Localization of Receptor Site on Insect Sodium Channel for Depressant β-toxin BmK IT2

Background BmK IT2 is regarded as a receptor site-4 modulator of sodium channels with depressant insect toxicity. It also displays anti-nociceptive and anti-convulsant activities in rat models. In this study, the potency and efficacy of BmK IT2 were for the first time assessed and compared among four sodium channel isoforms expressed in Xenopus oocytes. Combined with molecular approach, the receptor site of BmK IT2 was further localized. Principal Findings 2 µM BmK IT2 strongly shifted the activation of DmNav1, the sodium channel from Drosophila, to more hyperpolarized potentials; whereas it hardly affected the gating properties of rNav1.2, rNav1.3 and mNav1.6, three mammalian central neuronal sodium channel subtypes. (1) Mutations of Glu896, Leu899, Gly904 in extracellular loop Domain II S3–S4 of DmNav1 abolished the functional action of BmK IT2. (2) BmK IT2-preference for DmNav1 could be conferred by Domain III. Analysis of subsequent DmNav1 mutants highlighted the residues in Domain III pore loop, esp. Ile1529 was critical for recognition and binding of BmK IT2. Conclusions/Significance In this study, BmK IT2 displayed total insect-selectivity. Two binding regions, comprising domains II and III of DmNav1, play separated but indispensable roles in the interaction with BmK IT2. The insensitivity of Nav1.2, Nav1.3 and Nav1.6 to BmK IT2 suggests other isoforms or mechanism might be involved in the suppressive activity of BmK IT2 in rat pathological models.


Introduction
Voltage-gated sodium channels (VGSC) are key membrane proteins responsible for neuron excitability, consisting of an ionconducting a-subunit accompanied by one or more auxiliary subunits [1]. Generally, the a-subunit comprising four repeated domains (DI-DIV), each containing six transmembrane a-helixes (S1-S6) and a hairpin-like pore loop between S5 and S6 [2], split into an N-terminal part (SS1) and a C-terminal part (SS2). Despite the high structure similarity, various VGSC subtypes display distinct distribution, gating properties and function activities. Some neurotoxins can differentiate among them with preference for certain subtype(s) [3], thus providing clues about the structurefuction relationship of VGSCs and a potential molecule library for novel drug design or insecticide development.
Amongst the neurotoxins purified from scorpions, b-toxins shift the voltage dependence of VGSC activation to cause subthreshold channel opening, which can be enhanced when channels are preactivated by a depolarizing prepulse [4]. According to the phyletic-bioactivity, the b-toxins may be further divided into: bmammal toxins, depressant or excitatory insect-specific b-toxins and TsVII-like toxins acting on both mammals and insects [3].
The group of b-toxins is deemed to bind to a common receptor site-4 on VGSC a-subunits, which, however, shows a rather complex picture. The binding sites for b-mammal toxin CssIV (from Centruroides suffusus suffusus) and TsVII-like toxin Tz1 (from Tityus zulianus) have been mapped to DII S1-S2, DII S3-S4 and DIII SS2-S6 on mammalian VGSCs [4,5,6,7]. The effect of TsVII (i.e. Tsc from Tityus serrulatus) on reducing peak currents are also conferred by the S4 segments of DIII and DIV [8,9]. The excitatory and depressant b-toxins act distinctly, though they both target insect VGSCs [10,11]. DII of DmNa v 1 is implicated in the selective recognition of excitatory toxin AahIT (from Androctonus australis hector) [12], while several channel regions (DI S5-SS1, DI SS2-S6, DIII SS2-S6, and DIV SS2-S6) may be involved in the interacion with depressant toxin LqhIT2 (from Leriurus quinquestriatus hebraeus) [11]. Based on the results of mutation experiments applied on rat VGSCs and information provided by structure analysis of LqhIT2 [13,14], some possible interaction spots in DII S3-S4 of DmNa v 1 were deduced. However, no site-directed mutagenesis has been performed on insect VGSC yet as to dissect the receptor site for depressant b-toxins.
