2��1 currentntered fast inactivation slightly faster than NaV1.2 channelslone. The �1(R85C) subunit appears to be a complete loss ofunction in that none of the modulating effects of the wildype �1 were observed when it was co-expressed withaV1.2. Interestingly, the �1(R85H) subunit also failed toodulate fast kinetics, however, it shifted the voltage depen-ence of steady state slow inactivation in the same way ashe wild type �1 subunit. Immunohistochemical studies re-ealed cell surface expression of the wild type �1 subunit andndetectable levels of cell surface expression for both mu-ants. The functional studies suggest association of the1(R85H) subunit with the � subunit where its influence is

imited to modulating steady state slow inactivation. In sum-

Corresponding author. Tel: �61-3-8344-1957; fax: �61-3-9347-0446.-mail address: spetrou@unimelb.edu.au (S. Petrou).bbreviations: EGFP, enhanced green fluorescent protein; GEFS�,eneralized epilepsy with febrile seizures plus; HEK, human embry-

anic kidney; PBS, phosphate-buffered saline; SCN1A, sodium channel1 subunit; SCN1B, sodium channel �1 subunit; WT, wild type.

306-4522/07$30.00�0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2007.05.038

164

ary, the mutant �1 subunits essentially fail to modulate �ubunits which could increase neuronal excitability and un-erlie GEFS� pathogenesis. © 2007 IBRO. Published bylsevier Ltd. All rights reserved.

ey words: epilepsy, genetics, sodium channels, electro-hysiology, beta subunit, GEFS�.

eneralized epilepsy with febrile seizures plus (GEFS�) isfamilial epilepsy syndrome characterized by heteroge-

eous clinical phenotypes with the most common subtypeseing febrile seizures and febrile seizures plus (Scheffernd Berkovic, 1997). Inheritance is complex, with some

amilies segregating a major autosomal dominant geneSingh et al., 1999). GEFS� families have been identifiedith mutations in the genes encoding the regulatory so-ium channel �1 subunit (SCN1B), the pore-forming �ubunit (SCN1A) and other ion channels (Wallace et al.,998, 2001, 2002; Baulac et al., 2001; Harkin et al., 2002).ince the first discovery of a missense mutation in theCN1B gene, mutation analysis has revealed an additionalCN1B gene mutation (I70_E74del) on the same extracel-

ular domain which also causes febrile seizures and earlynset absence epilepsy (Audenaert et al., 2003).

Brain sodium channels are complexes of a large, cen-ral pore-forming � subunit of 260 kDa and one or twoomparatively smaller � subunits of 30–40 kDa (Isom andatterall, 1996; Isom, 2001). �1 Subunits are single trans-embrane domain glycoproteins composed of a large N-

erminal extracellular and a short C-terminal intracellularomains. � Subunits are not pore forming but interact with

he � subunit to modulate electrophysiological propertiesQu et al., 1995, 2001). Increases in sodium currents haveeen attributed to � subunit co-expression in some studiesTammaro et al., 2002) but not in others (Meadows et al.,002), although this latter study showed an increase inurface binding. Studies of the �1 subunit have yielded a

arge amount of information about the importance of thextracellular loop (Chen and Cannon, 1995; Makita et al.,996) containing the Cys121 and Arg85 residues implicated

n familial epilepsy. � Subunits also modulate slow inactiva-ion which has been shown to be important in cardiac andkeletal muscle physiology (Vilin et al., 1999). However, the

nformation on the role of the �1 subunit in modulation of slownactivation for brain sodium channels is limited.

The C121W missense mutation in human brainCN1B gene causes GEFS� (Wallace et al., 1998, 2002)

nd has been particularly well studied. This mutation dis-

ved.

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R. Xu et al. / Neuroscience 148 (2007) 164–174 165

upts the putative disulfide bridge of the extracellular loopmplicated in the maintenance of the immunoglobulin-likeold and important for interacting with sodium channel �ubunits (Meadows et al., 2002). When co-expressed withav1.3 in mammalian cells, C121W mutation caused a

ight shift of inactivation compared with the wild type (WT)1, potentially increasing channel excitability (Meadows etl., 2002). When it was co-expressed with skeletal muscleodium channel � subunit, C121W channels recoveredlower from fast inactivation (Tammaro et al., 2002).

