Báo cáo khoa học: Novel a-conotoxins from Conus spurius and the a-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors doc

Novel a-conotoxins from Conus spurius and the
a-conotoxin EI share high-affinity potentiation and
low-affinity inhibition of nicotinic acetylcholine receptors
Estuardo Lo
´
pez-Vera
1,
*
,
†, Manuel B. Aguilar
1,
*, Emanuele Schiavon
2
, Chiara Marinzi
2
,
Ernesto Ortiz
3
, Rita Restano Cassulini
2
, Cesar V. F. Batista
3
, Lourival D. Possani
3
,
Edgar P. Heimer de la Cotera
1
, Francesco Peri
2
, Baltazar Becerril
3

and Enzo Wanke
2
1 Laboratorio de Neurofarmacologı
´
a Marina, Departamento de Neurobiologı
´
a Celular y Molecular, Instituto de Neurobiologı
´
a, Universidad
Nacional Auto
´
noma de Me
´
xico, Campus Juriquilla, Queretaro, Me
´
xico
2 Dipartimento di Biotecnologie e Bioscienze, Universita
`
di Milano-Bicocca, Milan, Italy
3 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologı
´
a, Universidad Nacional Auto
´
noma de Me
´
xico, Cuernavaca,
Me
´
xico
Conotoxins are small, disulfide-rich peptides that

have been isolated from Conus, a large genus of
predatory marine snails. The primary structures of
more than 100 conotoxins have been determined and
classified into gene superfamilies on the basis of the
amino acid sequences of the signal peptides of their
Keywords
a-conotoxin; conotoxins; Conus spurius;
nicotinic receptor; potentiation
Correspondence
E. Wanke, Dipartimento di Biotecnologie e
Bioscienze, Universita
`
di Milano-Bicocca,
Piazza della Scienza, 2U3, 20126 Milan, Italy
Fax: +39 02 64483314
Tel: +39 02 64483303
E-mail:
*These authors contributed equally to this
work

Present address
Instituto de Ciencias del Mar y Limnologı
´
a,
Universidad Nacional Auto
´
noma de Mexico,
Mexico
(Received 4 April 2007, revised 3 June
2007, accepted 11 June 2007)

doi:10.1111/j.1742-4658.2007.05931.x
a-Conotoxins from marine snails are known to be selective and potent
competitive antagonists of nicotinic acetylcholine receptors. Here we des-
cribe the purification, structural features and activity of two novel toxins,
SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Chan-
nel, Mexico. As determined by direct amino acid and cDNA nucleotide
sequencing, the toxins are peptides containing 18 amino acid residues with
the typical 4 ⁄ 7-type framework but with completely novel sequences.
Therefore, their actions (and that of a synthetic analog, [c15E]SrIB) were
compared to those exerted by the a4 ⁄ 7-conotoxin EI from Conus ermineus,
used as a control. Their target specificity was evaluated by the patch-clamp
technique in mammalian cells expressing a
1
b
1
cd, a
4
b
2
and a
3
b
4
nicotinic
acetylcholine receptors. At high concentrations (10 lm), the peptides SrIA,
SrIB and [c15E]SrIB showed weak blocking effects only on a
4
b
2
and

a
1
b
1
cd subtypes, but EI also strongly blocked a
3
b
4
receptors. In contrast
to this blocking effect, the new peptides and EI showed a remarkable
potentiation of a
1
b
1
cd and a
4
b
2
nicotinic acetylcholine receptors if briefly
(2–15 s) applied at concentrations several orders of magnitude lower (EC
50
,
1.78 and 0.37 nm, respectively). These results suggest not only that the
novel a-conotoxins and EI can operate as nicotinic acetylcholine receptor
inhibitors, but also that they bind both a
1
b
1
cd and a
4

b
2
nicotinic acetyl-
choline receptors with very high affinity and increase their intrinsic cho-
linergic response. Their unique properties make them excellent tools for
studying the toxin–receptor interaction, as well as models with which to
design highly specific therapeutic drugs.
Abbreviations
a
1
b
1
cd, muscular nicotinic acetylcholine receptor; a
3
b
4
, peripheral nervous system nicotinic acetylcholine receptor; a
4
b
2
, central nervous
system nicotinic acetylcholine receptor; Acm, S-acetamidomethyl; ACN, acetonitrile; [c15E]SrIB, synthetic a-conotoxin from Conus spurius;
nAChR, nicotinic acetylcholine receptor; PTH, phenylthiohydantoin; SrIA, a-conotoxin IA from Conus spurius; SrIB, a-conotoxin IB from
Conus spurius.
3972 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS
precursors. In general, the members of each super-
family have a characteristic arrangement of their cys-
teine residues and a particular connectivity of their
disulfide bridges. Each gene superfamily comprises
one or more pharmacologic families: the O super-

family, containing x-conotoxins, j-conotoxins,
d-conotoxins, and lO-conotoxins; the M superfamily,
containing l-conotoxins, w-conotoxins, and jM-cono-
toxins; the S superfamily, containing r-conotoxins
and aS-conotoxins; the T superfamily, containing
e-conotoxins and v-conotoxins; the P superfamily,
containing the spasmodic peptides; the I superfamily,
containing several jI-conotoxins, and the A super-
family, containing a-conotoxins, aA-conotoxins and
jA-conotoxins [1].
Competitive antagonists of the nicotinic acetylcholine
receptors (nAChRs) belong to the a and aA families.
On the basis of the number of residues between the sec-
ond and third cysteines and on the spacing between the
third and fourth cysteines in the mature a-conotoxins,
these peptides have been divided into three groups: the
a4 ⁄ 7 subfamily, the a3 ⁄ 5 subfamily, and a heterogene-
ous group including peptides that do not belong to the
two previous groups. These groups have different
degrees of antagonistic effect on distinct nAChRs: a3 ⁄ 5
toxins block mostly muscular nicotinic acetylcholine
receptors a
1
b
1
cd subtypes, whereas a4 ⁄ 7 peptides, with
one exception, block neuronal subtypes [2].
In this article, we describe the purification, amino
acid sequence determination and cloning of the cDNA
encoding two novel peptides, SrIA and SrIB, found in

the venom of Conus spurius. The pattern and the spa-
cing of their cysteines indicate that they belong to the
a4 ⁄ 7 subfamily of conotoxins [3]. We also describe a
third peptide, [c15E]SrIB, synthesized by substituting
glutamate for the c-carboxyglutamate residue and used
for comparison together with the a-EI conotoxin
from Conus ermineus. We showed that results with
[c15E]SrIB were not significantly different from those
seen with the natural compounds, and then, owing to
the limited amounts of the natural toxins SrIA and
SrIB, used mainly this synthetic peptide for long-dur-
ation electrophysiologic tests.
The discovery of new agonists or antagonists is of
the utmost importance to widen the understanding of
alternative functions of nAChRs, which play a crucial
role in cellular and molecular mechanisms underlying
brain function.
Results
Purification of SrIA and SrIB
Fractionation of C. spurius venom by HPLC, as des-
cribed in Experimental procedures, gave the profile
shown in Fig. 1A. The fractions indicated as SrIA and
SrIB were repurified by RP-HPLC, yielding the two
pure peptides SrIA and SrIB (Fig. 1B,C), named follow-
ing the nomenclature proposed by Olivera & Cruz [1].
Amino acid sequences and cDNA cloning
Automated Edman sequencing of the native peptides
SrIA and SrIB unambiguously defined 12 and 13 resi-
dues, respectively. Low glutamine signals at positions
12 and 15 of SrIA and at position 15 of SrIB

