At high concentrations 10 lm, the peptides SrIA, SrIB and [c15E]SrIB showed weak blocking effects only on a4b2 and a1b1cd subtypes, but EI also strongly blocked a3b4 receptors.. Abbrevia
Trang 1a-conotoxin EI share high-affinity potentiation and
low-affinity inhibition of nicotinic acetylcholine receptors Estuardo Lo´pez-Vera1,*,†, Manuel B Aguilar1,*, Emanuele Schiavon2, Chiara Marinzi2,
Ernesto Ortiz3, Rita Restano Cassulini2, Cesar V F Batista3, Lourival D Possani3,
Edgar P Heimer de la Cotera1, Francesco Peri2, Baltazar Becerril3and Enzo Wanke2
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: enzo.wanke@unimib.it
*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 a1b1cd, a4b2 and a3b4 nicotinic acetylcholine receptors At high concentrations (10 lm), the peptides SrIA, SrIB and [c15E]SrIB showed weak blocking effects only on a4b2 and
a1b1cd subtypes, but EI also strongly blocked a3b4 receptors In contrast
to this blocking effect, the new peptides and EI showed a remarkable potentiation of a1b1cd and a4b2 nicotinic acetylcholine receptors if briefly (2–15 s) applied at concentrations several orders of magnitude lower (EC50, 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 a1b1cd and a4b2 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
a1b1cd, muscular nicotinic acetylcholine receptor; a3b4, peripheral nervous system nicotinic acetylcholine receptor; a4b2, 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.
Trang 2precursors 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 a1b1cd 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)1for 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
1 mLÆmin)1for 45 min.
Trang 3suggested 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
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.
Trang 4xyglutamic 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
a4b2 and peripheral nervous system nicotinic acetyl-choline a3b4 receptor subtypes As our present perfu-sion system is not sufficiently fast to resolve fast desensitizing currents such as those produced by a7 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
GIA ECCNPACGRHYSCGK 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 a3b2>> a7
PnIA GCCSLPPCAANNPDYC a a3b2>> a7
PnIB GCCSLPPCALSNPDYC a a 7 > a 3 b 2
EpI GCCSDPRCNMNNPDYC a a3b4, a3b2; a7
AuIA GCCSYPPCFATNSDYC a a3b4
Vc1.1 GCCSDPRCNYDHPEIC a a 3 a 7 b 4 , a 3 a 5 b 4
PeIA GCCSHPACSVNHPELC a a9a10, a3b2> a3b4> a7
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 a3b2>> a4b2, a3b4
MII GCCSNPVCHLEHSNLC a a 3 b 2 >> a 7 > a 4 b 2 , a 3 b 4
GID IR G c CCSNPACRVNNOHVC a 3 ⁄ b 2 , a 7 > a 4 ⁄ b 2
EI RDOCCYHPTCNMSNPQIC a a1b1cd, a3b4, a4b2
SrIA RTCCSROTCRMc YPcLCG a a4b2, a1b1cd
SrIB RTCCSROTCRMEYPcLCG a a 4 b 2 , a 1 b 1 cd
[c15E]SrIB RTCCSROTCRMEYPELCG a a4b2, a1b1cd
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)1for
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 posi(solu-tions 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.
Trang 5applications 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 a1b1cd receptor, as shown in Fig 4A,
and also for the a4b2and a3b4receptors [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
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 IC50of 99 ± 12 l M , 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 a4b2; 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: a1b1cd (open squares), 4.9 ± 0.25 s (n ¼ 5); a 3 b4(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 IC50 and Hill coefficient: for [c15E]SrIB,
46 ± 10 n M , 1 ± 0.1, and for EI, 187 ± 43 n M , 0.48 ± 0.06, respectively The number of experiments for each point ranged from three to 12.