Here we report a biophysical characterization of twoovel human mutations (R85C and R85H) in the SCN1Bene recently discovered in two families with GEFS�Scheffer et al., 2007). These mutations are likely to beignificant because R85 is evolutionarily conserved in so-ium channel �1 and �2 subunits and across differentpecies. We expressed NaV1.2 alone or in combinationith WT or mutant �1 subunits in human embryonic kidney

HEK)-293T cells to determine functional consequences.

EXPERIMENTAL PROCEDURES

lasmid pCD8-IRES-h�1 encoding human sodium channel auxiliary1 subunit was kindly provided by Dr. Al George (Vanderbilt Univer-ity, Nashville, TN, USA) (Lossin et al., 2002). Mutations creatingmino acid substitutions in position 85 of �1 subunit (R85C and85H) were introduced into pCD8-IRES-h�1 using the QuikChangeite-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and theollowing primers (mutated codons underlined): for R85C: GGAG-AGGATGAGTGCTTCGAGGGCCG (forward) and CGGCCCTC-AAGCACTCATCCTCCTCC (reverse); for R85H: GGAGGAG-ATGAGCATTTCGAGGGCCGCG (forward) and CGCGGCCCTC-AAATGCTCATCCTCCTCC (reverse). Successful mutagenesisas confirmed by DNA sequencing.

A plasmid encoding human NaV1.2 protein was created asollows. The NaV1.2 coding sequence as amplified from fetal humanrain cDNA library (Clontech, Mountain View, CA, USA) using longange PCR with the primers GGCCGGCCGCGGCCGCAAG-AGCTAAACAGTGATTAAAG and GCGCGTCTAGACCTCCTGT-GGAGTCCT, incorporating one NotI site and one XbaI site, re-pectively (underlined). Long range PCR was performed using theLONGase amplification system (Invitrogen, Carlsbad, CA, USA)ith a final Mg2� concentration of 3 mM. The resulting 6.1 kb PCRroduct was inserted into pcDNA3.1 (�) vector (Invitrogen) usingotI and XbaI restriction sites. The NaV1.2 coding sequence waserified by DNA sequencing.

ell culture and transfection

EK-293T cells (a HEK-293 cell line stably transfected with theV40 large T antigen) were maintained in Dulbecco’s Modifiedagle’s Medium (Invitrogen) supplemented with 10% (v/v) fetalovine serum (Invitrogen) for up to 20 passages. The cells wererown in six-well plates (Nunc, Roskilde, Denmark) to �60%onfluency, and then transfected in each well with 0.5 �g of theaV1.2 plasmid and 0.2 �g of a plasmid pEGFP in each well

Clontech) encoding enhanced green fluorescent protein (EGFP),sing Lipofectamine-2000™ (Invitrogen). For co-transfection ofT �1, �1(R85C) or �1(R85H), 0.5 �g was also used. EGFP was

sed as a marker of successful transfectants. Twenty-four hoursost-transfection, at least 60% of cells exhibited EGFP fluores-ence and cells were then split into Petri dishes. Approximately�l of Dynabeads (M-450 CD8, Dynal, Oslo, Norway) was also

dded into the Petri dish 0.5 h before electrophysiological analy-is. Since �1 subunit was cloned into the plasmid containing the

ell surface marker gene CD8 (pCD8-IRES-h�1), the attachment B

f the Dynabeads on to the cell membrane allows for selection ofhose cells that should also express the �1 subunit. Only cells thatere both EGFP positive and with three or more beads attachedere chosen for study.

lectrophysiological recording

ells placed into 35 mm Petri dishes were allowed to adhere forh prior to being mounted onto the stage of an epi-fluorescence-

quipped inverted microscope (Olympus IX71; Olympus, Tokyo,apan). Bath solution was perfused over the cells using a gravityriven perfusion system for at least 10 min, and then stoppeduring the actual recording. Electrodes were pulled using a Sutter-2000 puller (Sutter Instruments, Novato, CA, USA) from boro-ilicate micropipettes (World Precision Instruments, Sarasota, FL,SA) with an initial resistance of around 2 M�. All recordings wereade using an Axopatch 200B series amplifier (Axon Instruments,oster City, CA, USA) and a Digidata 1322A interface (Axon

