Supplemental Fig. 1. Primary sequences of Tap1a and Tap2a. (A) Homology comparison between TRTX-Tap1a and TRTX-Tap2a showing 55% identity in their amino acids. Both peptides display conserved domains for peptides belonging to the Family 1 of spider toxins.
Sequence alignment of peptides toxins showing at least 57% identity in their amino acids sequence with TRTX-Tap1a (B) and TRTX-Tap2a (C). The % of identity is shown relative to Tap1a and Tap2a. (D) Sequence alignment of peptides toxins previously characterized in the venoms of spider species evaluated in this work compared with TRTX-Tap1a and TRTX- Tap2a. The % of identity is shown relative to Tap1a. The activity reported for each peptide toxin is described in the far-right column. Source of peptides activity: Arachnoserver database.
TRTX-Tap1a DDCLGMFSSCDPNNDKCCPNRKCSRKDQWCKYQLW 35 100 Nav, CaV3 TRTX-Tb1c DDCLGMFSSCDPNNDKCCPNRVCRVRDQWCKYKLW 35 85 ND TRTX-Tb1b DDCLGMFSSCDPKNDKCCPNRVCRSRDQWCKYKLW 35 83 Kv4.2 TRTX-Tb1a AACLGMFESCDPNNDKCCPNRECNRKHKWCKYKLW 35 82 Kv4.2
B
C TRTX-Tap2a DCLGFMKPCDINNDKCCSSYVCGRNNHWCKFHL 33 100 NaV, CaV3 TRTX-Cm1b DCLGWFKSCDPKNDKCCKNYTCSRRDRWCKYYL 33 57 Nav TRTX-Cm1a DCLGWFKSCDPKNDKCCKNYTCSRRDRWCKYDL 33 57 Nav TRTX-Gr4a DCLGWFKGCDPDNDKCCEGYKCNRRDKWCKYKLW 34 57 KvAP
A TRTX-Tap1a DDCLGMFSSCDPNNDKCCPNRKCSRKDQWCKYQLW 35 100 Nav, CaV3 TRTX-Tap2a -DCLGFMKPCDINNDKCCSSYVCGRNNHWCKFHL 33 51 NaV, CaV3
D TRTX-Tap1a DDCLGMFSSCDPNNDKCCPNRK--CSRKDQWCKYQLW 35 100 Nav, CaV3 TRTX-Tap2a DCLGFMKPCDINNDKCCSSYV--CGRNNHWCKFHL 33 51 NaV, CaV3 TRTX-Pn3a DCRYMFGDCEKDED-CCKHLG--CKRKMKYCAWDFTFT 35 32 NaV TRTX-Hd1a ACLGFGKSCNPSNDQCCKSSSLACSTKHKWCKYEL 35 49 NaV TRTX-Ec2a YCQKFLWTCDTER-KCCEDMV--CEL---WCKLEK 29 29 BKCa TRTX-Ec2b YCQKFLWTCDTER-KCCEDMV--CEL---WCKYKE 29 31 BKCa TRTX-Ec2c YCQFKMWTCDSER-KCCEDMV--CRL---WCKLNL 29 31 BKCa TRTX-Df1a ECRWFLGGCSGGQT-CCEHLV--CHRKHQWCVWDWSF 34 28 Nav, CaV3 GTx-1-15 DCLGFMRKCIPDNDKCCRPNLV-CSRTHKWCQYVF 34 53 Nav, CaV3 ProTx-I ECRYWLGGCSAGQT-CCKHLV--CSRRHGWCVWDGTFS 35 24 Nav, CaV3, KV ProTx-II YCQKWMWTCDSERK-CCEGMV--CRL---WCKKKLW 30 34 Nav, CaV3 ProTx-III DCLKFGWKCNPRNDKCCSGLK--CGSNHNWCKLHI 33 43 NaV TRTX-Mb1a GVDKPGCRYMFGGCVQDDD-CCPHLG--CKRKGLYCAWDGT 38 31 Nav, CaV TRTX-Mb1b GVDKPGCRYMFGGCVQDDD-CCPHLG--CKRKGLYCAWDAS 38 31 Nav, CaV Cm2a GVDKEGCRKLLGGCTIDDD-CCPHLG--CNKKYWHCGWDGTF 39 23 Nav
Toxin name Sequence Identity Activity (%)Loop1 Loop2 Loop3 Loop4
Loop1 Loop2 Loop3 Loop4
Supplemental Fig. 2. The three-dimensional structures of Tap1a and Tap2a were calculated using the NMR structure of β-theraphotoxin-Ps1a (PaurTx3, PDB code 5WE3) and µ- theraphotoxin-Hhn1b (Hainantoxin-IV, PDB code 1RYV) as templates, respectively. (A) The cartoon models showed conserved inhibitory cysteine knot (ICK) structure with two antiparallel β-sheets and three Cys-bridges coloured in yellow. Loops 1 to 4 are coloured in blue, orange, green and red, respectively. (B) Surface representation of Tap1a and Tap2a structures with 180° rotation shown in blue: negatively charged, red: positively charged and yellow: hydrophobic residues. (C) Highlighted in blue (Tap1a) and red (Tap2a) are the differences in amino acids residues between these toxins. These residues are M6F, F7M, S8K, S9P, P12I, P19S, N20S, R21Y, K22V, S24G, K26N, D27N, Q28H, Y32F and Q33H for Tap1a compared to Tap2a, and D1 and W35 present only in Tap1a. Structures are shown in two orientations, rotated by 180°.
