Download Fluorescently-Labeled Toxins

Mini Review
Fluorescently-Labeled Toxins:
Novel Tools for Working with Live Cells
Nadia Sinai, B.Sc., Alon Meir, Ph.D. and Oren Bogin, Ph.D.
Venom proteins and peptides have emerged as invaluable tools for research, drug
discovery and drug development due to their small size, rigid structure, high potency and
selectivity. The use of labeled toxins will open up new research avenues which will help to
better understand ion channel function, with regard to biophysical and pharmacological
properties, tissue distribution, and to isolate specific channel activities in living cells.
Venom proteins and peptides have emerged as
invaluable tools for research, drug discovery
and drug development.1,2 Ion channel research
tools that are currently available include highly
specific antibodies and radiolabeled toxins.
Although Alomone Labs offers several antibodies
with extracellular epitopes, most work with
“live-cells” or native tissues is hampered by the
need to permeabilize the cells (for intracellular
epitopes) or by the high sequence homology
between ion channel extracellular domains, which
makes it difficult to isolate specific epitopes.
Using radiolabeled toxins requires working with
radioactivity, which limits the laboratories that
can use such techniques and another drawback is
that the resolution obtained by autoradiography
is low.3
The ability to produce toxins labeled with
a detectable probe (radioligands, biotin or
fluorescent dyes) has radically broadened the
use of toxins to include determination of ion
channel extracellular localization, distribution,
affinities, and number of binding sites, in live
cells. Detectable probes can be introduced into
the toxins by a number of techniques:
a) chemical synthesis at the terminal ends, or at a
specified location4,5
b) site directed mutagenesis to liable and active
amino acid residues 6-11
c) use of specific chemistries including Nhydroxysuccinimidyl ester chemistry (for
ε-amino groups of lysine), maleimide chemistry
(for sulphidryl moieties) and iodonization of
tyrosines.12-21
22
The last group of methods, although the most
common, are crude, and the detectable probe
can bind to the protein at unknown locations
and with unknown stochiometry. Even more
importantly, it may bind to crucial residues which
participate in the toxin-receptor binding interface.
Numerous papers have described the structure/
function of toxins, based on mutagenesis
experiments, molecular simulation, and structure
determination. The mode of action of these toxins
can be roughly divided into two major groups:
the “pore-plungers” and the “voltage-sensor
modulators” or “gating-modifiers”. “Poreplungers” bind to the pore region and physically
block the ion transport through the pore. For
example, the majority of K+ channel blockers
are characterized by the presence of a “dyad”
or “triad”, having a Lys in structural proximity to
either one or two hydrophobic residues (Phe, Tyr,
Trp) (Fig. 1).21 The Lys residue is “plunged” into
the pore and interacts with the channel’s Asp
residues inside the pore, while the hydrophobic
residues interact with hydrophobic residues on
the pore outer-surface.22,23 If the exposed Lys
is bound to a hindering dye moiety or the Tyr
residue is iodinated, the labeled protein may
be less active than the native form. This can
also occur with any bulky moiety at the toxinchannel interface, or with a secondary structural
perturbation due to dye or linker conjugation.
In light of these facts, dyes or radioligands
should be selectively introduced on the other
“face” (or “back”) of the toxin, at a specific
location, with well defined stochiometry. Sitespecific introduction of fluorescent dyes was
demonstrated for a number of toxins, including
Iberiotoxin, Hongotoxin, and ShK K+ -channels
blockers.3-5,10,11 Hafidi et al. were able to stain and
localize KCa1.1 (BK or Slo K+ channels) in cochlear
hair cells and tissues, using a mutated Asp19CysIberiotoxin labeled with Alexa488.10 Bingham et
al. chemically introduced a biotin derivative of Lys
at the same Asp19 of Iberiotoxin, and were able to
visualize KCa1.1 stably-transfected HEK-293 cells
using Streptavidin–Alexa488.4
Hongotoxin is a known blocker for K V1.1, K V1.2,
K V1.3 and K V1.6, with IC50 of 31pM, 170 pM,
86 pM and 6nM respectively.(see review in
this Modulator issue) By introducing Cys19Ala
of Hongotoxin (Ala19Cys-Hongotoxin, the
structural equivalent to Asp19 of Iberiotoxin)
Fig. 1: NMR Structure of Agitoxin-2
NMR structure of Agitoxin-2 (1AGT.pdb). Gly1, Thr9, Gly10,
Ser11, Arg24, Phe25, Lys 27, Met 29, Asn30 and Thr36
have been found to participate in the interaction with Kv1.3
(marked in green). 26 Asp20, located at the opposite face
of the protein was replaced with Cys (marked in red, see
article).Images were generated using the ViewerLite freeware
(Accelrys Inc.).