Fig. 1. Purification of SrIA and SrIB. (A)
Fractionation of the crude venom by means
of an analytical RP C18 HPLC column. Pep-
tides were eluted using a linear gradient of
5–95% solution B (dashed line) at a flow
rate of 1 mLÆmin
)1
for 90 min. Eluents
were: 0.1% v ⁄ v trifluoroacetic acid in water
(solution A), and 0.09% v ⁄ v trifluoroacetic
acid in 90% v ⁄ v ACN (solution B). (B, C)
Fractions indicated in (A) as SrIA and SrIB
were repurified using a gradient of 15–30%
buffer B (dashed line), at a flow rate of
1mLÆmin
)1
for 45 min.
E. Lo
´
pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs
FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3973
suggested the presence of c-carboxyglutamate residues
at these positions. Residues 3, 4, 9 and 17 of both pep-
tides were tentatively assigned as cysteine (Table 1), on
the basis of the absence of any amino acid signal at
these positions. This assumption was confirmed
directly by the experiments used to determine disulfide
bridges (see below). We obtained positive results
with PCR amplification of a-conotoxin-type cDNA,
reverse transcribed from C. spurius venom duct

total mRNA. Two primers known to match the con-
served signal peptide-coding region and the 3¢-UTR of
the a-conotoxin family, respectively [4], were success-
fully employed. Exactly the same sequence was
obtained from several colonies, which, together with
the demonstrated conservation of the signal and pro-
peptide regions, indicated that the amplification proto-
col was reliable. The deduced SrIA ⁄ SrIB precursor
sequence agreed with the results of direct peptide
sequencing and MS data (see below), and allowed us
to define the final unambiguous primary structure for
the mature toxins (Fig. 2). From the precursor
sequence, and on the basis of earlier observations by
our group with toxic peptides [5], we were also able to
predict the amidation of the C-terminal end of the
mature toxins. The primary structures of SrIA and
SrIB resemble those of previously isolated a-conoto-
xins with the cysteine framework 4 ⁄ 7 (Table 2).
MS
The chemical monoisotopic molecular masses of pep-
tides SrIA and SrIB determined by ESI MS are
2202.9 Da and 2158.8 Da, respectively (Table 1). The
agreement with the calculated masses (assuming two
disulfide bridges and an amidated C-terminus for each
peptide, plus one and two c-carboxyglutamate residues
for SrIB and SrIA, respectively) supports the Edman
sequence assignment for each peptide. The tentative
assignments of amidated C-termini, based on the struc-
ture of the precursor (see ‘cDNA cloning’), were con-
firmed by the ESI MS data.

Determination of disulfide bridges
Two major and more than 20 minor absorbing peaks
were observed during the chromatography of peptide
SrIA after partial reduction with Tris(2-carboxyethyl)
phosphine hydrochloride and alkylation with N-ethyl-
maleimide (Fig. 3). This high number of derivatives of
peptides alkylated with N-ethylmaleimide has been
observed in several studies [6], and it is thought to
reflect diastereoisomers resulting from the introduction
of a new chiral center in the maleimide ring after for-
mation of the S–C bond during alkylation. Another
factor that could generate additional derivatives is the
opening of the ring of the N-ethylsuccinimidocysteines
by hydrolysis [7]. Selected peptides were sequenced to
reveal the positions of the alkylated cysteines. The phe-
nylthiohydantoin (PTH) derivative of N-ethylsuccini-
midocysteine elutes between PTH-Pro and PTH-Met
in the HPLC system of the sequencer employed. The
presence of alkylated cysteines at positions 4 and 17 in
some peptides, and at positions 3 and 9 in other pep-
tides, clearly indicated that the connectivity of the two
disulfide bridges in peptide SrIA is of the type I–III,
II–IV. The absence of peptides with labeled cysteines
at positions 3 and 17 or 4 and 9 gives additional sup-
port to the proposed disulfide connectivity.
The synthetic peptide [c15E]SrIB
It has been reported recently that the c-carboxygluta-
mic residues present in toxin peptides may be
involved in the folding process but are not relevant
for their biological activity [8]. Starting from this

hypothesis, a peptide sequence was designed that was
analogous to those found for SrIA and SrIB, but
bearing glutamic acid residues in place of the c-carbo-
Table 1. Amino acid sequences and monoisotopic molecular mas-
ses of the peptides from C. spurius and of synthetic peptides
[c15E]SrIB and EI.
Peptide Sequence
Experimental
mass (Da)
Calculated
mass (Da)
SrIA RTCCSROTCRMcYPcLCG
a
2202.9 2202.8
SrIB RTCCSROTCRMEYPcLCG
a
2158.8 2158.8
[c15E]SrIB RTCCSROTCRMEYPELCG
a
2114.8 2115.0
EI RDOCCYHPTCNMSNPQIC
a
2075.4 2075.8
a
Amidated C-terminus; O, hydroxyproline; c, c-carboxyglutamate.
Fig. 2. The cloned cDNA sequence and the deduced amino acid sequence of the SrIA ⁄ SrIB conotoxin precursor. The residues present in the
mature toxins are underlined.
a-Conotoxins with potentiating effects on nAChRs E. Lo
´
pez-Vera et al.

3974 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS
xyglutamic residues at positions 12 and 15 (Table 1).
Testing the biological properties of such a peptide,
prepared by chemical synthesis and thus with a fully
defined chemical structure (including disulfide pat-
tern), would support the amino acid sequence and
folding of the native peptides proposed above, and
additional tests would not be limited by the availabil-
ity of the peptide, as might occur with the natural
toxins SrIA and SrIB. To obtain the desired folding
pattern (see Experimental procedures), we protected
the cysteine side chains with two orthogonal protect-
ing groups that can be removed selectively under
different conditions, allowing the formation of one
disulfide bridge at a time. For this purpose, Cys3 and
Cys9 were introduced as S-trityl-protected amino
acids, whereas S-(acetamidomethyl)cysteine was used
for positions 4 and 17. At the end of chain assembly
on the solid support, achieved using standard 2-(1-H-
benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexa-
fluorophosphate activation protocols for Fmoc
chemistry as previously described [9,10], the peptide
resin was treated with trifluoroacetic acid for cleavage
from the solid support and side chain deprotection,
with simultaneous liberation of the two thiol groups
in positions 3 and 9. The first disulfide bond was
then formed by air oxidation. Finally, the bis-aceta-
midomethyl-peptide generated was treated with
iodine, which caused removal of the protecting group
and simultaneous oxidation to disulfide, yielding the

fully folded sequence.
Physiologic effects of natural conotoxins and
their synthetic analogs
In order to explore the physiologic role of the novel
SrIA and SrIB conotoxins, we performed a series of
patch-clamp experiments on single cells from the line
TE671, which expresses the human muscle receptor
[11], and HEK293 lines stably transfected with the
human central nervous system nicotinic acetylcholine
a
4
b
2
and peripheral nervous system nicotinic acetyl-
choline a
3
b
4
receptor subtypes. As our present perfu-
sion system is not sufficiently fast to resolve fast
desensitizing currents such as those produced by a
7
re-
ceptors, we decided not to test our peptides on these
receptors, to avoid reporting putatively invalid data.
The experiments were done by voltage-clamping the
cells at ) 60 mV and comparing the responses to brief
Table 2. Amino acid sequence of SrIA, SrIB and [c15E]SrIB, com-
pared with some members of the a3 ⁄ 5, a4 ⁄ 3 and a4 ⁄ 7 subfamilies
[16,24,48].

