(Received for publication, June 7, 1995; and in revised form, January 12, 1996)
From the
The venom of the black widow spider (BWSV) (Latrodectus
mactans tredecimguttatus) contains several potent, high molecular
mass (>110 kDa) neurotoxins that cause neurotransmitter release in a
phylum-specific manner. The molecular mechanism of action of these
proteins is poorly understood because their structures are largely
unknown, and they have not been functionally expressed. This study
reports on the primary structure of -latroinsectotoxin
(
-LIT), a novel insect-specific toxin from BWSV, that contains
1214 amino acids.
-LIT comprises four structural domains: a signal
peptide followed by an N-terminal domain that exhibits the highest
degree of identity with other latrotoxins, a central region composed of
15 ankyrin-like repeats, and a C-terminal domain. The domain
organization of
-LIT is similar to that of other latrotoxins,
suggesting that these toxins are a family of related proteins. The
predicted molecular mass and apparent mobility of the protein (
130
kDa) encoded in the
-LIT gene differs from that of native
-LIT purified from BWSV (
110 kDa), suggesting that the toxin
is produced by proteolytic processing of a precursor. MALDI-MS of
purified native
-LIT revealed a molecular ion with m/z+ of 110916 ± 100, indicating that the native
-LIT is 991 amino acids in length. When the full-length
-LIT
cDNA was expressed in bacteria the protein product was inactive, but
expression of a C-terminally truncated protein containing 991 residues
produced a protein that caused massive neurotransmitter release at the
locust neuromuscular junction at nanomolar concentrations. Channels
formed in locust muscle membrane and artificial lipid bilayers by the
native
-LIT have a high Ca
permeability, whereas
those formed by truncated, recombinant protein do not.
The venom of the black widow spider (BWSV) ()(Latrodectus mactans tredecimguttatus) elicits
massive neurotransmitter release from vertebrate and invertebrate nerve
terminals(1, 2) , due to its content of high molecular
weight proteins, the latrotoxins (e.g. see (3) ).
-Latrotoxin (
-LTX) is well known for its potent ability to
trigger neurotransmitter release from vertebrate nerve
terminals(4, 5) , and has been an invaluable tool for
characterizing the mechanisms involved in presynaptic neurotransmitter
release(6, 7) . The mechanism of presynaptic action of
BWSV appears to be similar in vertebrates and
invertebrates(2, 8) ; however,
-LTX is inactive
at invertebrate nerve terminals, and other latrotoxins, the
latroinsectotoxins (LITs), specifically cause neurotransmitter release
at insect neuromuscular junctions(3, 8) . For example,
-LIT has no effect on frog motor nerve terminals at 50
nM, yet it induces a massive increase in transmitter release
from blowfly motor nerve terminals at 4 nM(8) . The
fact that the latrotoxins elicit similar physiological responses in
vertebrates and invertebrates suggests common mechanisms controlling
neurotransmitter release across phyla and emphasizes their utility as
tools for studying these mechanisms.
The primary structures of
-LTX and
-LIT have recently been
determined(9, 10) . The proteins are polypeptides of
more than 1300 amino acids and share 34% amino acid identity. Although
-LTX and
-LIT have been expressed in a recombinant
baculovirus expression system(11) , it is not clear whether the
full-length proteins are toxic. Also, when
-LTX was purified from
BWSV, it was tightly associated with an acidic low molecular weight
protein(12, 13) . Recombinant expression of a
latrotoxin is necessary for functional characterization of latrotoxins,
and is thus a prerequisite for site-directed mutagenesis studies that
may be informative in dissecting the mechanism of latrotoxin action at
nerve terminals. For this reason, we have cloned and sequenced the
structural gene for a novel insect-specific toxin,
-LIT, and have
determined the N and C termini of native
-LIT and the recombinant
protein. The full-length, recombinant protein is larger than the native
toxin and, unlike the latter, has no insect toxicity. However, when the
C-terminal domain of
-LIT was removed by site-directed
mutagenesis, the recombinant protein was highly toxic, causing
characteristic neurotransmitter release from locust motor nerve
terminals. We also show that the native and truncated, recombinant
toxins form channels in lipid bilayers and locust muscle membrane, but
that the channels differ greatly in terms of their Ca
selectivities.