nstruments). The bath solution was composed of the followingmM): 145 NaCl, 4 KCl, 1.0 MgCl2, 1.8 CaCl2 and 10 Hepes,djusted to pH 7.4 with NaOH. The osmolarity was adjusted to300 mosmol/L using sucrose and measured with an Osmomat30 (Gonotec, Berlin, Germany). The pipette solution consisted ofmM): 10 NaF, 110 CsF, 20 CsCl, 2 EGTA and 10 Hepes, with pHdjusted to 7.4 using CsOH and osmolarity to �300 mosmol/Lsing sucrose. Pipette and whole cell capacitance were fully com-ensated and the series resistance compensation was set topproximately 80% with the lag set to no greater than 15 �s. Inost cases the residual uncompensated series resistance wouldmount to 1 M� which would give a 4 mV error for a 4 nA current,hich is the extreme of the error we would anticipate. As mosturrent values measured were smaller than this we expect theoltage error to be less than 2 mV. All recordings were made 10in after obtaining the whole cell configuration to allow time foriffusion of pipette solution into the cell to remove contaminating� currents. We also monitored the change in access resistanceontinuously during the experiment. To reduce clamping artifactsaused by large currents we limited our analysis to cells whoseurrents were less than 4.0 nA in magnitude. The only exceptiono this was for the study on the effects of WT and mutant �1ubunits on the NaV1.2 channel expression by comparing theireak sodium currents in cells, as shown in Fig. 1, where weandomly chose eight cells for each group to measure currentensity. All recordings were acquired at 20 kHz with the low passlter set to 10 kHz on the Axopatch 200B amplifier. Clampex 9.0Axon Instruments) was used to run voltage protocols. The calcu-ated liquid junction potential was 5.8 mV and was not compen-ated. No hardware leak subtraction was performed.

All experiments were performed at room temperature (20–2 °C). Data are shown as means�S.E.M. with the number ofxperiments provided in the figures. Leak subtraction was per-ormed in software before the currents were normalized. Statisticalnalysis was performed using Student’s t-test and differencesere considered significant when P�0.05.

The voltage dependence of steady state fast activation wasnalyzed using a step protocol in which cells were depolarized torange of potentials from �100 mV to �50 mV in 5 mV incre-ents from a holding potential of �100 mV. Peak currents at

ndividual steps were measured and normalized to the maximumeak current and plotted against voltage. To calculate a reversalotential, the resulting I–V curve of each data set was individuallytted with the equation I�[1�exp(�0.03937·z·(V�V1/2)]�1·g·V�Vr), where I is the current amplitude, z is the apparent gatingharge, V is the potential of the given pulse, V1/2 is the half-aximal voltage, g is a factor related to the maximum number ofpen channels, and Vr is the reversal potential. Conductance washen calculated directly by using the equation G�I/(V�Vr), where

is conductance. The conductance values were fit with the

oltzmann equation G�1/(1�exp[(V�V1/2)/a]), where a is the

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R. Xu et al. / Neuroscience 148 (2007) 164–174166

lope at half maximum, V is the potential of the given pulse, and

1/2 is the potential for half-maximal activation.The voltage dependence of steady state fast inactivation was

etermined using a two-step protocol composed of a conditioningulse with depolarizing potential from �100 mV to �15 mV in 5V increments for 100 ms from a holding potential of �100 mVnd a test pulse at �5 mV for 20 ms. The peak current amplitudesuring the subsequent test pulses were normalized to the peakurrent amplitude during the first test pulse, plotted against theotential of the conditioning pulse, and fitted with the Boltzmannquation I�1/(1�exp[(V�V1/2)/a]), where I is equal to the testulse current amplitude, V is the potential of the conditioningulse, V1/2 is the voltage for half-maximal inactivation, and a is thelope factor.

Inactivation time constants were also determined usinglampfit 8.0 (Axon Instruments). For each recording, the peak ofurrents at all test potentials was aligned. The Chebyshev searchethod was used to fit each trace with an exponential equation

�A·exp[�(t�K)/�]�C, where I is the current, A is the relativeroportion of current inactivating with the time constant �, K is the

ime shift, and C is the steady-state persistent current.The recovery from inactivation protocol started with a condi-

ioning depolarization from a holding potential of �100 mV to �5V for 50 ms to fully inactivate channels. The voltage was then

eturned to the holding potential of �100 mV for variable intervalsevery 1 ms from 1 to 25 ms; and 5 ms from 25 to 50 ms). Finally,he voltage was stepped to �5 mV for 20 ms to test channelvailability. The peak current amplitude during the test potentialsas plotted as fractional recovery against the recovery period byormalizing to the maximum current during the conditioning po-entials.