Peptide Amino acid sequence Target
SI
ICCNPACGPKYSC
a
a
1
b
1
cd
SIA
YCCHPACGKNFDC
a
a
1
b
1
cd
GI
ECCNPACGRHYSC
a
a
1
b
1
cd
GIA
ECCNPACGRHYSCGK
a
a
1

b
1
cd
GII
ECCHPACGKHFSC
a
a
1
b
1
cd
MI
GRCCHPACGKNYSC
a
a
1
b
1
cd
CnIA
GRCCHPACGKYYSC
a
a
1
b
1
cd >> a
7
ImII ACCSDRRCR-WRC
a

a
7
, a
1
b
1
> a
3
b
2
AnIB GGCCSHPACAANNQDYC
a
a
3
b
2
>> a
7
PnIA GCCSLPPCAANNPDYC
a
a
3
b
2
>> a
7
PnIB GCCSLPPCALSNPDYC
a
a
7

> a
3
b
2
EpI GCCSDPRCNMNNPDYC
a
a
3
b
4
, a
3
b
2
; a
7
AuIA GCCSYPPCFATNSDYC
a
a
3
b
4
Vc1.1 GCCSDPRCNYDHPEIC
a
a
3
a
7
b
4

, a
3
a
5
b
4
Vc1a GCCSDORCNYDHPc IC
a
PeIA GCCSHPACSVNHPELC
a
a
9
a
10
, a
3
b
2
> a
3
b
4
> a
7
PIA RDPCCSNPVCTVHNPQIC
a
a
6
⁄ a
3

b
2
b
3
> a
6
⁄ a
3
b
4
>
a
6
b
4
, a
3
b
2
GIC GCCSHPACAGNNQHIC
a
a
3
b
2
>> a
4
b
2
, a

3
b
4
MII GCCSNPVCHLEHSNLC
a
a
3
b
2
>> a
7
> a
4
b
2
, a
3
b
4
GID IR GcCCSNPACRVNNOHVC a
3
⁄ b
2
, a
7
> a
4
⁄ b
2
EI RDOCCYHPTCNMSNPQIC

a
a
1
b
1
cd, a
3
b
4
, a
4
b
2
SrIA RTCCSROTCRMc YPcLCG
a
a
4
b
2
, a
1
b
1
cd
SrIB
RTCCSROTCRMEYPcLCG
a
a
4
b

2
, a
1
b
1
cd
[c15E]SrIB
RTCCSROTCRMEYPELCG
a
a
4
b
2
, a
1
b
1
cd
a
Amidated C-terminus; O, hydroxyproline; c,c-carboxyglutamate;
Y, sulfated tyrosine.
Fig. 3. Determination of the disulfide bridges of peptide SrIA. Deriv-
atives of peptide SrIA formed by partial reduction and alkylation
under acidic conditions were separated using two analytical RP C18
HPLC columns. Peptides were eluted using a linear gradient
(dashed line) of 10–30% solution B at a flow rate of 1 mLÆmin
)1
for
120 min. Eluents were: 0.1% v ⁄ v trifluoroacetic acid in water (solu-
tion A), and 0.09% v ⁄ v trifluoroacetic acid in 90% v ⁄ v ACN (solu-

tion B). Selected peptides were sequenced, and the positions at
which cysteines labeled with N-ethylmaleimide were observed are
displayed in the corresponding diagrams. The deduced connectivity
of the two disulfide bonds is indicated in the upper right inset.
E. Lo
´
pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs
FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3975
applications of 50 lm nicotine with those obtained
immediately after pretreatment with the different tox-
ins. The concentration of nicotine used during the
experiments was fixed at 50 lm, because this value is
well below the saturating region of the dose–response
curve for the a
1
b
1
cd receptor, as shown in Fig. 4A,
and also for the a
4
b
2
and a
3
b
4
receptors [12–14].
The pretreatment time and the concentration of each
toxin were varied in the range 3–150 s and 0.2 nm to
10 lm, respectively. A typical experiment performed on

a TE671 cell with a-conotoxin [c15E]SrIB at 1 lm is
shown in Fig. 4B. As indicated, the first 50 lm nicotine
control pulse produced a response that was strongly
reduced after 180 s of toxin perfusion. After 4 min of
washout, the application of an additional nicotine pulse
produced a recovery that was complete. As the amount
of purified toxins was limited, we did the majority of
the experiments with the synthetic toxin [c15E]SrIB and
used a known conotoxin [15], such as EI a-conotoxin,
as a control. In the case of the inhibitory effects des-
cribed in Fig. 4, the results obtained using natural or
synthetic peptides, at the same concentration, were not
A
BCE
D
Fig. 4. Blocking properties of a-conotoxins on different types of receptors. (A) Dose–response curve obtained with nicotine in TE671 cells.
The continuous line is the Boltzmann curve that best fits the data with the following parameters: an IC
50
of 99 ± 12 lM, and a Hill coefficient
of 1.98 ± 0.14 (n ¼ 12). The inset shows a representative example of the recorded currents in a single cell. (B) Inward currents recorded in
a TE671 cell during successive 50 l
M nicotine (nic) test pulses. The first and the last pulse are control and washout, respectively; the second
pulse was preceded by an 180 s pretreatment with a-conotoxin [c15E]SrIB (1 l
M). (C) Fractional blockade, at fixed toxin concentration
(10 l
M for 180 s), on the different subtypes of nAChR. *Statistically different at P < 0.05 as compared to a
4
b
2
; the numbers of experiments

are given in parentheses. (D) Normalized time course of the blockade, at 10 l
M EI, of the nicotinic response as a function of the toxin pre-
treatment time. Continuous curves are exponentials that best fit the data points with the following time constants: a
1
b
1
cd (open squares),
4.9 ± 0.25 s (n ¼ 5); a
3
b
4
(gray squares), 11 ± 1.9 s (n ¼ 5). Insets: superimposed traces of the nicotine responses obtained in a typical
TE671 cell and in an a
3
b
4
-expressing cell during control and toxin perfusion. Left inset: the traces show the block at 30 s and the recovery
after 40 s. Right inset: the traces show the block at 5 s and the recovery after 20 s. Scale bars: 2 s, 200 pA. (E) Fractional response data
obtained with a-conotoxin [c15E]SrIB and EI. The curves are best fitted with the following IC
50
and Hill coefficient: for [c15E]SrIB,
46 ± 10 n
M, 1 ± 0.1, and for EI, 187 ± 43 nM, 0.48 ± 0.06, respectively. The number of experiments for each point ranged from three to 12.
a-Conotoxins with potentiating effects on nAChRs E. Lo
´
pez-Vera et al.
3976 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS
significantly different, and only the data obtained with
the synthetic toxin are displayed.
Inhibitory actions

Figure 4C summarizes the data obtained at high toxin
concentrations (10 lm). It can be seen that the frac-
tional blockade obtained is both receptor-dependent
and toxin-dependent. [c15E]SrIB was ineffective on
a
3
b
4
receptors (n ¼ 4), and was a slightly better blocker
of the a
4
b
2
receptors (0.56 ± 0.04, n ¼ 7) than of
the a
1
b
1
cd receptors (0.39 ± 0.06, n ¼ 5, not statisti-
cally significant). On the other hand, EI toxin was
able to potently block muscle (0.95 ± 0.01, n ¼ 5) and
ganglionic (0.91 ± 0.03, n ¼ 4) receptors, but was less
potent for the central nevous system receptor
(0.61 ± 0.02, n ¼ 4). These data not only confirm that
EI is an inhibitor of muscle receptors [15], but they
also show that it is a strong inhibitor of the a
3
b
4
receptors and a relatively weak antagonist of the a