Alternatively, cells were collected, washed, and
resuspended in buffer containing 4 M urea, 100 mM NaCl, 50 mM Tris-HCl (pH 8.0) plus 2 mM EDTA and
5 mM DTT and sonicated on ice at high power settings (7
20 s with 20-s intervals). The cell lysate was diluted with 4
volumes of the same buffer, cleared by centrifugation (12,000
g; 30 min) followed by 0.22-µm filtration, and loaded onto
a Mono Q Sepharose column (Pharmacia Biotech Inc.) equilibrated in
lysis buffer. The column was washed with 5 volumes of lysis buffer
followed by 5 volumes of lysis buffer without urea and DTT. Proteins
were eluted using a linear gradient of 100-1000 mM NaCl in 50
mM Tris-HCl (pH 8.0) plus 1 mM EDTA. The fraction
containing recombinant protein was dialyzed against locust saline and
used for toxicity tests and electrophysiology studies.
The N terminus of the mature protein was
determined by amino acid sequencing and was found to be preceded by a
short sequence, viz. MHSKELQTISAAVARKAVPNTMVIRLKR, in the
-LIT cDNA. This sequence contains two in-frame Met residues
(-7 and -28) that can serve as translation initiation
sites. The nucleotide sequence surrounding Met(-7) (actatgg)
agrees with the classical Kozak consensus(27) , but the
nucleotide arrangement for Met(-28) (gaaatgc) resembles the
starting points for at least three other arachnid
proteins(12, 28, 29) . This Met residue is
preceded by an in-frame stop codon and is likely to be the initiation
codon. Although the resulting 28 amino acids stretch does not fit a
classical signal peptide consensus very well, the fact that it is
cleaved during synthesis of
-LIT, and that
-LIT is a secreted
protein, indicates that this sequence might serve as a signal peptide.
According to direct N-terminal sequence determination, the mature
protein starts from Asp (+1) in Fig. 1(lane 1).
The identity of this protein as -LIT was confirmed by showing that
it contains seven peptide sequences that are present in native
-LIT (determined by amino acid sequencing of tryptic peptides (Fig. 1)). This
-LIT cDNA encodes a protein with 1186 amino
acid residues and a predicted molecular mass of 132671 Da. Although
-LIT is a soluble protein, and hydrophobicity plots confirm its
hydrophilicity, it contains two hydrophobic regions (Fig. 1) in
its N-terminal region (residues 36-64 and 222-241).
Figure 1:
Alignment of the deduced amino acid
sequence of -LIT with
-LIT and
-LTX. Sequences are shown
in single-letter amino acid code, identified on the left and numbered
on the right. Underlined residues represent those obtained by
direct amino acid sequencing of N terminus and 7 tryptic peptides of
purified
-LIT. Hydrophobic segments are boxed. The arrows, R1-R22, mark the beginning of each of
the 22 consecutive ALRs. The vertical arrow marks the putative
site of C-terminal proteolytic processing of purified
-LIT.
Residues conserved in all three latrotoxins are shaded. Gaps
are introduced to improve the alignment of the deduced amino acid
sequences with
-LIT, and are indicated by a dash.
Figure 2:
Structural motifs of -LIT. A, dot matrix analysis. Dot matrix plots comparing the deduced
amino acid sequence of
-LIT with itself (top panel),
-LIT (middle panel), and
-LTX (bottom
panel). The axes refer to the numbering of amino acid residues.
Each dot represents a match (40% minimum identity) between windows of
20 residues. B, ALRs in latrotoxins. I, optimal
alignment of
-LIT ALRs (R1-R15). Amino acids
present in at least half of ALRs are in bold type and
summarized in a consensus shown below the alignment. A dash represents an amino acid residue where there is no consensus.