Modulator
No.21 Fall 2006 www.alomone.com
and binding to a number of fluorescent dyes,
Pragl et al. were able to obtain a toxin with the
same activity as the native toxin, which binds
to cells and colocalizes with Kv1.2 in brain
slices.3 Furthermore, Freudenthaler et al. were
able to visualize single channels on Jurkat T-cell
lymphocytes using ultrasensitive microscopic
methods to determine the dissociation and
off-rates of Hongotoxin, using the Cy5-Ala19CysHongotoxin.11 In another example, ShK, a toxin
isolated from the sea anemone Stichodactyla
helianthus, was found to be a highly potent Kv1.3
blocker with IC50 of 11pM.27 Beeton et al. have
introduced a number of fluorescent and biotin
tags to the N-terminal arginine residue, and used
these toxins to discriminate between activated
T-lymphocytes and resting T-lymphocytes, which
are differentiated by the high expression levels
of voltage-gated K V1.3 channels.5 Furthermore,
the authors reported detection at a level as low as
600 channels per cell.5
Agitoxin-2, a 38 amino acid toxin containing
3 disulfide bonds, isolated from the venom of
the scorpion Leiurus quinquestriatus hebraeus,
was successfully expressed and its NMR
structure solved (1AGT.pdb).24-25 Biochemical,
pharmacological and molecular simulation
experiments have identified the residues Gly1,
Thr9, Gly10, Ser11, Arg24, Phe25, Lys 27, Met 29,
Asn30 and Thr36 at the agitoxin-2/K V-channel
interface (Fig. 1).26 As was outlined previously,
point mutations were introduced at the other
non-interacting “face” of the toxin, thus reducing
the probability of hampering the toxin activity
after introduction of a detectable probe. Asp20
of Agitoxin-2 (the structural homolog of Asp19
Iberiotoxin and Ala19 Hongotoxin) was replaced
by Cys, and this mutant was recombinantly
expressed, purified, and reacted with TAMRAmaleimide, to form Asp20Cys-AgiTx-2-TAMRA
rAgitoxin-2-Cys-TAMRA (#RTA-420-T). rAgitoxin2-Cys-TAMRA was bioassayed for the inhibition of
K V1.3 currents, both in Xenopus oocytes and K V1.3
stably-transfected HEK cells, and it activity was
found to be identical to that of rAgitoxin-2 (#RTA420) (Fig. 4). Furthermore, rAgitoxin-2-Cys-TAMRA
was tested for its ability to fluorescently-label
K V1.3 stably-transfected HEK cells. As seen in
Fig. 2 and 3, rAgitoxin-2-Cys-TAMRA was able to
detect the functional K V1.3 channels in live cells
without fixation, and labeled the channels on the
cell membranes, as seen by the overlay picture,
in less than 30 minutes. Moreover, no labeling
was observed when the cells were pre-incubated
with a competing non-labeled rAgitoxin-2, and
no background binding was observed on nontransfected HEK cells (Fig. 2 and 3).
Fig. 2: Kv1.3 Expressing HEK-293 Cells Labeled with rAgitoxin-2-Cys-TAMRA
Confocal image of Kv1.3 expressing HEK-293 cells labeled with a fluorescent rAgitoxin-2-Cys-TAMRA (#RTA-420-T).
Kv1.3 expressing HEK-293 cells were grown on glass cover-slips, incubated in the presence of 50nM rAgitoxin-2-Cys-TAMRA for
30 minutes at room temperature and washed with PBS.
Left: Bright light field image of the cells.
Middle: Image acquired with a Rodamine filter.
Right: Merge of both images. As can be seen, rAgitoxin-2-Cys-TAMRA is localized with the membrane-anchored Kv1.3 K+ channel.
We would like to thank Mr. Vladimir Kiss and Dr. Noga Alagem from the Weizmann Institute of Science for their help with the
confocal microscope images.
Fig.3: rAgitoxin-2-Cys-TAMRA Binding to HEK Cells Stably Transfected with K V1.3 Channels
HEK K V1.3 transfected
50 nM rAgitoxin-2-Cys-TAMRA
10 µM rAgitoxin-2
+ 50 nM rAgitoxin-2-Cys-TAMRA
HEK non-transfected
50 nM rAgitoxin-2-Cys-TAMRA
Top: Staining of HEK-293 cells stably expressing K V1.3 channels with 50 nM of rAgitoxin-2-Cys-TAMRA (#RTA-420-T). Cells were
Alomone Labs is proud to launch rAgitoxin-2Cys-TAMRA as the first member in a new line of
fluorescently-labeled toxins, which we believe will
be invaluable tools for channel research both in
live cells and in tissues. Please check our website
for new labeled toxins.
Modulator No.21 Fall 2006 www.alomone.com
incubated for 30 minutes with the fluorescent toxin, washed with PBS and visualized. In the middle section the image acquired
with a Rodamine filter is shown and on the right it is merged with the bright light image of the same field to illustrate cellular
staining.
Middle: Displacement of labeled toxin by rAgitoxin-2 (#RTA-420). The same preparation as the one shown on top was
pre-incubated for 30 minutes with 10 µM of rAgitoxin-2, followed by another 30 minute incubation after addition of 50 nM
rAgitoxin-2-Cys-TAMRA.