4
b
2
central nervous system receptors, on which it had
never been tested before.
To further investigate these new EI data, we also
performed a series of kinetic experiments at a concen-
tration of 10 lm (see Fig. 4D). EI toxin blocked the
a
1
b
1
cd receptors with a s
on
of 4.9 ± 0.25 s (n ¼ 3),
and the a
3
b
4
receptor was blocked with a s
on
of
11 ± 1.9 s (n ¼ 3). Moreover, the s
off
values that we
observed for these receptors were 150 ± 13 s (n ¼ 4)
and 122 ± 6.5 s, respectively. Figure 4E shows the
dose–response curve for the [c15E]SrIB and EI a-cono-
toxins on the a
1

b
1
cd receptors. The estimated IC
50
and Hill coefficient obtained from these data are:
46 ± 10 nm and 1 ± 0.1 for [c15E]SrIB, and
187 ± 43 nm and 0.48 ± 0.06 for EI, respectively.
Because, at a toxin concentration [T], a simple
Clark’s model receptor theory predicts s
on
¼ s
off
⁄
(1 + [T] ⁄ K
D
), this relationship can be used to confirm
the previous IC
50
of Martinez et al. [15] on a
1
b
1
cd
receptors, which was 280 nm (low-affinity site) for the
mouse receptors, and to predict the unknown and
novel value of K
D
for the a
3
b

4
receptors. Indeed, we
found an IC
50
value of 187 nm for a
1
b
1
cd recep-
tors (Fig. 4E), which also agrees with the fractional
response of 0.04 at 10 lm EI and a s
off
of 150 s. For
the a
3
b
4
receptors, the above relationship results in a
K
D
value of about 1 lm.
On the whole, these experiments, designed to study
the antagonistic properties of the toxin [c15E]SrIB,
showed a narrower spectrum of specificity for nAChRs
than that of the EI a-conotoxin, owing to the null
effect of [c15E]SrIB on the a
3
b
4
subtype. In contrast,

EI was found to be a broad-spectrum a-conotoxin.
Potentiating effects
During the experiments designed to study inhibitory
actions of the two new peptides SrIA and SrIB, we dis-
covered that brief applications, at low toxin concentra-
tions, resulted in increased responses that were
immediately reversed after washout of the toxin. A
typical experiment performed on an a
1
b
1
cd-expressing
cell with different concentrations of a-conotoxin
[c15E]SrIB is shown in Fig. 5A. It can be seen that the
first and the last brief control pulses of 50 lm nicotine
produced very similar inward currents. However, if
15 s pretreatments with toxin were immediately fol-
lowed by the same brief nicotine pulses, currents
increased, and then decreased as a function of the drug
concentration.
In order to shed light on this novel action of the
a-conotoxins, we started to investigate whether the
various peptides exerted different levels of potentiation
on the same a
1
b
1
cd receptor. To clarify whether this
novel mechanism was peculiar to the new conotoxins
or common also to other, already known, conotoxins,

we chose the EI a-conotoxin, which is considered to be
an inhibitory conotoxin [15].
At a fixed toxin concentration of 10 nm, the relative
potentiation, (I
toxin
) I
control
) ⁄ I
control
, of the synthetic
[c15E]SrIB, the natural SrIB and SrIA peptides, and
the EI a-conotoxin, were as follows: 0.46 ± 0.09 (n ¼
10), 0.47 ± 0.08 (n ¼ 10), 0.44 ± 0.15 (n ¼ 9), and
0.54 ± 0.13 (n ¼ 9), respectively. These results suggest
that, at least for the a
1
b
1
cd receptor type and during
brief periods of time (15 s), pretreatment with a con-
centration of 10 nm toxin shows no clear differences
among these peptides. As the amount of natural toxin
available for experimentation is limited, and as no
significant differences were found when using the
synthetic peptide as compared to the native peptides,
we continued our assays using the two synthetically
prepared products, namely [c15E]SrIB and EI. The
results of these preliminary experiments, obtained only
with very low toxin concentrations (0.2 nm to 1 lm)
and brief time intervals, do not conflict with those

mentioned in Fig. 4, which were obtained with very
long pretreatments.
To investigate these mechanisms, the toxins were
studied in cells expressing various receptor types.
Unexpectedly, we discovered that their effects were
also receptor-dependent. To clarify the receptor specif-
icity, we used the two toxins ([c15E]SrIB and EI) on
three different receptors, namely a
1
b
1
cd, a
4
b
2
, and
a
3
b
4
, and the maximally observed relative potentiation
values are shown in Fig. 5B. Interestingly, whereas the
toxins were unable to produce potentiation in the
E. Lo
´
pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs
FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3977
ganglionic a
3
b

4
receptor (n ¼ 17), the mean fractional
potentiation in a
1
b
1
cd receptors for [c15E]SrIB
(0.75 ± 0.22, n ¼ 7) was higher than that obtained for
EI (0.35 ± 0.07, n ¼ 22, statistically different). The
effects of both toxins were found to be similar on the
a
4
b
2
receptor subtype.
Furthermore, we investigated the dose–response
curves of the maximal fractional potentiation produced
by the [c15E]SrIB and EI conotoxins on the a
1
b
1
cd
receptors. These data are shown in Fig. 5C, and were
fitted to dose–response curves with EC
50
values of
1.78 ± 1.9 and 0.37 ± 0.23 nm, for [c15E]SrIB and
EI, respectively. An example of this type of action
(15 s toxin pretreatment) is shown in Fig. 5D, in one
example of an a

1
b
1
cd-expressing cell, with both toxins
at two different concentrations (10 and 100 nm). In
this experiment, the two toxins were delivered alter-
nately to gain insight into the differences between their
sensitivities.
The kinetics of the development of the potentiated
response were very fast at concentrations higher than
2–5 nm, and it was almost impossible to determine its
time course, given that the rate of bath exchange
was < 1 s. However, by reducing the toxin concentra-
tion to 0.2 nm, we were able to follow, as a function of
the duration of the toxin perfusion, not only the expo-
nential increase in potentiation, but also the decay of
the potentiation response, up to the appearance of the
blockade. Indeed, if the pretreatment of the toxin las-
ted for more than 10–15 s, it was possible to observe
ABC
DEF
Fig. 5. Potentiation effects of a-conotoxins on different types of receptor. (A) Inward currents recorded in a TE671 cell during successive
50 l
M nicotine test pulses. The first and the last pulse are controls; the second, third, fourth and fifth pulses were each preceded by a 15 s
pretreatment with different concentrations of the a-conotoxin [c15E]SrIB. (B) Maximal relative potentiation [(I
tox
) I
control
) ⁄ I
control