Three amino acids of R1 are removed and placed outside in brackets to
improve the alignment. II, comparison of the consensus
sequences derived from ALRs in
-LIT,
-LIT,
-LTX, and
brain ankyrin. Gaps marked by periods were introduced to
facilitate alignment.
The dot matrix analysis also
shows that the middle region of -LIT is composed of repeated
units, a property that it shares with other latrotoxins. These regions
contain tandemly arranged imperfect copies of ALRs(30) . There
are at least 15 such repeats in
-LIT, some of which are rather
distant from the consensus sequence of ALR, but which are,
nevertheless, clearly identifiable (aligned in Fig. 2B). The ALR domain of
-LIT is shorter than
that of
-LIT and
-LTX, a difference that is reflected in the
asymmetric distribution of conserved segments in these regions (Fig. 2A, middle and bottom panels).
Four domains can be readily identified in the -LIT sequence and
those of other latrotoxins. The first is a presumptive signal sequence,
the second the N-terminal region, the third comprises the ALRs, and the
fourth is the C-terminal region. The N-terminal region is the best
conserved of these domains. Data base searches failed to detect
significant sequence similarities between the N- and C-terminal domains
of
-LIT (and those of the other two latrotoxins) and other
polypeptide structures. However, as expected, similarities to the ALRs
were detected in many proteins.The ALRs are relatively poorly conserved
between the three latrotoxins, although their positional conservation
is obvious (Fig. 3). By reanalyzing our previous data as
suggested in Bork(31) , we have identified at least 22 ALRs in
-LTX and
-LIT. The highest similarity to ALRs in other
proteins is detected in the N-terminal 11 repeats of
-LIT, except
for the first two repeats which are rather atypical. The ALR repeats
3-6 and 9-11 of
-LIT match those of human ankyrin very
well, but repeats 7, 8, and 12 exhibit the highest level of homology to
other ankyrin/cdc10 repeats-containing proteins ranging from bacterial
malate synthase G (accession no. P37330 in SWISS-PROT data base) to
human nuclear factor NF-
B (accession no. P19838). In general, the
sequence conservation of latrotoxin repeats is lower than in brain
ankyrins (Fig. 2B, II). Computer analysis,
using the Clustal method for dendrograms construction(32) ,
revealed that the ALRs are most homologous between the three
latrotoxins within the N-terminal 11 repeats, with the exception of 7th
repeat in
-LIT. The 7th repeat breaks a linear correlation between
-LIT repeat units and might play some specialized role for this
insectotoxin. There is less similarity between
-LIT repeats
12-15, and repeats 18-22 in
-LTX and
-LIT, than
between earlier repeats.
Figure 3:
Structural domain model of the
latrotoxins. Domains I and IV represent the signal
sequence and the C-terminal sequence respectively. N-terminal domain II
contains two hydrophobic segments (HS1 and HS2) shown
in black. Percentage numbers indicate amino acid identities
between the respective domains in -LIT. Domain III is
composed of ALRs. The vertical arrow indicates the estimated
site of C-terminal processing of
-LIT. The scale below shows
number of amino acid residues.
Figure 4:
Estimation of the relative molecular mass
of native and recombinant -LIT. A, Western blot with an
antibody against
-LIT. Bacteria (E. coli BL21 (DE3))
transfected with the vector, pT7-7(16) , or pT7.
FL or
pT7.
M, were treated with vehicle(-) or
isopropyl-1-thio-
-D-galactopyranoside (+), and
protein extracts were prepared after 2 h. The samples, with BWSV as a
standard, were blotted and detected by an antibody specific for
-LIT. B, MALDI-MS of native
-LIT. MALDI-MS of
-LIT purified from BWSV(3) . The mass/charge ratio of
selected peaks are indicated;
-LIT+ represents the
-LIT molecular ion.