Use dependence was determined by stepping to �5 mV from100 mV for 17.5 ms at 39 Hz. The protocol was carried out for.56 s and data were analyzed by a baseline subtraction beforeormalization. Individual peak current amplitudes were normal-

zed to the peak current amplitude during the first depolarization

ig. 1. Sodium currents recorded in HEK-293T cells. (A) RepresenaV1.2��1(R85C) and NaV1.2��1R85H channels using the voltaverage peak current densities for each of the four experimental g

nd plotted against time. 1

The voltage dependence of slow inactivation was determinedsing a two step protocol. The channels were conditioned by 60 sulses from �120–0 mV in steps of 5 mV. They were thentepped back to �120 mV for 20 ms to ensure they had recoveredrom fast inactivation. This was then followed by a test step to �5V for 20 ms to determine availability (Spampanato et al., 2003).ntry into slow inactivation was also measured using a two-steprotocol. Channels were conditioned with variable duration stepso �10 mV, followed by a return to �120 mV to recover from fastnactivation. Finally a step to �5 mV for 20 ms was performed toetermine availability. To determine recovery from slow inactiva-ion, channels were stepped to �10 mV for 60 s. They were theneturned to �120 mV for a variable duration and this was followedy a test step to �5 mV for 20 ms to determine availability. Steadytate voltage dependence of slow inactivation was fitted to aoltzmann with an offset I�(1�I0)/(1�exp[(V�V1/2)/a])�I0.

1 immunofluorescence

o investigate the cellular fate of �1 subunits, HEK 293T cellsere transfected with either WT or mutant �1 subunits and labeledsing anti-�1ex antibody (kindly provided by Dr. L. Isom). At leasthree repeats with duplicates for each group were analyzed in-luding controls. HEK cells were transfected and cultured for 24 hn 0.5% poly-L-lysine-coated coverslips, fixed on ice with 2%araformaldehyde in 0.1 M phosphate-buffered saline (PBS) pH.4, for 20 min then rinsed three times in PBS. The cells were

ncubated overnight at 4 °C in anti-�1ex antibody (1:2000) dilutedn PBS and 10% CAS-block (Invitrogen) for non-permeabilizedells, or permeabilized with 0.3% Triton-X 100. Cells were washedhree times in PBS and incubated with an Alexa 488–conjugatedoat anti-rabbit IgG (1:500) (Molecular Probes, Eugene, OR,SA) for 1 h at room temperature. Cells were rinsed three times

n PBS, and mounted using fluorescent mounting medium (Dako-ytomation, Pty Ltd., Botany, NSW, Australia) and sealed withlear nail varnish. Images were captured using a BioRad MRC

ces obtained from cells expressing NaV1.2 alone, NaV1.2�WT �1,dence of steady state fast activation protocol. (B) Comparison ofown with S.E.M. For each experimental group n�8 cells.

tative tra

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R. Xu et al. / Neuroscience 148 (2007) 164–174 167

Zeiss Axioplan 2 microscope (Carl Zeiss, Oberkochen, Ger-any) and an oil immersion 63�1.4 NA objective. Images werenalyzed using Confocal Assistant 4.02.

RESULTS

85C and R85H do not alter current density

ypical macroscopic currents recorded from HEK-293T cellsxpressing human NaV1.2 alone, NaV1.2 with WT or mutant1 subunits are shown in Fig. 1A. In this experiment, dataere analyzed from cells randomly selected irrespective of

heir maximum current. Simple I–V protocols were run toetermine peak currents and cell capacitances were mea-ured using the Membrane Test mode in Clampex 9.0. Cur-ent densities determined for each of the four groups (NaV1.2lone, NaV1.2��1WT, NaV1.2��1(R85C) and NaV1.2�1(R85C)) and were not significantly different (Fig. 1B).hese results indicate that the WT �1 subunit does not alterurrent density and the mutant �1 subunits do not appear toause detectable changes in trafficking or expression of func-ional channels in HEK-293T cells.

utant �1 subunits fail to modulate ratef fast inactivation

ig. 2 compares the time constant (�) of fast inactivation atrange of voltages in the four experimental groups. The

oltage range in the figure is limited to 10–50 mV tomphasize the range in which there were differences.