] for different
receptor types ([c15E]SrIB, line pattern; EI, gray pattern). The maximal concentration used was 100 n
M, and pretreatment lasted for 15 s.
The number of experiments is shown in parentheses on the bars. *Statistically different at P < 0.05 as compared to the EI effect. (C) Dose–
response relationships for potentiation, observed in a
1
b
1
cd receptors, for a-conotoxins [c15E]SrIB (open squares), and EI (gray squares). Con-
tinuous lines are dose–response curves fitting the experimental data with the following values of IC
50
(nM) (maximal): for [c15E]SrIB,
1.78 ± 1.9, 0.93 ± 0.11; for EI, 0.37 ± 0.23 n
M, 0.46 ± 0.1. Each point represents a variable number of experiments from three to 11. (D) In
the same cell, the two toxins were applied alternately, each for 15 s pretreatment intervals at different concentrations as indicated. (E, F)
The potentiation ⁄ blockade (open squares) kinetics on a
1
b
1
cd receptors, for [c15E]SrIB (E) at 0.2 nM and EI (F) at 0.2 and 1 nM. Continuous
curves are exponentials with the following time constants: [c15E]SrIB, s
on
7.07 ± 0.1.1, s
off
31 ± 2.3 s; EI, s
on
(0.2 nM) 6.03 ± 0.32, s
off
(0.2 nM) 16.4 ± 1.3 s; s
off

(1 nM) 9.4 ± 1.5 s. See text.
a-Conotoxins with potentiating effects on nAChRs E. Lo
´
pez-Vera et al.
3978 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS
an exponentially decaying depotentiation process. We
show two examples obtained by using the two different
toxins on the a
1
b
1
cd receptor. Figure 5E,F shows the
potentiation ⁄ blockade (I
toxin
⁄ I
control
) data versus dur-
ation of toxin pretreatment obtained from experiments
done at 0.2 nm [c15E]SrIB or 0.2 and 1 nm EI, respect-
ively (n ¼ 3). Note the different time scales in Fig. 5E
and Fig. 5F. Potentiation data at 1 nm are not shown
for [c15E]SrIB, because they were too fast to be
resolved. On the contrary, data at 1 nm for EI,
although fast (but not fitted to exponentials), are
shown because they illustrate the interesting depotenti-
ation with a time constant different from that observed
at 0.2 nm. From these experiments, it can be seen that
both the development of potentiation and the depoten-
tiation or block are dependent on the toxin type and
concentration. These data suggest a very complex

mechanism of toxin–receptor interaction that warrants
additional study. Unfortunately, this was beyond the
scope of this study.
On the whole, these results suggest that the potentia-
tion described here could be a property of different clas-
ses of a-conotoxins. On the other hand, we do not
exclude the possibility that this effect could be confined
to the conotoxins that act on both neuronal and muscu-
lar receptor subtypes, as those used in this work are the
only ones reported to be active on both targets. On
the a
1
b
1
cd receptor, the synthetic toxin [c15E]SrIB was
less potent than EI, but the latter was less efficient.
Discussion
Biochemical characterization of SrIA and SrIB
The primary structures of peptides SrIA and SrIB iso-
lated from the worm-hunting snail C. spurius reflect
post-translational modifications of proline and gluta-
mine residues, together with the amidation of the
C-terminus of a shared toxin precursor. From analysis
of the cDNA sequence, the C-terminus, including the
last cysteine, is: CGGRR. This sequence is typically
present in peptides processed post-translationally.
Several rules have emerged from matching the
sequences of the mature peptides with the nucleotide
sequences of the cDNAs encoding scorpion toxins. If
one or two basic residues are present at the C-termi-

nus, they are removed post-translationally. If a glycine
precedes the basic residue(s), it is used to amidate the
residue preceding the glycine [5]. The MS analyses of
toxins SrIA and SrIB showed that these peptides are
in fact amidated.
The amino acid sequences indicate that the peptides
share structural features typical of the a-conotoxin
family. The two peptides contain four and seven resi-
dues between the second and the third cysteines, and
between the third and the fourth cysteines, respectively
(CCX
4
CX
7
C). This spacing defines the subfamily of
the a4 ⁄ 7-conotoxins (Table 2), the most widespread
category of nicotinic antagonists present in cone snail
venoms [2]. The a4 ⁄ 7-conotoxins have a conserved
proline in loop I, which comprises residues between
the second and the third cysteines. Together with Vc1a
[16], peptides SrIA and SrIB are the only known a4 ⁄ 7-
conotoxins in which this constant proline is post-trans-
lationally modified to hydroxyproline (Table 2). This
derivative has been found in l-conotoxins, x-conot-
oxins, j-conotoxins, jA-conotoxins, aA-conotoxins,
w-conotoxins, e-conotoxins, v-conotoxins, r-conotox-
ins, jM-conotoxins, d-conotoxins, and I-conotoxins
[17]. It was also discovered in the a4 ⁄ 7-conotoxin GID
[18], although not at the conserved proline of loop I.
Another unusual characteristic of SrIA and SrIB is the

presence of c-carboxyglutamate residues. This post-
translational modification has been described in Conus
peptides such as the conantokins, the c-conotoxins, the
I-conotoxins, and the e-conotoxins [17], and in the
N-terminal region of the a4 ⁄ 7-conotoxin GID [18].
However, Vc1a and peptides SrIA and SrIB are the
only a-conotoxins in which c-carboxyglutamate
residues occur in loop II, which comprises residues
between the third and the fourth cysteines.
Peptides SrIA and SrIB have 18 amino acids and an
amidated C-terminus. They are predicted to have
charges of 0 and + 1, respectively, at physiologic pH.
It has been pointed out that a-conotoxins specific for
neuronal subtypes of nAChR are neutral or negatively
charged [19], whereas a-conotoxins that target muscle
receptors have a net positive charge [20]. Because,
according to these authors, peptide SrIA could be
considered a potential antagonist of neuronal nAChR,
and toxin SrIB a probable antagonist of muscle
nAChR, we decided to test peptides SrIA and SrIB in
biological preparations separately expressing neuronal
(central, a
4
b
2
, and ganglionic, a
3
b
4
) and muscle

(a
1
b
1
cd) subtypes of nAChR. Unexpectedly, peptides
SrIA and SrIB were active on both central and muscle
types of the nAChR, which constitutes a novel activity
profile of the conserved a4 ⁄ 7-conotoxin-type scaffold.
Even more surprising was the finding that peptides
SrIA and SrIB have nAChR-potentiating activity, in
contrast to all previously studied a4 ⁄ 7-conotoxins.
It has been postulated that divergence within a sin-
gle superfamily to produce functionally different famil-
ies is one of the neuropharmacologic strategies
employed by the Conus genus, and may account in
part for its success in nature [21].
E. Lo
´
pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs
FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3979
Structure–function relationship for SrIA, SrIB,
and EI
Peptides EI, SrIA and SrIB contain structural
elements of the two types of conotoxins that act
differentially on neuronal and muscle nAChR. Toxin
EI [15] (present study) and peptides SrIA, SrIB and
[c15E]SrIB are the only conotoxins with a type I
cysteine scaffold known to act on muscle nAChR.
Except for SrIA, they have positive net charges that
might contribute to their activity on muscle receptors