Figure 5:
The truncated, recombinant -LIT
increases mEPSP discharge. Effect of truncated, recombinant protein on
mEPSPs recorded intracellularly from metathoracic extensor tibiae
muscle fibers of adult locust. A, mEPSP discharge before
protein application. A brief burst of mEPSPs is seen. The average mEPSP
frequency for a 5-min recording epic was 1.2 s
. B-E, mEPSP discharges 18, 20, 22, and 41 min,
respectively, after application of 10
M protein. Similar data for 10
M native
-LIT were obtained from 10 other
preparations.
Figure 6:
Influence of divalent cations on changes
in mEPSP discharge induced by truncated, recombinant protein (also
native toxin; data not shown). Histograms of mEPSP frequency versus time; bins are number of mEPSPs recorded during successive 1-min
periods. The arrows indicate time of application of truncated,
recombinant protein. A, increase in mEPSP discharge induced by
10M protein. Note delay in increase of
mEPSP frequency and then marked variations in mEPSP frequency. B, rapid onset of action of 10
M protein. Typical example of data from 7 preparations. C,
saline containing 10 mM MgCl
delayed the onset of
action of 10
M protein. Also, note that
increase in mEPSP frequency was reduced compared with B.
Example of data from three preparations. D, 10
M native
-LIT induced a small miniature discharge
in saline lacking divalent cations. Data in A-D are
typical of those obtained from nine
preparations.
A further
comparison of the truncated, recombinant protein and native -LIT
was made by investigating their channel-forming properties. Previous
studies have shown that latrotoxins are channel-forming proteins (e.g. see (34) ). The recombinant protein formed
channels of
20 pS in artificial bilayers (n = 15
experiments) (Fig. 7A). The bilayers were exposed on
both sides to 190 mM NaCl. The saline on the cis side
contained 2 mM CaCl
; the trans CaCl
concentration was 0.2 mM. When 10
M truncated, recombinant protein was applied to the cis side, channel openings appeared 5-30 min later. The
channels exhibited a slight inward rectification with a conductance
ratio of
0.8. The rectification was reversed when recombinant
protein was applied to the trans side. These observations
suggest that
-LIT inserts into an artificial bilayer and that its
insertion is polarized and Ca
-insensitive. Changes in
Ca
concentration (n = 19) did not
affect either the rectification or the reversal potential (V
= 0 mV) of the single channel current,
thus suggesting that the channel formed by recombinant
-LIT does
not select for Ca
(Fig. 7A). Also, V
was unchanged when SO
was substituted for Cl
on one side of a
bilayer. Although not tested systematically, the probability of
occurrence of channels formed by the recombinant protein did not appear
to be affected by membrane potential. How do the results for the
recombinant toxin compare with those for native
-LIT? In lipid
bilayers, native
-LIT formed channels with a maximal slope
conductance of
40 pS (n = 33) (Fig. 7B). This compares with a conductance of
5
pS for channels induced in bilayers by
-LIT(34) , but the
lipids and saline solutions were different. The channels formed by
native
-LIT also inwardly rectified (the conductance ratio was
0.5-0.6). The rectification was insensitive to changes in
Ca
concentration (Fig. 7B). However, V
for the single channel current varied with the
Ca
concentration gradient across the bilayer (n = 5). Analysis of the Ca
concentration
data using a modified Goldman equation for divalent ions gave a
permeability (P) ratio for P
:P
of
60:1. In other
words, the channels formed by native
-LIT were
Ca
-selective. Shatursky et al.(34) have shown that native
-LIT forms channels that
are selective for divalent cations. Channels also formed in inside-out
patches of membrane excised from locust extensor tibiae muscle fibers
using patch pipettes containing 10
M
-LIT (n = 17) or 10
M protein (n = 12) (Fig. 8). The conductances
and ion selectivities of these channels were similar to those observed
in the bilayer experiments, except that channels formed by the
recombinant protein inwardly rectified (Fig. 8A),
whereas those formed by native
-LIT exhibited outward
rectification (Fig. 8B). In terms of mechanism, it is
important to know whether channels are formed when a latrotoxin is
applied to the cytoplasmic face of a natural membrane, but we have not
yet developed a suitable protocol to test this. Despite this, it is
clear from the results of the studies with artificial membranes and
locust muscle membrane patches that if recombinant
-LIT inserts to
form channels, then its insertion is not influenced by net surface
charge, lipid content of membrane, and presence of surface sugars etc.