BmK IT2, a depressant b-toxin from the scorpion Buthus martensi Karsch, can induce strong insect toxicity [15]. Like other depressant toxins, such as LqhIT2 [11,16], BmK IT2 possesses two non-interacting binding sites (the high/low-affinity binding sites) on insect nerve membranes [17,18]. Despite typical antiinsect features of depressant b-toxins, BmK IT2 displayed antinociceptive and anticonvulsant activities in rat models [19], which were attributed to the specific modulation on brain VGSCs [20]. Such effects against mammals have also been observed in other depressant b-toxins [21,22,23] and explained as a consequence of adaptive evolution of these toxins. However, the binding affinity of BmK IT2 to rat brain synaptosomes was quite low [17,18] and the specific target is still unidentified.
To forward the understanding for the binding features of depressant b-toxins and their intriguing functional diversity, in the present study, we attempted to address the following issues: 1) Can BmK IT2 modulate the mammalian VGSC subtypes from central neuronal system (i.e. Na v 1.2, Na v 1.3 and Na v 1.6)? 2) What is the selectivity of BmK IT2 between these mammal subtypes and insect VGSC DmNa v 1? 3) What is the binding/recognition site on insect VGSC for BmK IT2?

Materials
BmK IT2 was purified by column chromatography from the crude venom of the Asian scorpion Buthus martensi Karsch as described previously [15]. The purity of the toxin was confirmed by mass spectrometry.
The genes encoding the sodium channel a-subunit DmNa v 1 (P35500.3) from Drosophila paralytic temperature-sensitive and the auxiliary TipE subunit were kindly provided by J. Warmke (Merck, New Jersey, USA) and M. S. Williamson (IACR-Rothamsted, UK), respectively. Plasmids in combination with cDNAs of rat/mouse VGSC a-isoforms i.e. rNa v 1.2 (CAA27287), rNa v 1.3 (CAA68735) and mNa v 1.6 (Q9WTU3.1), as well as b1 subunit were originally from Dr. Alan L. Goldin (University of California, USA).  Table S1). All clones were verified by DNA sequencing according to their wild-type sequences (See Figure S1). Plasmid DNAs were harvested and isolated from XL1-blue E. coli (Stratagene, USA).

Voltage-gated sodium channel expression and Electrophysiological studies
Mammalian VGSCs rNa v 1.2a, rNa v 1.3a and mNa v 1.6a were expressed in Xenopus oocytes accompanied with auxiliary subunit b1 while the insect VGSC DmNa v 1 was coexpressed with TipE for generating robust Na + currents.
Two-electrode voltage-clamp recordings were performed at room temperature (18u-22uC) using the TURBO TEC-03X amplifier (npi Instruments, Germany) and Cellwork E5.5 software (npi electronic Instruments). Voltage and currents electrodes were filled with 3 M KCl. Currents were filtered at 1.3 kHz and sampled at 10 kHz with a four-pole Bessel filter. Bath solution composition was (in mM): NaCl 96, KCl 2, CaCl 2 1.8, MgCl 2 2 and HEPES 5 (pH 7.4). Toxin BmK IT2 were diluted with bath solution and applied directly to the bath at desired concentration.
From a holding potential of 2100 mV, oocytes were depolarized with a three-step protocol [25]. The first and last test depolarization of 25 ms duration ranged from 270 mV to +70 mV in steps of 10 mV. The second depolarization (PP) to a voltage of 210 mV was used to prime the channels ensuring maximal binding of the b-toxin to the channel. The third segment of 25 ms at 2120 mV ensured recovery from inactivation. Repetition interval was 2 seconds. The peak currents elicited in the test depolarizations were plotted as a function of voltage, resulting in current/conductance-voltage relationships (I/G-V curves). This approach provided an assessment of the BmK IT2 effect on channel activation with and without a depolarizing prepulse (PP) in one experiment.

Data analysis
Data were acquired by Cellworks Reader 3.6 (NPI electronic Instruments) and analyzed with Origin 7.5 (Northampton, USA) software.
Only recordings with leakage below 0.10 mA and fluctuation within 0.05 mA were selected in statistical analysis. The results are shown as means 6 SEM with the number of experiments provided as n in the talble legends.
Mean conductance (G) was calculated from peak current/ voltage relations using the equation G = I/(V2V rev ), where I is the peak current elicited upon depolarization, V is the membrane potential, and V rev is the reversal potential. The voltage dependence for the activation of I was fit with the Boltzmann relation, G/G max = 1/[1+exp(V 1/2 2V)/k m ], where V 1/2 is the voltage for half-maximum activation and k m is the slope factor. The EC 50 values were determined by measuring the currents induced by BmK IT2 at the voltage of channel activation threshold (240 mV).