[20], and they (except EI) share with most of the
a3 ⁄ 5-conotoxins (blockers of a
1
b
1
cd nAChR) a tyro-
sine at position 4 of loop II that is not present in
any of the a4 ⁄ 7 conotoxins known previously
(Table 2). This tyrosine has been found to make an
important contribution to the affinity of toxin MI
for the a ⁄ d subunit interface of the muscle nAChR
[22]. The three peptides have threonines and methio-
nines at position 4 of loop I and position 2 of
loop II, respectively. These residues are not present
at these positions in any of the other a4 ⁄ 7 toxins
studied to date, with the exception of Met10 in toxin
EpI (Table 2). It seems probable that these threo-
nines and methionines are somewhat involved in the
binding and ⁄ or activity with muscle nAChR. Alter-
natively, the nonpolar methionine residue at position
2 of loop II might be involved in binding to neuron-
al nAChR subtypes, because all known a4 ⁄ 7-cono-
toxins have a nonpolar residue at this position
(Table 2). Peptides EI, SrIA, SrIB and [c15E]SrIB
have very similar hydrophobic aliphatic residues
occupying position 7 of loop II (isoleucine in toxin
EI; leucine in peptides SrIA, SrIB, and [c15E]SrIB);
aliphatic residues (leucine, isoleucine, or valine) also
occur at this position in toxins MII, PeIA, GIC,
Vc1.1, PIA, and GID, which target diverse neuronal

subtypes (including a
3
b
4
and a
4
b
2
) with variable
affinities (Table 2). Thus, it is probable that hydro-
phobic aliphatic residues at position 7 of loop II
contribute to the binding and ⁄ or activity of peptides
EI, SrIA, SrIB and [c15E]SrIB with a
3
b
4
and ⁄ or
a
4
b
2
nAChRs. Finally, except for toxin GID,
peptides SrIA, SrIB, and [c15E]SrIB are the only
a4 ⁄ 7-conotoxins known to have an arginine at posi-
tion 1 of loop II (Table 2). In GID, this residue has
been demonstrated to contribute to the block of the
a
4
b
2

subtype [18], which is consistent with the biolo-
gical activity of peptides SrIA, SrIB and [c15E]SrIB
on a
4
b
2
nAChRs. So far, the toxin with the highest
affinity (IC
50
¼ 152 nm) for the a
4
b
2
subtype is GID,
and it blocks the a
3
b
2
and a
7
subtypes with  40-
fold higher affinities [18].
The physiologic role of the SrI and EI
a-conotoxins
In the present article, we have defined the weak antag-
onist properties of the novel C. spurius a-conotoxins,
and of a synthetic analog of one of them, on three of
the more important types of acetylcholine receptor.
Moreover, while comparing these properties with those
of the well-known a-conotoxin EI, we discovered that

it has a selectivity spectrum somewhat different from
that known previously. a-Conotoxin EI had been
considered a specific blocker of a
1
b
1
cd nAChRs
[15,17,23,24], but our results show that it also may
block the a
3
b
4
and a
4
b
2
neuronal subtypes.
This part of our results emphasizes the importance of
testing conotoxins not only on the expected subtypes of
the known molecular target (based on the toxin
sequence and on the current pharmacologic knowledge
in the field), but also on other target subtypes and even
on nonrelated targets. Recently, toxin ImII has been
found to inhibit both a
7
and a
1
b
1
cd nAChRs to similar

extents [25], whereas the a3 ⁄ 5-conotoxin CnIA not only
inhibits fetal muscle nAChRs, but also blocks the neur-
onal a
7
subtype, although with an  80-fold lower affin-
ity [26]. One surprising and distinct activity associated
with the same protein scaffold of the a4 ⁄ 7-conotoxins
has been reported for toxin q-TIA from C. tulipa;it
inhibits the a
1
-adrenoreceptor, and has the same disul-
fide connectivity as ‘classic’ a-conotoxins [27]. Like
toxin q-TIA, which has an extended N-terminal
sequence, peptides SrIA and SrIB have sequence fea-
tures (hydroxylated proline in loop I and c-carboxyglut-
amate residues in loop II) that differ considerably from
those of other a4 ⁄ 7-conotoxins.
The second part of our results reveals a novel cono-
toxin-induced functional nAChR state consisting of a
potentiation of the response; the potentiation can be
detected both with the new toxins and with EI. It can
be observed and quantitatively characterized at extre-
mely low concentrations and with brief applications.
Interestingly, longer applications produced either a
null effect or an inhibitory effect, as expected from the
kinetic data shown in Fig. 5E,F and the affinity of the
inhibitory process, which were evaluated with pro-
longed pretreatments.
The a-conotoxins described in this communication
showed that they can regulate the nAChR response. It

is known [28] that nAChRs are subjected to a variety
of actions, including the increase or decrease of the
affinity of the receptor for nicotinic ligands, a phenom-
enon that may occur in the absence of agonist, and
possibly results from stabilization of the desensitized
state [29]. Numerous examples of positive and negative
a-Conotoxins with potentiating effects on nAChRs E. Lo
´
pez-Vera et al.
3980 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS
allosteric effectors acting at neuronal nAChRs have
been reported, illustrating the importance of the allos-
teric nature of this protein. For example, it was shown
that progesterone and 17-b-estradiol act as negative
and positive effectors, respectively, of the a
4
b
2
receptor
subtype [30,31]. Atropine and zinc are reported to have
similar effects on some nAChRs [32,33], although the
required concentrations of these drugs were higher by
more than two orders of magnitude than those of our
peptides. Interestingly, the same mixed partial agonist
and antagonist behavior was observed for the well-
known blocker d-tubocurarine [34]. It has been repor-
ted recently [35] that a-conotoxin PnIA and a synthetic
derivative of it ([A10L]PnIA) weakly potentiate acetyl-
choline-activated currents in the wild-type a
7

nAChR;
these authors also reported that on mutant (a
7
-L247T)
receptors, [A10L]PnIA potentiated the acetylcholine-
evoked current and acted as an agonist by itself. The
mechanisms involved in these processes may be related
to previous findings that a-conotoxin MI binds to two
distinct sites on the a
1
b
1
cd nAChR, one at the ad
interface, and another at the ac (or ae) interface [36].
Concluding remarks
As it is unknown how and where the peptides studied
in this work bind the different receptor assemblies, it is
premature to suggest any hypothesis regarding the
structure–function mechanisms underlying the peptide
binding. Single-channel studies are in progress using
mutagenized peptides and cells expressing specific
nAChRs.
These peptides are promising tools for studies at a
detailed molecular level of the structure–activity rela-
tionship that underlies the action of the nAChR-target-
ing conotoxins. Considering that nAChRs are
implicated in brain diseases such as schizophrenia, noc-
turnal frontal lobe epilepsy [37], and Alzheimer’s dis-
ease, these new peptides are also candidate models to
develop potentially therapeutic drugs of major import-

ance [38]; for example, peptides SrIA, SrIB and
[c15E]SrIB might lead to the development of a
4
b
2
-select-
ive enhancers, which are beginning to be discovered [39].
Experimental procedures
Specimen collection and venom extraction
Specimens of C. spurius were collected in the Yucatan
Channel, Mexico. The venom was obtained by dissection of
the venom ducts. The ducts were homogenized in 10 mL of
0.1% v ⁄ v trifluoroacetic acid and 40% v ⁄ v acetonitrile
(ACN). The homogenate was centrifuged at 17 000 g for
30 min at 4 °C using a Beckman Coulter Avanti J20 centri-
fuge with JA-20 rotor. The supernatant, containing the pep-
tides, was subsequently processed.
Peptide purification by RP-HPLC
HPLC was performed on a n Agilent 1100 Series LC System
(G1322A Degasse r, G1311A Quaternary Pump, G1315B Diode
Array Detector, G1328A Man ual Injector; Hewlett-Packard,
Waldbronn, German y). The venom e xtract was fractionated
with a Vydac (Toluca, Mexico) C18 analytical reverse-phase
column (2 18TP54, 5 lm, 4.6 · 250 mm) equipped with a
Vydac C18 guard column (218GK54, 5 lm, 4.6 · 10 mm).
Peptides were eluted with a linear g radient of 5–95% solution B
at a fl ow rate of 1 m LÆmin
)1
over 90 min, where solution A is
0.1% v ⁄ v aqueous trifluoroacetic acid and solution B is 0 .09%