However, this does not seem to be true for the native toxin, because
the channels that it forms exhibit opposite rectification properties in
artificial and natural membranes.
Figure 7:
Channel formed by truncated, recombinant
protein and native -LIT in lipid bilayers. Current/voltage
characteristics for a lipid bilayer located at the tip of a patch
pipette and exposed, via the pipette solution, to 10
M recombinant protein (A) and 10
M native
-LIT (B). Note slight inward
rectification in each case (ratio of inward and outward conductances
= 0.75).
, the patch pipette saline contained 0.2 mM CaCl
and 190 mM NaCl; the bath contained 2
mM CaCl
and 190 mM NaCl.
, the
patch pipette contained 2 mM CaCl
and 190 mM NaCl; the bath contained 0.2 mM CaCl
and 190
mM NaCl. Note that only V
for channels
formed by native
-LIT was sensitive to the change in CaCl
distribution. The voltage is the pipette potential; the saline
bath into which the pipette tip was dipped was at earth potential (0
mV). Data points are mean single channel current amplitudes obtained
from frequency histograms of channel current amplitudes constructed
from at least 200 channel openings at each pipette potential. The
standard deviations of the means are smaller than the symbols. The
solid lines are linear regression fits to the data. Insets: A, inward single channel currents (openings upwards) of
2
pA recorded at a pipette potential of 60 mV; B, similar to A, but pipette potential was 40 mV. The single channel data
were filtered at 0.5 kHz cut-off.
Figure 8:
Channel formed in locust muscle membrane
patches. Current/voltage characteristics for channels formed by
10 M truncated, recombinant protein (A)
and 10
M native
-LIT in inside-out patches
excised from plasma membrane of locust metathoracic extensor tibiae
muscle fibers. Note slight inward rectification of channels formed by
recombinant protein (A) and strong outward rectification of
channels formed by native toxin (B).
, the patch pipette
saline contained 2 mM CaCl
and 190 mM NaCl; the bath contained 0.2 mM CaCl
and 190
mM NaCl.
, the patch pipette contained 2 mM CaCl
and 190 mM NaCl; the bath contained 40
mM CaCl
and 190 mM NaCl. Note that as in Fig. 7B, only V
for channels
formed by native
-LIT was sensitive to the change in CaCl
distribution. Data analysis and presentation same as for Fig. 7. Insets in A are single-channel records
obtained at pipette potentials of 100 mV (top; openings are
upwards) and -100 mV (bottom). Insets in B are single-channel records obtained at -45 and -56 mV.
The records were filtered at 0.5 kHz
cut-off.
In this study, we have used amino acid sequences obtained
from a novel insect-specific toxin, -LIT, to clone its cDNA, and
we have raised a specific antibody to native
-LIT to confirm the
identity of the clone as
-LIT. The data described herein
demonstrate that the primary structure of
-LIT exhibits features
in common with those of other cloned
latrotoxins(9, 10) , indicating that the latrotoxins
have evolved from a single ancestral gene. The greatest similarity
between these toxins is to be found in their N-terminal domains, where
the most noticeable structural feature is two hydrophobic segments that
are positionally well conserved (Fig. 1). The second hydrophobic
segment, which is shorter and has better defined borders, is one of the
most conserved regions between the latrotoxins and may, therefore, be
involved in an important aspect of latrotoxin function. Sequences
preceding the N-terminal domain, and which are removed during
maturation, differ greatly among
-LIT,
-LTX, and
-LIT.
These sequences are identified as separate domains in Fig. 3and
are probably signal sequences. The only structural feature common to
these sequences is a cluster of basic amino acid residues at positions
-1 to -4 that represents a potential endopeptidase cleavage
site, and is followed by an acidic N terminus of Glu and Asp residues.