Bottom: rAgitoxin-2-Cys-TAMRA does not stain non-transfected HEK-293 cells. The procedure was as in the top panel.
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Fig. 4: rAgitoxin-Cys-TAMRA Retains the Inhibitory Characteristics of the Wild Type Toxin
References:
1. Bogin, O. (2005) Venom Peptides and their Mimetics as Potential
A
B
Drugs, Modulator Issue #19 www.alomone.com.
2. Lewis, R.J. and Garcia M.L (2003) Nat. Rev. Drug. Discov. 2, 790.
3. Pragl, B. et al. (2003) Bioconjug. Chem. 13, 416.
4. Bingham, J.P. et al. (2006) Bioconjug. Chem. 17, 689.
5. Beeton, C. et al. (2003) J. Biol. Chem. 278, 9928.
6. Wanner, S.G et al. (1999) Biochemistry 38, 5392.
7. Koschak, A. et al. (1997) Biochemistry 36, 1943.
8. Grunnet. M. et al. (1999) Am. J. Physiol. 277, G22.
9. Shimony, E. et al. (1994) Protein Eng. 7, 503.
10. Hafidi, A. et al. (2005) Neuroscience 130, 475.
11. Freudenthaler, G. et al. (2002) Histochem. Cell Biol. 117, 197.
12. Darbon, H. and Angelides, K.J. (1984) J. Biol. Chem. 259, 6074.
13. Angelides, K.J. (1989) Methods Cell Biol. 29, 29.
14. Jones, O. T. et al. (1989) Science 244, 1189.
15. Cohen, M.W. et al. (1991) J. Neurosci. 11, 1032.
16. Angelo, K. et al. (2003) Pflugers Arch. 447, 55.
17. Massensini, A.R et al. (2002) J. Neurosci. Methods 116, 189.
18. Sekine-Aizawa, Y. and Huganir, R.L. (2004) Proc. Natl. Acad. Sci.
U.S.A. 101, 17114.
19. Wang, H. et al. (2003) Nature 421, 384.
20. Racape, J. et al. (2002) J. Biol. Chem. 277, 3886
21. Jimenez-Gonzalez, C. et al. (2003) Eur. J. Neurosci. 18, 2175.
22. Mouhat, S. et al. (2005) J. Pept. Sci. 11, 65.
23. Rodrıguez de la Vega, R.C. et al. (2003) Trends Pharmacol. Sci. 24,
222.
24. Garcia, M.L. et al. (1994) Biochemistry 33, 6834.
25. Krezel, A.M. et al. (1995) Protein Sci. 4, 1478.
26. Gao, Y.D. and Garcia, M.L (2003) Proteins 52, 146.
27. Kalman, K et al. (1998) J. Biol. Chem. 273, 32697.
A. K V current inhibition by rAgitoxin-2 (#RTA-420) (100nM, pink) and rAgitoxin-2-Cys-TAMRA (#RTA-420-T) (100nM, blue) in
K V1.3 transfected HEK-293 cells.
Top: Time course of K V1.3 current amplitude is presented. Currents were elicited from holding potential of –100 mV by 200
ms voltage pulses to +50 mV, every 10 seconds. Periods of toxin perfusions are indicated according to the color code defined
Related Products
Compound
Product #
above.
Toxins
Bottom: Superimposed traces taken from the same experiment shown on top, before (black) or during toxin perfusion (pink or
rAgitoxin-2 ____________________________________
rAgitoxin-2-Cys ________________________________
rAgitoxin-2-Cys-TAMRA _________________________
rCharybdotoxin _______________________________
blue).
B. Dose response of K V1.3 current inhibition in Xenopus oocytes for rAgitoxin-2, rAgitoxin-2-Cys-TAMRA and rAgitoxin-2-Cys
(#RTA-420-C).
Top: time course of current amplitude, upon toxin perfusion. Currents were elicited from resting potential of –100 mV, by a 100 ms
test pulse to 0 mV, delivered every 10 sec. The bars above the plot represent periods of toxin perfusion, for each toxin sequential
RTA-420
RTA-420-C
RTA-420-T
RTC-325
rHongotoxin-1 _________________________________ RTH-400
rIberiotoxin ___________________________________ RTI-400
Stichodactyla (ShK) toxin _______________________ S-400
applications of the following concentrations: 1, 10, 50 and 100 nM, where the toxin species is indicated.
Antibodies
Middle: Superimposed traces representing currents under control conditions (black) and during perfusion of 100 nM toxin:
Anti-Kv1.3 (extracellular)-FITC ___________________ APC-101-F
Anti-hK V11.1 (HERG) (extracellular)-FITC _________ APC-109-F
Anti-P2X7 (extracellular)-FITC ___________________ APR-008-F
rAgitoxin-2-Cys-TAMRA (orange), rAgitoxin-2 (blue) and rAgitoxin-2-Cys-TAMRA (green).
Bottom: Dose response of toxins on current inhibition (n=3 for each toxin, mean and SD). See inset for color coding.
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No.21 Fall 2006 www.alomone.com