ig. 2. Time constant of fast inactivation plotted against voltage. Cur

otentials (as shown in the inset) and a time constant was determined. Statistica

hree groups.

xpression of �1WT produced a small, but statisticallyignificant, decrease in the time constant of fast inactiva-ion compared with the NaV1.2 alone at voltages between0 and 50 mV demonstrating the modulatory function ofhe �1 subunit (there were no differences below 10 mV, dataot shown). The time constant measured for channel cur-ents containing �1(R85C) subunits was similar to NaV1.2lone and significantly different to the �1WT, suggesting thathis mutation cannot modulate the rate of fast inactivation.lthough differences between the �1(R85H) and �1WT mea-urements were not statistically significant, the �1(R85H)ata are similar to the measurements for NaV1.2 alone.

1(R85C) and �1(R85H) fail to modulate steady statectivation and fast inactivation

ormalized conductance and steady state fast inactivationor each of the four groups are plotted as functions ofoltage in Fig. 3. Expression of WT �1 causes a significantightward shift in the normalized conductance curve, sug-esting that WT �1 modulates NaV1.2 channel function byecreasing the number of open channels at a given volt-ge (Fig. 3A). In contrast, both R85C and R85H mutations

n �1 subunits failed to display this modulation of normal-zed conductance, suggesting a loss of this function.

Similarly, WT �1 modulates the voltage dependence ofast inactivation of NaV1.2 as demonstrated by the signif-cant right shift of the inactivation curve (Fig. 3B). However,either �1(R85C) nor �1(R85H) modulated fast inactiva-

tivation for each cell was fit to a single exponential at a range of test

rent inac l comparisons were made between NaV1.2 plus WT �1 and the other

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R. Xu et al. / Neuroscience 148 (2007) 164–174168

ion, again suggesting a loss of function for these muta-

ig. 3. Voltage dependence of normalized peak conductance and steas a function of voltage. (B) Steady state fast inactivation for each of to pooled averages and plotted (see Table 1 for V1/2 values). Statistichree groups with significance as indicated. Protocol schemes are sho

ions. p

The normalized conductance and the fast inactivation data

nactivation. (A) Normalized conductance shown for each of the groupss as a function of voltage. For both graphs Boltzmann curves were fitrisons were made between the NaV1.2�WT �1 group and the othere insets.

dy state ihe groupal compa

oints were also fit to the Boltzmann equation and the V1/2

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R. Xu et al. / Neuroscience 148 (2007) 164–174 169

alues were obtained (Table 1). The V1/2 for NaV1.2 subunitlone is significantly different from the other groups reflecting thebservations made by comparison of the individual data points.

1(R85C) and �1(R85H) fail to modulate recoveryrom fast inactivation

ormalized current as function of time following an inacti-ating voltage step is plotted in Fig. 4 for each of thexperimental groups. Cells expressing NaV1.2 alone re-overed significantly slower than those expressingaV1.2�WT �1 subunits, suggesting that the �1 subunitodulates the rate of recovery from fast inactivation. Theutant channels recovered from fast inactivation similar toaV1.2 alone and did not modulate recovery like the WT1, indicating that the mutations result in �1 subunits thatave lost this modulatory function.

able 1. Biophysical parameters for activation and fast inactivation

hannel type Voltage-dependence of activation

V1/2 (mV) Slope

av1.2 �14.2�1.4* 7.5�0.2av1.2 �1 �10.0�1.0 6.6�0.2av1.2 R85C �14.1�1.5* 6.9�0.4av1.2 R85H �14.2�1.4* 7.5�0.3

Values presented are mean�standard error. Comparison was madStatistical significance marked as P�0.05.

ig. 4. Recovery of channel availability from fast inactivation plotted a

ere made between the WT �1 group and the other three groups with significand axis scale change.

1(R85C) and �1(R85H) fail to modulate useependence

igh frequency voltage steps can be used to predictehavior of Na� channels during rapid action potentialring. Therefore we stimulated cells with 39 Hz pulserains, as this has been shown to reveal differencesetween channel isoforms (Spampanato et al., 2001).ells expressing NaV1.2 alone showed �20% less avail-bility after the first pulse compared with those coex-ressing NaV1.2 and WT �1 subunits (Fig. 5 ). However,oexpression of NaV1.2 and �1(R85C) or �1(R85H) pro-uced currents that responded in a manner similar toaV1.2 alone, suggesting that the mutant channelsave increased use dependence relative to WT �1ubunit.