The central domain of
-LIT is comprised of cdc10/ALR, but the ALR
domain is shorter in
-LIT than in other latrotoxins and the number
of the repeated units is reduced. The presence of ALRs in a large
number of functionally different proteins (e.g. cell cycle
proteins, enzymes, and transcription factors), which are widespread
from prokaryotes to human(31) , suggests that they do not have
a unique function but rather serve as a general structural domain. The
data on several of these proteins, particularly ankyrin itself,
indicate that they could mediate protein-protein
interactions(35, 36) . Although individual members of
an ALR may have distinct functions(37) , it seems likely that
an ALR domain acts as an integral structural unit and that the spatial
organization of its repeats is a functional determinant. Interestingly,
the vast majority of other proteins containing ALRs are either
intracellular or membranous (where the ALR domain is intracellular),
but the ALRs of
-LIT are extracellular. The role, if any, of the
ALRs in latrotoxin toxicity has not yet been established, although they
may be involved in the binding of toxin to presynaptic
receptors(6, 38) .
The structure of the protein
encoded in the -LIT gene predicts a molecule that is larger than
the mature toxin isolated from the venom, i.e. that there is a
disparity between the molecular weight of the toxin, as deduced from
the cDNA sequence, and the relative mobility of
-LIT purified from
BWSV. While the N terminus of
-LIT was identified unambiguously by
protein sequencing, the precise position of the C terminus was
difficult to determine, and it was unclear whether the apparent
molecular mass of the purified, native toxin on SDS-PAGE reflected an
electrophoretic artifact. Expression of the full-length
-LIT cDNA
in bacteria revealed that its calculated molecular mass is accurately
reflected in the relative mobility of the protein on SDS-polyacrylamide
gel electrophoresis and clearly showed for the first time that
functionally active latrotoxin in BWSV derives from proteolytic,
C-terminal processing. The full-length recombinant protein was not
insectotoxic, whereas C-terminal truncation produced a protein that
exhibited many of the properties of native
-LIT, a result that may
be relevant to other latrotoxins. Comparison of the primary structure
of the C-terminal domain of
-LIT,
-LTX ,and
-LIT shows
that the three latrotoxins are similar in this region, which suggests
that this domain may have an important biological function. Our
prediction that the cDNA of
-LIT encodes a protein that is
substantially larger than the toxin purified from BWSV (3) is
supported by the experimental data described herein. In view of the
conservation of the C-terminal domain among latrotoxins, we propose
that C-terminal processing is required for all members of this family.
In support of this contention, it has recently been shown by MALDI-MS
that native
-LTX is C-terminally truncated. (
)
Although it has been firmly established that
latrotoxins cause a massive release of transmitter from axon terminals,
the mechanism underlying this phenomenon is not well understood. A
putative receptor for -LTX has been
purified(39, 40) . This comprises two structurally
similar protein subunits (200 and 160 kDa, respectively)(40) .
The subunits are thought to form a complex with others that do not bind
-LTX. Is this receptor involved in the formation by
-LTX of
ion channels in axon terminal membrane? Support for this possibility
comes from studies of channel formation by
-LTX in the surface
membrane of Xenopus laevis oocytes.
-LTX channels were
formed in oocytes injected with RNA extracted from rat brain, but not
in uninjected oocytes(41) . However, latrotoxins form channels
in artificial membranes lacking other proteins (this
study)(34) , and in native membranes as well, such as locust
muscle membrane, in which one might not expect to find latrotoxin
receptors. Nevertheless, although in these cases latrotoxin receptors
are not essential to the formation of channels by latrotoxins, their
presence may greatly enhance this phenomenon. The involvement of
receptors in latrotoxin-induced release of neurotransmitter is an
attractive proposal because it provides a ready explanation for the
phylum specificities of the latrotoxins. It is difficult to understand
how a single
-LIT molecule could form an ion channel because,
according to hydropathy profile analysis, it contains only two
hydrophobic regions that are of a sufficient length to constitute a
conventional membrane-spanning
-helix. Also, the primary structure
of
-LIT does not reveal direct sequence homology with proteins
that form channels. However, it has been shown that
-LTX molecules
aggregate in solution(42) , so it is possible that a latrotoxin
ion channel is formed from several toxin molecules.
