fluorophores representing 
 a variety of scaffolds and excitation / emission spectra were selected 
 for this research . using a modified fischer condensation reaction , 
 brevenal ( 1 )
was reacted with fluorophore hydrazides 
 to give the brevenal fluorophore conjugates 25 and biotinylated brevenal ( 6 ) ( scheme 1 ) .
the brevenal conjugates were then evaluated for their 
 affinity 
 for the brevenal binding site using the radioligand binding assay . 

the dansyl derivative ( 2 ) did not bind effectively to 
 the brevenal binding site and was eliminated from further research . 

the coumarin ( 3 ) , texas red ( 4 ) , and bodipy 
 fl ( 5 ) fluorescent conjugates and the biotin analogue 
 ( 6 ) showed good affinity for the binding site ( ki = 236 , 156 , 340 , and 318 nm , respectively ) , 
 as compared to brevenal ( ki = 149 nm ) . to determine the optimal excitation and emission wavelengths for 
 the brevenal conjugates , the fluorescent brevenal ligands were diluted 
 to a concentration of 10 m in assay buffer , and the absorbance 
 and emission spectra
optimal excitation ( ex ) and emission 
 ( em ) wavelengths were chosen as follows : ex:438 nm;em:474 nm for coumarin - brevenal 
 ( 3 ) , ex:495 nm;em:525 nm for bodipy fl - brevenal ( 5 ) , and ex:585 nm;em:620 nm for texas red - brevenal ( 4 ) . in order to characterize binding and 

determine the equilibrium 
 dissociation constant ( kd ) of compounds 35 , saturation binding experiments were 
 performed using serial dilutions of the fluorescent ligands in the 
 presence and absence of brevenal ( 10 m ) .
all three fluorescent 
 brevenal ligands bound to rat brain synaptosomes ( figure 1 ) . total binding of the conjugates was concentration - dependent 
 and nonlinear , while nonspecific binding of each was concentration - dependent 
 and linear ( figure 1a  c ) .
subtraction 
 of nonspecific binding from total binding yielded the specific saturation 
 binding component .
nonspecific binding was determined to be highest 
 for the texas red - brevenal conjugate ( 4 ) and lowest for 
 bodipy fl - brevenal ( 5 ) .
to ensure saturation had been 
 achieved , the concentrations were increased for each fluorescent ligand 
 to at least 1 m , but there was no resulting increase in specific 
 binding curves ( data not shown ) .
saturation binding and inhibition of texas 
 red - brevenal ( a ) , coumarin - brevenal 
 ( b ) , and bodipy fl - brevenal ( c ) binding to rat brain synaptosomes . 

total binding without competition (  ) and nonspecific binding 
 with competition from 10 m brevenal (  ) was measured 
 using relative fluorescence units ( rfus ) .
specific binding (  ) 
 was calculated from the difference between mean total binding and 
 mean nonspecific binding at each indicated concentration of the fluorescent 
 brevenal ligands .
( d ) competition 
 for binding was assayed using texas red - brevenal (  , dotted 
 line , 10 nm , n = 6 ) , coumarin - brevenal (  , 
 dashed line , 100 nm , n = 6 ) , and bodipy fl - brevenal 
 (  , solid line , 10 nm , n = 12 ) against varying 
 concentrations of unlabeled brevenal ( 10 pm to 10 m ) to determine 
 inhibition of binding . to identify which fluorescent ligand would be best for competition 
 assays , given the nonspecific binding of each fluorescent ligand , 
 we next measured the percent of total binding for brevenal displacement 
 of texas red - brevenal ( 10 nm ) , coumarin - brevenal ( 100 nm ) , and bodipy 
 fl - brevenal ( 10 nm ) .
as shown in figure 1d , 
 texas red - brevenal ( 4 ) exhibited the highest background 
 and/or nonspecific binding interference , while bodipy fl - brevenal 
 ( 5 ) exhibited the lowest background and/or nonspecific 
 binding interference .
in addition , coumarin - brevenal was the only 
 fluorescent ligand that could not be measured with a competition assay 
 at 10 nm , likely due to lower brightness and thus less fluorescence 
 signal at this concentration of fluorescent ligand , coupled with the 
 lower limit of detection by the fluorescence plate reader . because 
 of the limitations for the texas red- and coumarin - brevenal derivatives 
 and the associated lower nonspecific binding of the bodipy fl - brevenal 
 derivative to rat brain synaptosomes , we chose bodipy fl - brevenal 
 as the ligand for further characterization and comparison to the radioligand 
 binding assay .
h nmr spectra for the compounds ( cd3od , 500 mhz ) can be viewed in the supporting 
 information .
nonlinear regression analysis of the specific 
 binding component of the saturation assays indicated that bodipy fl - brevenal 
 bound to rat brain synaptosomes with a kd of 264  46 nm ( n = 15 ) . in order to confirm the accuracy and precision 
 of the fluorescent ligand binding assay ,
a series of inhibition binding 
 experiments were performed using biotinylated brevenal and various 
 naturally occurring pbtx analogues ( pbtx-1 , pbtx-2 , pbtx-3 , and pbtx-9 ) 
 compared to unlabeled brevenal itself . as shown in figure 2 , both biotinylated brevenal and unlabeled brevenal 
 competed for binding to synaptosomes .
none of the naturally occurring 
 pbtx analogues were able to displace bodipy fl - brevenal from synaptosomes . 