v ⁄ v trifluoroacetic acid in 90% v ⁄ v aqueous ACN. The same
column was also employed to repurify the components of the
venom, using a linear gradient of 15–30% of solution B at a
flow rate of 1 m LÆmin
)1
for 4 5 min.
Amino acid sequence
Peptides were adsorbed onto polybrene-treated (Biobrene
Plus; Applied Biosystems, Foster City, CA) glass fiber fil-
ters, and the amino acid sequence was determined by auto-
mated Edman degradation using an automatic instrument
(Procise 491 Protein Sequencing System; Applied Biosys-
tems) by the pulsed-liquid method.
MS analysis
Native peptides were applied directly into a Finnigan
LCQ
DUO
ion trap mass spectrometer (Finnigan, San Jose,
CA). The LCQ mass spectrometer is coupled to a Surveyor
syringe pump delivery system. The eluate at 20 lLÆmin
)1
was split to allow only 5% of the sample to enter the nano-
spray source (1.0 lLÆmin
)1
). The spray voltage was set to
1.6 kV, and the capillary temperature was set to 130 °C.
All spectra were obtained in the positive-ion mode. The
acquisition and deconvolution of data were performed with
xcalibur software (Thermo Electron Corp., Nashville, TN)
on a Windows NT PC system.

Determination of disulfide bridges
The connectivity of the cysteines of toxin SrIA was deter-
mined by partial reduction with Tris(2-carboxyethyl) phos-
phine hydrochloride and alkylation with N-ethylmaleimide.
The peptide (11.8 nmol) was dissolved in 10 lL of denatur-
ing buffer (0.1 m sodium citrate containing 6 m guanidine
hydrochloride, pH 3.0), and 27 l L of 0.1 m Tris(2-carboxy-
ethyl) phosphine hydrochloride in the same buffer was
added. The mixture was incubated for 15 min at room
E. Lo
´
pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs
FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3981
temperature. Sixty-eight microliters of 0.1 m N-ethylmalei-
mide dissolved in denaturing buffer was added, and the
sample was incubated for 30 min at room temperature [40].
The mixture was diluted with 900 lL of 10% solution B,
and injected into two, reverse-phase analytical columns in
tandem (Sephasil Peptide C8, 5 lm, 4.6 · 100 mm; Phar-
macia Biotech, Uppsala, Sweden; Eclipse XDB-C8, 5 lm,
4.6 · 150 mm; Agilent Technologies, Palo Alto, CA) provi-
ded with a MetaGuard Nucleosil C8 precolumn (5 mm,
4.6 · 10 mm; MetaChem, Torrance, CA). A linear gradient
from 10% to 30% solution B was developed at 1 mLÆmin
)1
for 120 min at room temperature; the absorbance of the
eluate was monitored at 220 nm. The absorbing peaks not
present in the corresponding reagent blank were collected
and taken to dryness for automatic sequence analysis.
cDNA cloning

The venom duct and gland from one C. spurius specimen
was dissected, and total RNA was isolated using the RNA-
gents Total RNA Isolation System (Promega, Madison,
WI), according to the supplier’s instructions. Reverse tran-
scription of mRNA was primed by oligo-p(dT)
22
NN, where
p(dT)
22
annealed with the polyA tail of mRNA, and NN
localized the primer to the border between the polyA tail
and the 3¢-UTR of the mRNA. Reverse transcription was
performed using the 1st Strand cDNA Synthesis Kit for
RT-PCR (avian myeloblastosis virus) (Roche Diagnostics
Corporation, Indianapolis, IN) as follows:  1 lg of total
RNA and 20 pmol of oligo-p(dT)
22
NN were added to a vol-
ume of 20 lL containing 10 mm Tris ⁄ HCl (pH 8.3), 50 mm
KCl, 5 mm MgCl
2
,1mm dNTP mix, 50 units of RNase
inhibitor, and  20 units of AMV reverse transcriptase. The
reaction was incubated at 25 °C for 10 min, and then at
42 °C for 60 min. The reverse transcriptase was inactivated
by incubation at 95 °C for 5 min after the reaction was
completed. Conotoxin cDNA was amplified by Vent DNA
Polymerase (New England Biolabs, Inc., Beverly, MA),
using reverse transcription products as templates, and oligo-
nucleotides corresponding to the conserved signal peptide-

coding region and the 3¢-UTR of a-conotoxin cDNAs as
primers. The primers were: P2, 5¢-TCTGCGAATGGG
CATGCGGATGATGTT-3¢, corresponding to the signal
peptide-coding region; and P3, 5¢-TGCTCCAACGTCGT
GGTTCAGAGGGTC-3¢, corresponding to the 3¢-UTR [4].
PCR was carried out as follows: to a total 100 lL reaction
volume containing 1 · ThermoPol Reaction Buffer [20 mm
Tris ⁄ HCl (pH 8.8), 10 mm KCl, 10 mm (NH
4
)
2
SO
4
,2mm
MgSO
4
, 0.1% Triton X-100] and 0.2 mm dNTP mix was
added 5 lL of reverse transcription product and 20 pmol of
each primer. The mixture was incubated in the thermal
cycler at 95° for 5 min, and then 2 units of Vent DNA
Polymerase were added (Hot Start). The parameters for
thermal cycling were: 95 °C for 5 min; then 95 °C for 30 s,
47 °C for 30 s, 72 °C for 30 s for 35 cycles; then 72 °C for
5 min (GeneAmp PCR System 2400; Perkin-Elmer, Welles-
ley, MA). The PCR products were purified with a QIAquick
Gel Extraction Kit (Qiagen GmbH, Hilden, Germany), and
blunt-end ligated with EcoRV-digested pBluescript II
KS (+) vector (Stratagene, La Jolla, CA). The ligation
products were electrotransformed into electrocompetent
DH5a Escherichia coli cells, and plated on agar plates con-

taining 200 lgÆmL
)1
5-bromo-4-chloroindoyl-3-yl b-d-gal-
actoside ⁄ isopropyl thio-b-d-galactoside ⁄ ampicillin to select
for recombinant colonies. White colonies were picked, and
their plasmids were screened by size on a 1.2% agarose gel
[41]. Plasmid DNA was purified with a High Pure Plasmid
Isolation Kit (Roche Diagnostics Corporation), and cloned
DNA was sequenced with a 3100 DNA Sequencer (Applied
Biosystems), using primers T3 and T7, which anneal in the
flanking regions of the cloning site of the vector.
Synthesis of peptides [c15E]SrIB and EI
Solid-phase peptide synthesis
N
a
-Fmoc-amino acids, 2-(1-H-benzotriazol-1-yl)-1,1,3,3-
tetramethyl uronium hexafluorophosphate and Rink amide
MBHA resin were from Novabiochem (Laufelfingen, Swit-
zerland). All other reagents and solvents were from Sigma-
Aldrich (St Louis, MO). Mass spectra were recorded on a
Fourier Transform Ion Cyclotrone Resonance instrument
(model APEXII; Bruker Daltonics, Billerica, MA) equipped
with a 4.7 T cryomagnet (Magnex, Oxford, UK). RP-HPLC
was performed on a Waters 515 system using a Waters
(Milano, Italy) Symmetry 300 C-18 analytical column (5 lm,
4.6 · 150 mm) or a Merck (Darmstadt, Germany) LiChro-
CART 250 C-18 semipreparative column (10 lm,
10 · 250 mm). Linear gradients of ACN in water ⁄ 0.1% tri-
fluoroacetic acid were used for peptide elution, with flow
rates of 1 mLÆmin