Results
Efficacy of BmK IT2 on VGSC isoforms from insect and mammalian central neuronal system Using the two-electrode voltage clamp recording, BmK IT2 was subjected to a comparative study for the effects on four VGSC subtypes, rNa v 1.2/b1, rNa v 1.3/b1, mNa v 1.6/b1 and DmNa v 1/ TipE expressed in Xenopus oocytes (Fig. 1). The voltage-dependent channel activation was investigated by a three-step protocol (see Materials and Methods). 2 mM BmK IT2 induced significant subthreshold currents (at 250 mV) in DmNa v 1/TipE channels with a depolarizing prepulse (PP) of 25 ms (Fig. 1A). The halfmaximal activation voltage (V 1/2 ) of DmNa v 1/TipE was shifted by about 211 mV and the slope factor (k m ) was increased from 3.72 to 7.97 mV (p,0.001, n = 10) by 2 mM BmK IT2 (EC 50 = 2.960.36 mM, Table 1). This shift was also observed only in the presence of a prepulse ( Fig. 1B-C). In contrast, rNa v 1.2/b1, rNa v 1.3/b1, mNa v 1.6/b1 were totally insensitive to BmK IT2 at concentrations of 2 mM ( Fig. 1A and B) and even up to 20 mM (Fig. S2). Prolonging the PP duration to 50 ms was unable to enhance the efficacy of BmK IT2 (data not shown). Though the activation of rNa v 1.3/b1 eventually responded to BmK IT2 at a rather high concentration (50 mM; DV 1/2 = 24.84 mV, data not shown), rNa v 1.2/b1 and mNa v 1.6/b1 still remained insensitive. Whether or not b1 subunit was coexpressed with these mammalian VGSC subtypes did not influence the action of BmK IT2 (not shown).
On all investigated VGSC subtypes, a small depression of current amplitude was observed (,10% for mammalian VGSCs and ,20% for DmNa v 1/TipE, Fig. 1C) after application of BmK IT2. There were no significant BmK IT2-induced changes in inactivation process of channels (data not shown). The results suggest that BmK IT2 exhibited distinguished subtype selectivity on sodium channels, preferring the insect target rather than mammalian central neuronal isoforms.

Mutations in DII S3-S4 impacted BmK IT2 function on insect VGSC
Previous reports demonstrated that substitutions introduced to DII (e.g. E 779 Q in DII S1-S2, and E 837 Q, L 840 C, G 845 N in DII S3-S4 of rNa v 1.2a; G 658 N in DII S3-S4 of rNa v 1.4) reduced the effects of the b-toxins Css4 and Tz1 [4,5,6,7]. As for the case of depressant toxin, structural bioinformatics analysis deduced three analogous residues in DmNa v 1 (E 896 , L 899 and G 904 ) might also be crucial in the interaction with LqhIT2 [13,14]. Based on these studies and considering the high homology between LqhIT2 and BmK IT2, mutations of D 838 , E 896 , L 899 and G 904 (corresponding to E 779 , E 837 , L 840 and G 845 in rNa v 1.2, Fig. 2A), were individually introduced into DmNa v 1.
The mutants were co-expressed with TipE subunit ensuring the functional expression and currents were recorded in the same condition as that of wild type DmNa v 1. The gating property of all mutants was not altered with respect to those of wild-type channels, thus the subsequent electrophysiological analysis was not ''contaminated'' by mutagenesis. The normalized conductancevoltage relationship of mutants were assessed in the absence and presence of 2 mM BmK IT2 with a 25 ms-PP. Mutant D 838 C showed the similar response to BmK IT2 as wild type DmNa v 1 (Fig. 2B), whereas the mutations of Glu 896 , Leu 899 and Gly 904 totally abolished negative shift of voltage-dependent activation induced by 2 mM BmK IT2 (DV 1/2 ,2.0 mV, Dk m ,1.0 mV, n = 7 or 8, Fig. 2C-E, Table 2). Besides, the mutants DmE 896 C, DmL 899 C and DmG 904 N were also resistant to BmK IT2 at higher concentrations (Talbe 1). This result verified that residues E 896 , L 899 and G 904 in DII S3-S4 of DmNa v 1 play critical roles in responding to BmK IT2.