Our initial
hypothesis that the multifunctional activity of a latrotoxins requires
the presence of a low molecular weight protein (LMWP) is clearly
incorrect because the truncated, recombinant protein was toxic and
induced transmitter release in the absence of a LMWP. However, the
different permeability properties of the channels induced by native
-LIT on the one hand and truncated, recombinant
-LIT on the
other hand require an explanation. Possibly, there are differences in
posttranslational modifications of the two toxins, although native
-LTX is neither glycosylated nor phosphorylated. When
-LTX is
purified from BWSV by conventional biochemical methods it is always
tightly associated with a LMWP (7947 Da) (12) called
lactrodectin(13) . There are other LMWPs in BWSV, and,
according to immunochemical studies, at least one of these (LMWP2) is
present in venom fractions containing latroinsectotoxins(43) .
Although there is no direct evidence that a LMWP is associated with
native
-LIT, its presence could account for the Ca
selectivity of the channel formed by this protein. It could also
account for the higher slope conductance of the channel that it forms
in both artificial and natural membranes (almost twice that of channels
formed by the recombinant protein) and for the outward rectification of
the channel that it forms in locust muscle membrane patches. Although
the presence of a LMWP is clearly not essential for the induction of
transmitter release from locust motor nerve terminals by the
recombinant protein, further comparative studies of native
-LIT
and the recombinant protein are required to determine whether there are
subtle differences in their properties in this respect.
It has been
noted previously that the presence of Ca is not
essential for the presynaptic action of
latrotoxins(8, 44) , although other divalent cations
may be required if Ca
is absent. In the studies
reported herein,
-LIT and the truncated, recombinant protein
increased mEPSP frequency in saline containing no divalent cations.
However, chelators were not used, so we cannot be certain that the
extracellular concentrations of Ca
and Mg
were indeed zero. Nevertheless, this result is essentially
similar to that obtained by Magazanik et al.(8) in
their studies of
-LIT in which divalent cation-free saline
containing EGTA was used. Normally, extracellular Ca
plays a primary role in the action of the latrotoxins on motor
nerve terminals; but Mg
can substitute for
Ca
. Perhaps entry of Mg
into an
axon terminal through channels induced by a latrotoxin slowly exchanges
with Ca
in intracellular stores and this leads to
enhanced transmitter release. However, the concentration of
Ca
in synaptosomes, as measured by Fura-2
fluoresence, does not rise when transmitter release is induced by
-LTX in Ca
-free saline(44) . In the
present study, when the extracellular concentration of MgCl
was raised while keeping the concentration of CaCl
constant (2 mM), the increase in mEPSP frequency induced
by
-LIT was delayed. Competition between Mg
and
Ca
for transport through the toxin-induced channels
could account for this.
By virtue of their potent and specific
action on transmitter release from nerve terminals, the latrotoxins
have considerable potential as tools for investigating mechanisms of
transmitter release in nervous systems. The ability to synthesize a
functional, recombinant latrotoxin offers exciting opportunities for
using site-directed mutagenesis to identify those structural features
that contribute to the mode of action of -LIT. The structural and
functional information on cloned and native
-LIT presented herein
has emphasized the common structural organization of the latrotoxins,
although it is not yet clear from their sequence differences how they
exhibit distinct phylum specific toxicities. It is clear, however, that
the latrotoxins have a common domain organization and that only two
domains participate in their biological function. Nevertheless, the
parts of these proteins that are responsible for high affinity binding,
multimerization and formation of channels remain to be determined. The
important question of how the unique insect specificity of
-LIT is
achieved could be addressed in future experiments using hybrids between
different latrotoxins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X92679[GenBank].