this is in agreement with previous findings using the radioligand 
 brevenal receptor binding assay .
competition for binding was assayed using bodipy fl - brevenal ( 10 nm ) 
 against varying concentrations of nonfluorescent ligands : brevenal 
 (  ) , biotinylated brevenal (  ) , pbtx-1 (  ) , pbtx-2 
 (  ) , pbtx-3 (  ) , and pbtx-9 (  ) from 1 pm to 
 10 m in order to determine inhibition of bodipy fl - brevenal 
 binding to rat brain synaptosomes .
the thick solid line denotes brevenal 
 binding ( n = 12 ) , the dashed line denotes biotinylated 
 brevenal binding ( n = 12 ) , and the thin lines denote 
 the lack of binding curves generated from the various pbtx analogues 
 ( n = 6 ) . using the kd of bodipy fl - brevenal 
 determined from saturation experiments , ki values were calculated for brevenal and biotinylated brevenal ( table 1 ) .
in addition , a series of radioligand binding 
 experiments were also performed in order to validate the fluorescent 
 ligand binding assay . as shown in table 1
, 
 there was no significant difference in ki values calculated for brevenal or biotinylated brevenal when using 
 either the fluorescent ligand or radioligand binding assays ( p > 0.05 ; student s t - test ) . 

this 
 indicates that the fluorescence - based assay is a viable substitute 
 for the radioligand - based assay .
additionally , on the basis of time 
 to complete an assay ( 2 days for radioligand versus less than 2 h 
 for fluorescent ligand ) , this new method will allow a much higher 
 throughput of samples .
fluorescent - based assays may be able to detect 
 natural product 
 ligand  receptor interactions that may be overlooked in the 
 traditional radioligand assays , because of the higher background associated 
 with radioligands .
the most recently 
 described radioligand binding assay for brevenal used a more tedious centrifugational method because all 
 attempts at using a simplified , high - throughput multiwell filter plate 
 format resulted in an assay with extraordinarily high background . 

the fluorescent - based multiwell filter plate assay described in this 
 report has relatively low background and therefore may provide a platform 
 that can discern more subtle ligand  binding site interactions . 