DIII from DmNa v 1 conferred BmK IT2 sensitivity to rNa v 1.2 Although E 896 , L 899 and G 904 positively support the action of BmK IT2, sequence alignments ( Fig. 2A) indicate these residues are also conserved in corresponding positions of all BmK IT2insensitive mammalian VGSCs investigated. It appears that they are necessary, but not sufficient to fulfill the interaction with BmK IT2, suggesting additional channel region(s) might be involved.
To find out the region(s) responsible for BmK IT2 recognition, four chimeras (ChD1, ChD2, ChD3 and ChD4; Fig. 3A  the channel activities were not impaired by cross-species domain substitution. Like rNa v 1.2a,most chimeric channels were regulated by mammalian b1 subunit but not TipE from insect (data not shown). The only exception was ChD4 that seemed insensitive to either b1 or TipE. The activation of chimeras ChD1, ChD2 and ChD4 were hardly modified by 2 mM BmK IT2 (Fig. 3B), like wild type Na v 1.2a/b. In contrast, ChD3 gained the response to 2 mM BmK IT2, which caused a statistically significant shift of channel activation (DV 1/2 = 25.64 mV, p,0.005, n = 10) (Fig. 3B, Table 2). The increased sensitivity in ChD3 (EC 50 = 22.566.65 nM, Table 1) also suggested DIII seemed to play a necessary role in the interaction between insect sodium channel and BmK IT2.
Residues in DIII SS2-S6 critical for the sensitivity of DmNa v 1 to BmK IT2 To further clarify the possible interaction site in DIII, a series of mutations have been perfomed in DIII SS2-S6 pore loops of rNa v 1.2a and DmNa v 1. The mutagenesis design was based on the previous report that suggested DIII SS2-S6 might be involved in the binding of LqhIT2 [11]. First, to verify whether this region accounted for BmK IT2 binding, the DIII SS2 loops were compared (Fig. 4A) and exchanged between DmNa v 1 and rNa v 1.2 (Fig. 4B), giving rise to two loop chimeras: L(Dm)Na v 1.2 and L(1.2)DmNa v 1. Unexpectedly, the whole loop replacement in DmNa v 1 (I 1512 to I 1534 ) by that of rNa v 1.2 (M 1425 to L 1447 ) resulted in channels hardly expressed in Xenopus oocytes even accompanied by TipE subunit. Thus for generating robust Na + currents, two residues in rNa v 1.2-type loop had to be restored as present in DmNa v 1 (I 1529 /R 1530 ) (See Material and Methods). Double mutant DmI 1529 K/R 1530 Y was then produced as the compensation of the incomplete loop substitution.
Similar to the case of chimera ChD3, in the presence of 2 mM BmK IT2 and a 25 ms prepulse, the voltage-dependent activation of L(Dm)Na v 1.2 displayed a mild but significant shift with DV 1/2 of about 25 mV (p,0.05, n = 8, Fig. 4C and Table 2). As for L(1.2)DmNa v 1 (Fig. 4D), the substitution by most part of the DIII SS2 loop from rNa v 1.2 could not prevent BmK IT2-induced shift in the voltage of half-maximal activation (DV 1/2 = 212.48 mV). Interestingly, however, unlike wild type DmNa v 1, the slope factor of its activation curve was barely affected by BmK IT2 (L(1.2)DmNa v 1: Dk m ,1 mV, n = 8; DmNa v 1: Dk m = +4.25 mV, n = 10; Table 2). It was noticeable that double-mutant DmI 1529 K/ R 1530 Y exhibited largely attenuated sensitivity to 2 mM BmK IT2 as the toxin-induced DV 1/2 decreased to 25.06 mV with the slope factor (k m ) unchanged ( Table 2). The results indicats that the DIII SS2-S6 pore-loop of DmNa v 1 plays a major role in BmK IT2 interaction and it was the main contributor in conferring BmK IT2 sensitivity to rNa v 1.2.