Voltage-dependence of fast inactivation

V1/2 (mV) Slope n

5 �59�1* �6.8�0.3 146 �53�2 �5.7�0.3 102 �59�1* �6.3�0.3 156 �58�2* �7.1�0.4 14

n Nav1.2��1 and other channels.

n of time for each of the experimental groups. Statistical comparisons

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R. Xu et al. / Neuroscience 148 (2007) 164–174170

1(R85H) modulates voltage dependence of slownactivation while �1(R85C) is without effect

ig. 6A compares the steady state slow inactivation in theour groups and reveals that �1(R85H) modulates voltageependence in a manner similar to WT �1. Interestingly,1(R85C) did not shift voltage dependence away from thateen by expression of NaV1.2 alone. These differenceshere also borne out by the V1/2 values (Table 2). Exam-

nation of the rate of entry into and recovery from slownactivation showed that WT �1 did not modulate eitherf these slow kinetic parameters and not surprisingly thewo mutant �1 subunits were also without effect (Fig. 6Bnd C).

utant �1 subunits fail to reach the membrane andannot be detected intracellularly

ells were transfected with WT or mutant subunit genes,abeled with a �1 antibody and imaged using a confocal

icroscope. Transfection of empty vector alone did noteveal any organized membrane delimited signal (Fig. 7A).

T subunits gave a strong signal that was localized to theell membrane (Fig. 7B). In contrast, we could not detectny cell surface expression of the �1(R85C) or the1(R85H) subunits which gave a background signal similar

o that of empty vector control (Fig. 7C, D).

ummary

ur data demonstrate that the WT �1 subunit modulates

ig. 5. High frequency use dependence. Cells were stimulated with a trotocol scheme is shown in the inset.

aV1.2 by: i) shifting steady state activation (normalized d

onductance) in the positive direction; ii) shifting steadytate fast inactivation in the positive direction; iii) mar-inally speeding entry into fast inactivation at higheroltages; iv) speeding recovery from fast inactivation; v)ecreasing use dependence; and vi) shifting voltageependence of slow inactivation in the positive direction.he R85C mutation provided none of these functions,hile the R85H mutation modulated voltage depen-ence of slow inactivation. vii) Mutant subunits were notetectable using a �1 antibody either at the cell surfacer intracellularly.

DISCUSSION

ue to its wide distribution and high expression in excitableembranes the �1 subunit has been studied extensively.

t regulates a number of sodium channel functions byltering channel gating, surface expression and cell adhe-ion (Isom et al., 1994). Since the SCN1B gene has poly-orphic trinucleotide repeats in its introns an early study

uggested that this gene might be a good candidate forausing hereditary disorders (Makita et al., 1994). Linkagenalysis identified SCN1B as a cause of GEFS� (Wallacet al., 1998) and now four GEFS� mutations have been

dentified (Wallace et al., 2002; Audenaert et al., 2003;cheffer et al., 2007). The clinical profile of individuals with85C and R85H mutations are similar to those carrying the121W mutation, with febrile seizures and febrile seizureslus as the major phenotypes. In the present study, we

ltage steps (39 Hz) and normalized current was plotted for each pulse.

emonstrated that, for the most part, these new mutations

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R. Xu et al. / Neuroscience 148 (2007) 164–174 171

xhibit functional losses in the fast kinetics similar to thosebserved in the C121W mutation. In addition, we foundhat the R85C mutation alters slow inactivation but the85H mutation does not, demonstrating a key role for the

esidue at position 85. The changes caused by mutationst this position are likely to cause hyperexcitability in the

ig. 6. Analysis of slow inactivation. (A) Voltage dependence of steads continuous curves. (B) Entry into slow inactivation. (C) Recoverifferences, where found, are indicated. Note break and axis scale ch

eurons which may, in turn, lead to seizures. c

1 subunit coexpression modulates NaV1.2 function

1 Subunit expression significantly increases sodium cur-ents in Xenopus oocytes (Isom et al., 1992, 1995), how-ver, this effect is not consistently observed in mammalianell lines. For example, �1 subunit co-expression in-

w inactivation (see Table 2 for V1/2 values). Boltzmann fits are shownlow inactivation. Protocol scheme is shown in the inset. StatisticalB and C.

y state sloy from s

reased currents in HEK-293 cells expressing NaV1.4

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R. Xu et al. / Neuroscience 148 (2007) 164–174172

Tammaro et al., 2002) but did not increase currents inhinese hamster ovary or Chinese hamster lung cellsxpressing NaV1.2, even in the presence of a demonstra-le increase in sodium channel binding (Meadows et al.,002). Interestingly, this latter study also showed that over-xpression of �1(C121W) in oocytes actually decreasedodium currents. In our experiments, �1 subunit had noffect on peak current density (Fig. 1).