)1
for analytical or 8 mLÆmin
)1
for semi-
preparative purposes. The peptides were prepared by
manual, solid-phase peptide synthesis using standard 2-(1-H-
benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluoro-
phosphate activation protocols for Fmoc chemistry as
previously described [9,10]. Couplings were carried out with
a five-fold excess of activated amino acid for a minimum of
30 min, and monitored by the 2,4,6-trinitrobenzenesulfonic
acid test [42]. At the end of chain assembly, the resin was
washed with dimethylformamide ⁄ dichloromethane, dried
under nitrogen, and treated with a mixture of cation scaven-
gers in trifluoroacetic acid with magnet stirring for 2 h to
detach the peptides from the resin and simultaneously to
deprotect amino acid side chains. The crude peptides were
precipitated in hexane ⁄ diethylether 1 : 1 (v ⁄ v), centrifuged at
3290 g using a Hereus Sepathec centrifuge with 2251 rotor,
dissolved in 20% aqueous ACN, and purified by semiprepar-
ative HPLC [43,44].
The S-acetamidomethyl (Acm)-protected precursors of
the [c15E]SpIB and EI peptides had the follow-
ing sequences: RTCC(Acm)SROTCRMEYPELC(Acm)G-
a-Conotoxins with potentiating effects on nAChRs E. Lo
´
pez-Vera et al.
3982 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS
NH
2

, and RDOCC(Acm)YHPTCNMSNPQIC(Acm)-NH
2
,
respectively.
Mass analysis of purified peptides gave the following
results: peptide RTCC(Acm)SROTCRMEYPELC(Acm)G-
NH
2
, MS 2262.0 (calculated 2262.5); peptide RDOCC(Ac-
m)YHPTCNMSNPQIC(Acm)-NH
2
, MS 2237.3 (calculated
2237.8).
Formation of the first disulfide bond
The lyophilized peptides were dissolved in 10 mm aqueous
ammonium bicarbonate to a final peptide concentration of
5mm. The pH was adjusted to 8 by adding 1.5 m Tris ⁄ HCl
buffer (pH 8.8). The resulting solution was stirred for 48 h
and then concentrated. The bis-Acm-peptides containing
disulfide bridges between Cys3 and Cys9 in the case of
[c15E]SrIB or between Cys4 and Cys10 in the case of EI
were purified by semipreparative HPLC.
Mass analysis gave the following results: Bis-Acm-
[c15E]SrIB precursor, MS 2260.6 (calculated 2260.8); Bis-
Acm-EI precursor, MS 2235.1 (calculated 2235.4).
Acm group removal and formation of the second
disulfide bond
The bis-Acm peptides were dissolved in 80% aqueous acetic
acid to a final peptide concentration of 1 mm. Three equiv-
alents (eq.) of 37% hydrochloric acid were added. Iodine

(10 eq., methanolic solution) was added dropwise, and the
resulting mixture was stirred for 2 h. The reaction was then
quenched by adding 1 m ascorbic acid until the orange
color disappeared. The mixture was concentrated, and the
final product was isolated by HPLC and lyophilized. This
procedure resulted in the formation of disulfide bridges
between Cys4 and Cys17 in [c15E]SrIB and between Cys5
and Cys18 in EI. Mass analysis gave the following results:
[c15E]SrIB, MS 2114.8 (calculated 2115.0); EI, MS 2075.4
(calulated 2075.8).
Electrophysiology
Cell culture
Cells of the rhabdomyosarcoma TE671 clone [45] were
routinely cultured in DMEM containing 4.5 gÆL
)1
of
glucose and 10% fetal bovine serum. The cells were
incubated at 37 °C in a humidified atmosphere with
5% CO
2
. Cells expressing a
3
b
4
nAChRs [46] were kindly
provided by D. Feuerbach (Novartis Pharma AG, Basel,
Switzerland). Cells expressing a
4
b
2

nAChRs were kindly
provided by G. Casari (S. Raffaele Institute, Milan, Italy).
Solutions
The standard extracellular solution contained: 130 mm
NaCl, 5 mm KCl, 2 mm CaCl
2
,2mm MgCl
2
,10mm
Hepes ⁄ NaOH buffer, and 5 mmd-glucose (pH 7.40). The
standard pipette solution at [Ca
2+
]
i
¼ 10
)9
m (pCa 9) con-
tained: 130 mm K
+
-aspartate, 10 mm NaCl, 2 mm MgCl
2
,
0.2 mm CaCl
2
,10mm EGTA ⁄ KOH, 10 mm Hepes ⁄ KOH
buffer, and 1 mm ATP (Mg
2+
salt) (pH 7.30). Fresh nico-
tine and conotoxins were added every 2 h to the extra-
cellular medium from a stock solution in distilled water.

Patch-clamp recordings
Currents were recorded on an MC700A patch-clamp ampli-
fier (Axon Instruments, Union City, CA) at room tempera-
ture, as previously described [47]. Pipette resistance
(1–2 MW), cell capacitance and series resistance errors were
carefully compensated (85–95%) before each voltage-clamp
protocol was run. The extracellular solutions were ejected
through a nine-hole (0.6 mm) remote-controlled linear posi-
tioner (average response of 0.5–1 s) placed near the cell
under study or by the similar rotating perfusion system
(Biologic, Grenoble, France). Tygon rather than silicon
tubing (Tygon-R3603; Cole-Palmer, Vernon Hills, IL) was
used for extracellular solutions, to prevent nicotine adsorp-
tion. We used nicotine instead of acetylcholine to exclude
the action of the latter on muscarinic receptors that are
potentially present on the cell membrane, and we verified
that the responses described in the TE671 cells could be
blocked by 500 nm a-bungarotoxin (not shown). The flow
rate was constant at 120 lLÆmin
)1
. The holding potential
was set at ) 60 mV. Data acquisition and analysis were per-
formed with pclamp 8.2 (Axon Instruments) and origin
7.1 (Microcal Software, Inc., Northampton, MA) software,
respectively.
Acknowledgements
We thank Maria Eugenia Ramos for the collection
of C. spurius specimens, Patricia Villalobos and
Andre
´

s Falco
´
n for technical assistance, Leopoldo
Martı
´
nez for artwork, and Dorothy D. Pless for
revision of the manuscript. This work was supported
by grants from Ministero dell’Universita
`
e della
Ricerca Scientifica e Tecnologica (MURST-COFIN
2001-03 and 2003-05; FISR-Neurobiotecnologie:
Fisiopatologia del sistema nervoso; FIRB-2001) to
E. Wanke, Consejo Nacional de Ciencia y Tecnologı
´
a
(CONACYT) to E. P. Heimer de la Cotera (41477-
Q) and M. B. Aguilar (43754-Q), and Programa de
Apoyo a Proyectos de Investigacio
´
n e Innovacio
´
n
Tecnolo
´
gica-UNAM (PAPIIT-UNAM) to E. P. Hei-
mer de la Cotera (IN-204403). R. Restano Cassulini
and E. Schiavon are PhD students in Physiology at
Milano-Bicocca University, Department of Biotechno-
logy and Biosciences.

E. Lo
´
pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs
FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3983
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