Trang 6significantly 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
a3b4receptors (n¼ 4), and was a slightly better blocker
of the a4b2 receptors (0.56 ± 0.04, n¼ 7) than of
the a1b1cd receptors (0.39 ± 0.06, n¼ 5, not
statisti-cally significant) On the other hand, EI toxin was
able to potently block muscle (0.95 ± 0.01, n¼ 5) and
ganglionic (0.91 ± 0.03, n¼ 4) receptors, but was less
potent for the central nevous system receptor
(0.61 ± 0.02, n¼ 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 a3b4
receptors and a relatively weak antagonist of the a4b2
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
a1b1cd receptors with a son of 4.9 ± 0.25 s (n¼ 3),
and the a3b4 receptor was blocked with a son of
11 ± 1.9 s (n¼ 3) Moreover, the soff values that we
observed for these receptors were 150 ± 13 s (n¼ 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 a1b1cd receptors The estimated IC50
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 son¼ soff⁄
(1 + [T]⁄ KD), this relationship can be used to confirm
the previous IC50 of Martinez et al [15] on a1b1cd
receptors, which was 280 nm (low-affinity site) for the
mouse receptors, and to predict the unknown and
novel value of KD for the a3b4 receptors Indeed, we
found an IC50 value of 187 nm for a1b1cd
recep-tors (Fig 4E), which also agrees with the fractional
response of 0.04 at 10 lm EI and a soff of 150 s For
the a3b4 receptors, the above relationship results in a
KDvalue 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 a3b4 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 a1b1cd-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 a1b1cd 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, (Itoxin) Icontrol)⁄ Icontrol, of the synthetic [c15E]SrIB, the natural SrIB and SrIA peptides, and the EI a-conotoxin, were as follows: 0.46 ± 0.09 (n¼ 10), 0.47 ± 0.08 (n¼ 10), 0.44 ± 0.15 (n ¼ 9), and 0.54 ± 0.13 (n¼ 9), respectively These results suggest that, at least for the a1b1cd 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 a1b1cd, a4b2, and
a3b4, and the maximally observed relative potentiation values are shown in Fig 5B Interestingly, whereas the toxins were unable to produce potentiation in the
Trang 7ganglionic a3b4 receptor (n¼ 17), the mean fractional
potentiation in a1b1cd receptors for [c15E]SrIB
(0.75 ± 0.22, n¼ 7) was higher than that obtained for
EI (0.35 ± 0.07, n¼ 22, statistically different) The
effects of both toxins were found to be similar on the
a4b2receptor subtype
Furthermore, we investigated the dose–response
curves of the maximal fractional potentiation produced
by the [c15E]SrIB and EI conotoxins on the a1b1cd
receptors These data are shown in Fig 5C, and were
fitted to dose–response curves with EC50 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 a1b1cd-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 funcconcentra-tion 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
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 [(Itox) 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 a1b1cd 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 (n M ) (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 n M and EI (F) at 0.2 and 1 n M 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 n M ) 6.03 ± 0.32, s off (0.2 n M ) 16.4 ± 1.3 s; soff(1 n M ) 9.4 ± 1.5 s See text.
Trang 8an exponentially decaying depotentiation process We
show two examples obtained by using the two different
toxins on the a1b1cd receptor Figure 5E,F shows the
potentiation⁄ blockade (Itoxin⁄ Icontrol) 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 (n¼ 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 a1b1cd 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 (CCX4CX7C) 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-conotx-conot-oxins, jA-conotx-conot-oxins, aA-conotx-conot-oxins, w-conotoxins, e-conotoxins, v-conotoxins, r-conotox-ins, jM-conotoxr-conotox-ins, d-conotoxr-conotox-ins, 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, a4b2, and ganglionic, a3b4) and muscle (a1b1cd) 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]
Trang 9Structure–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 a1b1cd 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 methiothreo-nines 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 neuron-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 a3b4 and a4b2) 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 a3b4 and⁄ or
a4b2 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
a4b2 subtype [18], which is consistent with the
biolo-gical activity of peptides SrIA, SrIB and [c15E]SrIB
on a4b2 nAChRs So far, the toxin with the highest
affinity (IC50¼ 152 nm) for the a4b2 subtype is GID,
and it blocks the a3b2 and a7 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 a1b1cd nAChRs [15,17,23,24], but our results show that it also may block the a3b4and a4b2neuronal 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 a7and a1b1cd nAChRs to similar extents [25], whereas the a3⁄ 5-conotoxin CnIA not only inhibits fetal muscle nAChRs, but also blocks the neur-onal a7subtype, although with an 80-fold lower affin-ity [26] One surprising and distinct activaffin-ity associated with the same protein scaffold of the a4⁄ 7-conotoxins has been reported for toxin q-TIA from C tulipa; it inhibits the a1-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
Trang 10allosteric 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 a4b2receptor
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 a7 nAChR;
these authors also reported that on mutant (a7-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 a1b1cd 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 a4b2
-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 4C 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 an Agilent 1100 Series LC System (G1322A Degasser, G1311A Quaternary Pump, G1315B Diode Array Detector, G1328A Manual Injector; Hewlett-Packard, Waldbronn, Germany) The venom extract was fractionated with a Vydac (Toluca, Mexico) C18 analytical reverse-phase column (218TP54, 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 gradient of 5–95% solution B
at a flow rate of 1 mLÆmin)1over 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 mLÆmin)1for 45 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 LCQDUO 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 130C All spectra were obtained in the positive-ion mode The acquisition and deconvolution of data were performed with xcalibursoftware (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 containdenatur-ing 6 m guanidine hydrochloride, pH 3.0), and 27 lL 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