The �1 subunit right shifted the voltage dependence ofctivation (Fig. 3A), which is consistent with other observa-ions on the effect of �1 on NaV1.2 channels in HEK cells (Qut al., 2001). On its own, this would reduce number of openhannels at any given membrane potential and reduce excit-bility in neurons. The �1 subunit also right shifted the volt-ge dependence of fast inactivation and this is also consis-ent with previous findings (Qu et al., 2001). A right shift in fastnactivation, on its own, would increase excitability. The �1

able 2. Biophysical parameters for slow inactivation

hannel type V1/2 (mV) Slope n

av1.2 �59�3* �6.8�0.3 6av1.2��1 �52�2 �5.8�0.3 9av1.2��1 (R85C) �59�3* �6.4�0.4 9av1.2��1 (R85H) �52�2 �5.3�0.3 7

Significantly different from both Nav1.2��1 and Nav1.2��1 (R85H),�0.05.

ig. 7. Cell surface expression of beta subunits in HEK293 cells. (A) HEK cells1 with clear membrane delimitation. (C, D) Cells expressing �1(R85C) (C) or

ubunit also caused a right shift in voltage dependence of slownactivation (Fig. 6A), which would similarly increase excitability.

�1 Subunit coexpression sped up the rate of entry intoFig. 2) and rate of recovery from fast inactivation (Fig. 4).owever, both these effects were small and, in the case ofntry into fast inactivation, only apparent over a limited volt-ge range. Coexpression of the �1 subunit also caused lessundown in response to high frequency stimulation (Fig. 5).aster recovery and decreased use dependence cause an

ncreased availability of channels. The overall consequencesf these changes are likely be increased excitability during longvents involving several action potentials, such as bursts.

It is intriguing that �1 subunit coexpression decreasedteady state slow inactivation by shifting the voltage sen-itivity by �7 mV. This excitability modulating effect wouldause an increase in available channels and would beonsistent with �1 subunits increasing the excitability ofodium channels. Although it is known that the �1 subunitodulates slow inactivation in heart and skeletal muscle

odium channels (Vilin et al., 1999), this seems to the firstbservation implicating the �1 subunit in slow inactivationf brain sodium channels.

1(R85C) and �1(R85H) subunits do not modulatehe fast kinetics of the � subunit

he R85C mutation failed to produce any of the modula-ory effects of the WT �1 subunit discussed above. The

transfected with an empty vector. (B) Cell surface expression of WT�1(R85H) (D) did not show membrane specific fluorescence.

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R. Xu et al. / Neuroscience 148 (2007) 164–174 173

85H mutation showed essentially the same deficits ex-ept in its effect on the voltage dependence of steady statelow inactivation where it modulated channel functions inhe same way as the WT �1 subunit. As with the other twoEFS� mutations (C121W and I70_E74del) in SCN1Bene, position 85 is in the extracellular domain which isecessary for modulation of the � subunit (Chen and Can-on, 1995; Makita et al., 1996). The loss of function of85C and R85H with respect to fast kinetics is similar to

hat found with the C121W mutation (Meadows et al.,002; Tammaro et al., 2002) suggesting that there may ben important common element for these mutations toause GEFS�. In the case of the R85C mutation, ourxperiments cannot determine the cause of this loss.or example, �1(R85C) may not be properly transcribed orxpressed, trafficked or inserted into the membrane or itay not associate correctly with the NaV1.2 subunit. In the

ase of the R85H mutation, however, the modulating ef-ects of �1(R85H) on slow inactivation are similar to WT �1ubunit, presumably because of association with NaV1.2 onhe cell surface. Thus, the loss of the modulatory effects onast kinetics cannot be due to lack of expression or associa-ion but is likely to be due to a direct consequence of thehange in the residue from arginine to histidine. This sug-ests that the �1(R85C) may associate with NaV1.2 but fail toodulate channel function. This is in contrast to the impair-ent caused by the C121W mutation where a partial rescuef function was seen when �1(C121W) was overexpresseduggesting that the lack of the association caused by theutation was the cause (Meadows et al., 2002).