however , it is important to note that fluorescence - based assays can 
 be problematic if the addition of a bulky fluorescent probe causes 
 binding issues ; therefore , optimization of the fluorescence - based 
 assays is critical .
interestingly , we have previously found that the 
 addition of the bodipy - fl molecule to brevetoxin actually increased 
 binding affinity when compared to the radioactive brevetoxin .
while the mechanisms behind this observation 
 are currently unknown , other studies have shown that conjugating bodipy 
 molecules to various ligands may slightly increase or decrease binding 
 affinity for the receptors .
even though there were small changes in 
 the binding affinity of the bodipy derivatives , they still remained 
 effective in establishing binding constants . in this study ,
bodipy fl - brevenal was found to bind with a higher 
 inhibition constant than brevenal alone on the radioligand assay ( 149 
 nm vs 340 nm , respectively ) .
while the addition of the bodipy fl molecule 
 did affect the binding affinity , it was still found to bind with sufficient 
 affinity to be used as a suitable ligand for the development of a 
 fluorescence - based receptor binding assay .
furthermore , a fluorescent 
 ligand with lower affinity for the brevenal binding site could conceivably 
 be more sensitive in detecting binding by brevenal derivatives , as 
 it is easier to displace .
indeed , most modifications to the brevenal 
 structure have been found to decrease affinity to the binding site .
to further 
 examine the nature of the brevenal binding site , a series of inhibition 
 experiments were performed using other site - specific vssc ligands , 
 including tetrodotoxin ( site 1 ) , veratridine and aconitine ( site 2 ) , 
 scorpion toxins from leiurus quinquestriatus ( site 
 3 ) and centruroides sculpturatus ( site 4 ) , and deltamethrin 
 ( site 7 ) .
as shown in table 2 , none of these 
 ligands were able to displace bodipy fl - brevenal from rat brain synaptosomes , 
 suggesting that brevenal does not bind to vssc sites 15 or 
 7 .
these data are in agreement with previous findings using the radioligand 
 brevenal binding assay . to determine if brevenal binds to vsscs , a receptor 
 extraction 
 experiment was performed to isolate the protein and/or protein complex(es ) 
 that bind to brevenal under native conditions .
these experiments were 
 conducted in nondenaturing conditions to preserve the binding site 
 of brevenal , as it could involve several incongruent sites along the 
 primary protein sequence when it is folded together in a secondary 
 or tertiary structure . as shown in figure 3a ,
total protein was isolated using the biotin - brevenal conjugate , 
 resulting in an intense protein band at approximately 250 kda in the 
 membrane extract .
this band was confirmed as positive for the alpha 
 subunit of vsscs using western blot analysis ( figure 3b ) .
the smaller band at 40 kda in the membrane extract of 
 the western blot is unknown at this time , but it could be nonspecific 
 binding by the antibody to another protein , or it could be a piece 
 of the alpha subunit that was cleaved by native proteases in the tissue 
 during protein extraction .
these results , in conjunction with the 
 receptor binding screening of other vssc ligands , suggest that brevenal 
 elicits its action through a novel mechanism , which may be through 
 a presently unknown binding site on vsscs or another protein in the 
 vssc complex ( e.g. , a beta subunit or other protein associated with 
 noncovalent interactions to the alpha subunit of the vssc ) .
generally , 
 the alpha subunit of the vssc is associated with up to three different 
 beta subunits .
in addition , modulation of vsscs 
 by other receptors ( e.g. , acetylcholine receptors , receptor tyrosine 
 kinases ) and secondary messenger systems ( e.g. , calcium / calmodulin , 
 g - protein subunits g , protein kinases , tyrosine 
 phosphatases ) indicates association of vsscs with other cellular and 
 membrane proteins and/or extracellular matrix proteins .
ongoing work in our laboratory is directed toward identifying the 
 brevenal binding site in vsscs .
coomassie stain and western blot of the 
 brevenal receptor protein 
 isolated from rat brain .
protein was extracted from rat brains , and 
 the brevenal receptor protein complex was isolated using the biotin - brevenal 
 conjugate and streptavidin - conjugated magnetic beads .
isolated protein 
 ( cytosolic compartment , lanes 1 and 4 ; membrane compartment , lanes 
 2 and 5 ; nuclear compartment , lanes 3 and 6 ) was denatured , separated 
 by size , and stained with coomassie brilliant blue ( panel a ) to determine 
 total protein bands , as well as stained with an antisodium channel 
 antibody in western blot analysis ( panel b ) .
brevenal binds to a protein 
 complex of different sized proteins ( panel a ) , of which the approximate 
 250 kda band stains positively for the alpha subunit of vsscs ( panel 
 b ) .
because 
 of the hazards associated with radioactive 
 material usage , there exists considerable need in drug discovery and 
 natural product research for nonradioactive assays to examine receptor  ligand 
 interactions .
the development of novel fluorescence - based techniques 
 has allowed for the elucidation of complex receptor interactions for 
 a variety of important targets , such as the g - protein coupled receptors .
fluorescence - based assays have several advantages over radioligand 
 assays , including lower nonspecific binding and background , production 
 of far less hazardous waste , and simpler and faster protocols .
given 
 the advantages of the fluorescent platform , we developed a fluorescence - based 
 receptor binding assay for the brevenal binding site on rat brain 
 synaptosomes .
this assay has already shown utility in screening for 
 selective agonists for the brevenal binding site .
in addition , work 
 is ongoing in our laboratory to use compounds screened by the fluorescence - based 
 brevenal receptor binding assay to aid in the further identification 
 of the brevenal binding site and identify novel ligands for this binding 
 site .
brevenal , pbtx-1 , pbtx-2 , 
 pbtx-3 , and pbtx-9 were purified from cultures of karenia 
 brevis ( wilson strain ) as previously described .
reagent grade sucrose , sodium phosphate dibasic , trizma base , hepes , 
 choline chloride , glucose , egta , bovine serum albumin ( bsa ) , glycine , 
 protease inhibitor cocktail , veratridine , aconitine , leiurus 
 quinquestriatus scorpion venom , and centruroides 
 sculpturatus scorpion venom were purchased from sigma - aldrich . 

reagent grade potassium chloride , magnesium 
 sulfate , etoh , sodium dodecyl sulfate ( sds ) , glacial acetic acid , 
 and acs certified petroleum ether were purchased from fisher scientific . 