A Tap1a B C
Tap2a
Tap1a Tap1a
Tap2a
Tap2a
N C
N C
N
N
C
C N C
N C
Supplemental Fig. 3. Recombinant production of TRTX-Tap1a (rTap1a) and TRTX-Tap2a (rTap2a). (A) A synthetic optimized gene coding for the peptide Tap1a or Tap2a was transformed in BL21(DE3) cell line and expression induced with IPTG. After semi-purification of the recombinant fusion protein 6xHis-MBP-peptide (Line 1), the 6xHis-MBP tag was removed using TEV protease cleavage (Line 2). (B) RP-HPLC purification of cleaved rTap1a or rTap2a was performed in Vydac218TP C18 using a two-step gradient of acetonitrile/0.05%
trifluoroacetic acid (5-50% B/45 min and 50-80% B/8 min; dashed line). Mass spectrometry analysis of recombinant Tap1a and Tap12a peaks (orange filled area) showing masses 4237 Da and 3901 Da, respectively (red traces). This mass agrees with native peptide mass plus one additional N-terminal glycine remaining from the TEV protease cleavage site.
20 25 30 35
0 500 1000 1500
Retention time (min)
Absorbance (214nm)
20 25 30 35
0 500 1000 1500
Retention time (min)
Absorbance (214nm)
MBP-Toxin MBP rTap1a rTap2a
1 2 1 2
rTap1a
A B rTap2a
3901 Da
4237 Da
Supplemental Fig. 4. Representative current traces of NaV1.1-1.7 (A-G) in the absence (black dashed traces) and presence of rTap1a and rTap2a (green and orange traces). Concentrations used are described. Both rTap1a and rTap2a simultaneously slowed the fast inactivation and reduced the peak current of the NaV1.3 subtype, and induce only reduction in the peak current for the subtypes NaV1.1, NaV1.2, NaV1.6 and NaV1.7. Weak or absent inhibitory activity were observed for the subtypes NaV1.4 and NaV1.5.
B
rTap1a
111 nM 1000 nM Saline
rTap2a 333 nM 1000 nM Saline NaV1.2
A
C
D
E
F
G
rTap1a
333 nM 1000 nM Saline
rTap2a
333 nM 1000nM Saline NaV1.1
rTap1a 111 nM 1000 nM Saline
rTap2a
111 nM 1000 nM Saline NaV1.3
rTap2a 1000 nM Saline rTap1a
1000 nMSaline NaV1.4
rTap1a 1000 nM Saline
rTap2a 1000 nM Saline NaV1.5
rTap1a Saline 111 nM 1000 nM
Saline 167 nM 500 nM rTap2a NaV1.6
Saline 111 nM 1000 nM rTap1a
1000 nM Saline 111 nM rTap2a NaV1.7
Supplemental Fig. 5. Representative current traces of CaV3.1–3.3 (A-C) in the absence (black dashed traces) and presence of rTap1a and rTap2a (green and orange traces). Concentrations of rTap1a and rTap2a assayed are described. The IC50 values calculated are described in Table 1.
rTap2a
333 nM 9000 nM Saline
CaV3.3 CaV3.2
CaV3.1 rTap1a
111 nM Saline 1000 nM
rTap1a
9000 nM Saline
rTap2a
9000 nM Saline
rTap2a
9000 nM Saline rTap1a
3000 nM 9000 nM Saline
A
B
C
Supplemental Fig. 6. Tap1a does not alter compliance during graded bladder distension. The pressure/volume relationship during graded bladder distension (compliance) is unchanged with intravesical instillation of Tap1a (ns; P≥0.05, two-way ANOVA. N=6 mice).
0 8 16 24 32 40
0 100 200 300 400
Pressure mmHg
Volume (µl)
Control 1µM Tap1a 10µM Tap1a
Peptide Ion channel
ΔV50 I-V (mV) Mean ± SEM N
ΔV50 SSI (mV) Mean ± SEM N
Tap1a hNaV1.1 -2.3 ± 2 3 -28.2 ± 3.5 3
hNaV1.6 -5 ± 1.5 5 -7 ± 2 4
hNaV1.7 -1.4 ± 0.5 3 -11 ± 2.8 3
hCaV3.1 -4 ± 1.4 4 -2 ± 2 3
hCaV3.2 -1.8 ± 0.7 5 -5 ± 1.4 5
Tap2a hNaV1.1 -4.3 ± 1.3 4 -10.3± 1.8 6
hNaV1.6 -6 ± 1.6 5 -7.2 ± 1.4 5
hNaV1.7 -1.2 ± 2 3 -11.5 ± 2.8 3
hCaV3.1 -0.5 ± 1.3 6 3.2 ± 1 3
Supplemental Table 1. rTap1a and rTap2a behaved as gating modifiers. The voltage- dependence of activation and steady-state inactivation for hNaV1.1, hNav1.6 and hNaV1.7, and CaV3.1 and CaV3.2 were recorded via automated patch-clamp electrophysiology of cell lines expressing NaV and CaV3 channels subtypes. Na+ and Ca2+ currents were measured before and after addition of rTap1a or rTap2a, and the calculated ΔV50 are indicated for each NaV and CaV3 subtype. Data are Mean ± SEM from three to six independent experiments.