85 mutations and membrane expression

oth mutations showed altered membrane expression levelsonsistent with the general lack of functional modulation.educed intracellular signal relative to WT may be due to

ncreased degradation of mutant subunits. Neither mutationeems to be acting via a dominant negative effect and theost likely cause of Na� channel dysfunction would be due

o a rapid degradation of the mutant subunits such that theyre unavailable for interaction with the � subunit.

85 mutations and slow inactivation

ur data have demonstrated a mutation specific differencen modulating the voltage dependence of slow inactivation.he R85C mutation fails to produce the �7 mV shift in the

1/2 of slow inactivation produced by the WT �1 subunit orhe R85H mutation. The role of slow inactivation in neuralring is not well understood but may be involved in con-rolling burst duration (Elliott, 1997) or setting basal excit-bility (Ulbricht, 2005). In terms of these two potentialffects on neuronal function, the �7 mV shift, from typicalesting potentials, caused by the WT �1 would be to in-rease basal availability and place the resting membraneotential on a shallower part of the inactivation curve which

n turn would reduce slow inactivation during burst firing.he net effect of the changes caused by WT �1 would be

o increase excitability. It is somewhat puzzling that reduc-ion in cell surface expression of the R85H appears to be as

evere as the R85C yet WT slow inactivation is retained. This 4

ould be explained if very low levels of R85H, below theetection limit of our immunohistochemical method, are suf-cient to interact with the � subunit to modulate slow inacti-ation but insufficient to alter fast kinetics.

85 mutations and epilepsy

t has been shown that the common effect of epilepsy-inked mutations in sodium channels is to cause electro-hysiological changes that may change excitability in theeuronal networks (Spampanato et al., 2004). However,

ncreased excitability is not the only cause of epilepsy asome mutations cause a complete loss of sodium channelunction which can only lead to reduced firing (Rhodes etl., 2005). As reported very recently, a mutation (R859C) inhe sodium channel SCN1A related to GEFS� in factecreases channel excitability (Barela et al., 2006). In thisontext, disinhibition from interneurons may override anyeduced excitability in principal neurons expressing mutanthannels. GEFS� mutations that cause electrophysiolog-

cal changes have been discovered frequently in SCN1And once in a SCN2A (Sugawara et al., 2001; Meisler andearney, 2005; Ito et al., 2006). Although beta subunitsssociate with alpha subunits it is not known which partic-lar alpha subunit partners with beta1 subunits to causeEFS� and more studies are needed to resolve this issue.urthermore, the exact morphological, anatomical or neu-on type distribution of each of the � subunits would alsoontribute to the overall consequences of a given mutation.

What is the overall effect of the R85C mutation, givenhat voltage dependence of activation, fast inactivation andlow inactivation are left shifted compared with WT? Thehift in activation causes increased current at a givenotential and will decrease the membrane potential re-uired to fire an action potential and hence increase excit-bility. The left shifts in the inactivation curves have thepposite effects, decreasing availability and making theeurons less excitable. The overall effect is less clear,owever given that all three activation curves shift in theame direction it is as if the sodium channels are experi-ncing a more depolarized membrane potential and hencere likely to be more excitable. A computer modeling studyonfirmed this (Thomas et al., 2007). The mutations alsoaused small changes in rates of entry into and exit fromast inactivation, however computer modeling showed thathanges in gating rates produce only very small changes

n excitability compared with changes in activation curves.

CONCLUSION

n summary, the R85C and R85H mutations result in a lackf �1 subunit protein such that the excitability modulatingroperties of �1 are absent. Such changes in neuronsould result in an increased excitability that may lead toeizures. A more comprehensive understanding of theathophysiological consequences of these mutations willave to await the creation of mouse models.

cknowledgments—The work was support by a program grant

00121 from Australian National Health and Medical Research

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R. Xu et al. / Neuroscience 148 (2007) 164–174174

ouncil (NHMRC) and Bionomics Limited. R. Xu and E. A.homas are NHMRC Peter Doherty Fellows. The authors arerateful to Prof. Al George for provision of the �1 subunit andr. C. Lossin for helpful discussions.

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(Accepted 30 May 2007)(Available online 12 July 2007)