To determine the residue(s) in this region critical for the interaction with BmK IT2, a series of site-directed mutations of DmNa v 1 were produced (see Materials and Methods). All mutants displayed gating parameters (Table 2) similar to those of wild type DmNa v 1, ruling out the possibility that the alteration of gating behavior was involved in variation of BmK IT2 sensitivity. Subsequent analysis demonstrated that among all the mutants (Fig. 5)

VGSC subtype-selectivity of BmK IT2
BmK IT2 was classified into the group of b-depressant insect toxin because: 1) it shares high sequence similarity with other welldefined depressant anti-insect toxins, such as LqhIT2, LqqIT2 and BjIT2 [26]; 2) BmK IT2 is toxic to insect but not mammals [27,28]. This insect-selectivity was also observed in binding experiments tested on cockroach nerve cords which displayed a 200-300 fold higher affinity with BmK IT2 than rat brain synaptosomes [17]. However, like some other depressant b-toxins [21,22,23], BmK IT2 also evolves function against mammals, e.g. antinociceptive and anticonvulsant activities in rat models [19]. As recent studies have mostly focused on the pharmacological phenotype of BmK IT2, the underlying mechanism and molecular target in rat brain remain unintelligible. In this study, the efficacay and selectivity of BmK IT2 was assayed for the first time among independently cloned VGSCs from insect (DmNa v 1) and mammalian central nervous system (i.e. rNa v 1.2, rNa v 1.3 and mNa v 1.6) expressed in Xenopus oocytes.
Results showed that the main effects of BmK IT2 on DmNa v 1 included a decrease of peak Na + current (by ,20%) and a significant hyperpolarizing shift of the activation. These are typical effects for scorpion depressant b-toxins. The increase of the slope value of activation curve, reflecting the decreased voltage dependence of activation process and a larger subthreshold channel open probability, is also observed in previous reports The values of half-maximum activation voltage V 1/2 and corresponding slope factor (k m ) were determined in the absence and presence of 2 mM BmK IT2. Application of BmK IT2 shifted channel activation by DV 1/2 . The data were represented as the mean 6 SEM and n is the number of independent experiments. doi:10.1371/journal.pone.0014510.t002 characterizing the function of depressant b-toxins LqqIT2 and LqhIT2 [29,30]. In contrast, three mammalian VGSCs were totally insensitive to BmK IT2. The low affinity of BmK IT2 to rat brain synaptosomes can be explained by the insensitivities of Na v 1.2 and Na v 1.6,which are dominant VGSC subtypes spreading throughout CNS [31,32], to BmK IT2. It is noteworthy that BmK IT2 was capable of inhibiting the total Na + currents in rat dorsal root ganglion (DRG) neurons [20]. According to our results that Na v 1.2, Na v 1.3 and Na v 1.6 are BmK IT2-insensitive, the action of BmK IT2 on Na + currents of DRG neurons may be a result of selective modulation on other neuronal VGSC subtypes, most likely Na v 1.7, Na v 1.8 and/or Na v 1.9 channels. Thus, it may allow us to speculate that peripheral nerve VGSC subtypes might be the major targets responsible for BmK IT2-induced anti-nociception and anticonvulsant effects in rat models, though the subtypes or other membrane proteins that are possibly involved in the working mechanism of BmK IT2 still need to be further characterized.

Construction of insect-mammalian chimeric channels
Since the insect and mammalian VGSCs are highly similar in both structural and functional properties, insect-mammalian chimeras could be constructed to determine the regions responsible for the toxin recognition and interaction. Previously for localizing the insect VGSC domain that binds b-excitatory toxin AahIT, a chimeric channel was constructed from rat brain rNa v 1.2 in which DII was replaced by that of Drosophila [12]. Here we also chose rNa v 1.2 as backbone of chimeric channels that accepted insect VGSC domains considering that: 1) rNa v 1.2 channel is insensitive to BmK IT2 at very high concentration (e.g. 20 mM); 2) as a typical VGSC subtype from mammalian nervous system, rNa v 1.2 channel has been well characterized in Xenopus oocytes and displays an excellent performance in expression level. The four resulting insect-mammalian chimeras were all expressed functionally and identified to be TTX-sensitive VGSCs. Chimeric channels could be regulated by b1 subunit except ChD4 that seemed insensitive to either b1 or TipE subunit. The low current density of ChD4 was improved only by prolonging the expression time duration. These results agreed with the finding that the binding site for b1 was localized to DIV in rNa v 1.2 [33] and implicated that TipE might not regulate DmNa v 1 through DIV.
To directly reveal the BmK IT2 binding region(s) in DmNa v 1 channel and confirm the results obtained in Na v 1.2 backbone chimeras, we also attempted to generate the mammalian-insect chimeras in which the independent domains of DmNa v 1 were replaced by those of rNa v 1.2. Unfortunately, due to the rather low expression level, these chimeras failed to serve as satisfying candidates for the subsequent pharmacological analysis.
The binding feature of BmK IT2 on DmNa v 1 The classical voltage-sensor trapping model indicates that btoxins function as a stablizer of activated state of VGSCs by trapping the outward DII S4 and hereby shift the activation threshold to more hyperpolarized potentials [4].
In this study, mutations of G 904 , E 896 , L 899 in DII S4 of DmNa v 1 completely abolished the action of BmK IT2, suggesting that, like other b-toxins (e.g. CssIV and Cn2), BmK IT2 functionally interacts with DmNa v 1 through DII S4 as described in the voltage-sensor trapping model (Fig. 6). However, these residues could not serve as a major determinant to BmK IT2 sensitivity as they are well conserved in the BmK IT2-insensitive mammalian channels like rNa v 1.2, rNa v 1.3 and mNa v 1.6. The subsequent study revealed that DIII rather than DII could confer BmK IT2 insect-preference to mammalian sodium channel. The channel epitope that interacts with BmK IT2 was further narrowed down to residues around the N-part of DIII SS2-S6 loop (I 1512 /Q 1513 /N 1516 /D 1517 /I 1519 ) as well as the hydrophobic I 1529 and the positive R 1530 , implying the hydrophobic and electrostatic interactions may both be decisive for toxin binding. Although the residue alterations at positions 1512-1526 and at position 1530 had minor impact on toxin efficacy, the exchange of hydrophobic Ile at position 1529 in DmNa v 1 to the Lys present in rNa v 1.2 largely impaired the toxin-channel interaction. Thus the central role of I 1529 seemed to support the hydrophobic interaction in toxin-channel inter-recognition (Fig. 6).
Apparently, the receptor site for BmK IT2 involves at least two channel regions: 1) DII S3-S4 linker, for mediating toxin functional interaction with voltage-sensor; 2) DIII SS2 loop: the determinant for BmK IT2 specific targeting.This is different from the case for excitatory b-toxin: the receptor site for AahIT was found to reside mainly in DmNa v 1 DII [12]. Our result confirms that the receptor sites for excitatory and depressant b-toxins are not identical on insect VGSC [10,34], however, they have an overlapping region, i.e. DII. That is in concordance with the fact that excitatory toxins can compete with depressant toxins for the high-affinity binding site on insect nerve membrane [11,34].
Interestingly, despite targeting VGSCs from different phyla, the binding features of BmK IT2 and Tz1, a b-like toxin that can strongly affect the activation of muscular Na v channel but was incapable of affecting the activation of cardiac and peripheral nerve Na v channels [5], appear very similar: toxins recognize and bind to the pore loop of DIII and then are capable of trapping the outward movement of voltage-sensor in DII, thus lowering the threshold for channel activation.

Conclusion
The insect-selectivity of BmK IT2 was highlighted in this study when differentiating between heterologously expressed VGSC subtypes from insect and mammalian central nervous system. The results suggested Na v 1.2, Na v 1.3, and Na v 1.6 channels were not involved in mediating the BmK IT2-induced antinociceptive and anticonvulsant effect in rat models. The study revealed the receptor site on insect VGSC DmNa v 1 for depressant b-toxin BmK IT2 consisted of at least two regions, i.e. DII and DIII. The recognition epitope for insect-preference were localized to the hydrophobic residues within DIII pore-loop SS2-S6. Finally, the inter-species chimeric channels employed here may provide a promising operation for identifying putative binding site(s) in VGSCs targeted by other specific modulators. Figure S1 Sequences of the DmNa v 1 mutants indicating the mutated residues in DII and DIII. The loop chimera L(Dm)Na v 1.2 was produced by replacing the diversed residues within DIII SS2-S6 loop of rNa v 1.2 by those from DmNa v 1 (underlined residues) correspondingly. In addition, single-or multiple-mutagenesis were also employed on DmNav1, giving rise to the loop-chimera or mutants listed below. Black dots in loop-chimera/mutants indicated the unchanged residues compared to the sequence of L(Dm)Na v 1.2 (or DmNa v 1).