hplc grade meoh 
 and ch3cn and lc - ms grade ch3cn with 0.1% formic 
 acid were purchased from honeywell burdick and jackson .
sterile pbs and the 
 fluorophores bodipy fl hydrazide ( d2371 ) , diethylaminocoumarin-3-carboxylic 
 acid hydrazide ( d355 ) , texas red hydrazide ( t6256 ) , and dansyl hydrazine 
 ( d100 ) were all purchased from life technologies .
the fluorescent dyes bodipy fl hydrazide ( d2371 ) , 
 7-diethylaminocoumarin-3-carboxylic acid hydrazide ( d355 ) , and texas 
 red hydrazide ( t6256 ) , dansyl hydrazine ( d100 ) , and ( + ) -biotin hydrazide 
 were conjugated to brevenal using a modified fischer reaction .
a one - pot reaction was performed for each fluorophore 
 using a 1:1 molar ratio of brevenal to fluorophore in dmf .
the reaction 
 conditions were carried out using a catalytic amount of tungstophosphoric 
 acid at 60 c for 4 h with stirring .
the dmf was evaporated to 
 dryness under vacuum , and the residual material was resuspended in 
 a small amount of meoh and filtered ( 0.2 m nylon syringe filter ) 
 ( fisher scientific ) .
compounds 2 , 3 , 5 , and 6 were purified using 
 a phenomenex luna c18 column , with mixtures of meoh and 
 h2o as the eluents .
compound 4 was purified 
 using an agilent zorbax c18 column using a mixture of meoh 
 and h2o modified with 0.1% formic acid as the eluent .
structures of 
 the fluorescent brevenal conjugates were confirmed using h nmr experiments and were performed on a bruker avance 500 mhz spectrometer 
 equipped with a 1.7 mm txi probe at 298 k. pale yellow solid 
 in 61% yield ; h nmr ( cd3od ) ,  0.89 ( 1h , 
 t , j = 7 hz ) , 0.92 ( 3h , d , j = 7 
 hz ) , 1.04 ( 3h , s ) , 1.12 ( 3h , s ) , 1.15 ( 3h , s ) , 1.45 ( 4h , m ) , 1.65 
 ( 2h , m ) , 1.74 ( 6h , m ) , 1.89 ( 5h , m ) , 2.02 ( 5h , m ) , 2.16 ( 4h , m ) , 2.86 
 ( 7h , m ) , 3.24 ( 2h , m ) , 3.32 ( 2h , m ) , 3.49 ( 1h , m ) , 3.66 ( 1h , m ) , 3.72 
 ( 1h , m ) , 3.95 ( 1h , m ) , 5.09 ( 1h , m ) , 5.19 ( 1h , dd , j = 16 and 2 hz ) , 5.44 ( 1h , m ) , 5.78 ( 1h , m ) , 6.03 ( 1h , m ) , 6.69 ( 1h , 
 m ) , 7.26 ( 1h , d , j = 8 hz ) , 7.58 ( 1h , m ) , 7.93 ( 1h , 
 d , j = 9 hz ) , 8.26 ( 1h , d , j = 7 
 hz ) , 8.38 ( 1h , d , j = 9 hz ) , 8.56 ( 4h , m ) ; hresims m / z 904.5140 [ m + h ] ( calculated 
 for c51h74n3o9s , 905.5140 ) .
pale yellow solid 
 in 99% yield ; h nmr ( cd3od ) ,  0.90 ( 4h , 
 t , j = 7 hz ) , 0.97 ( 3h , d , j = 7 
 hz ) , 1.04 ( 3h , s ) , 1.13 ( 3h , s ) , 1.20 ( 3h , s ) , 1.25 ( 6h , t , j = 7 hz ) , 1.37 ( 2h , m ) , 1.57 ( 3h , m ) , 1.64 ( 3h , m ) , 1.76 
 ( 5h , m ) , 1.89 ( 3h , s ) , 1.92 ( 2h , s ) , 2.05 ( 3h , m ) , 2.09 ( 3h , s ) , 2.26 
 ( 3h , m ) , 3.26 ( 2h , m ) , 3.34 ( 2h , m ) , 3.55 ( 5h , m ) , 3.97 ( 1h , m ) , 4.10 
 ( 3h , m ) , 5.09 ( 1h , d , j = 10 hz ) , 5.19 ( 1h , d , j = 17 hz ) , 5.43 ( 1h , m ) , 5.95 ( 1h , m ) , 6.03 ( 1h , t , j = 11 hz ) , 6.42 ( 1h , d , j = 10 hz ) , 6.60 
 ( 1h , d , j = 2 hz ) , 6.70 ( 1h , m ) , 6.85 ( 1h , dd , j = 9 and 2 hz ) , 7.59 ( 1h , d , j = 9 hz ) , 
 8.40 ( 1h , m ) , 8.54 ( 1h , m ) , 8.72 ( 1h , s ) ; hresims m / z 914.5525 [ m + h ] ( calculated for 
 c53h76n3o10 , 914.5525 ) . pink solid in 
 44% yield ; h nmr ( cd3od ) ,  0.56 ( 2h , 
 t , j = 7 hz ) , 0.90 ( 4h , t , j = 6 
 hz ) , 0.95 ( 3h , d , j = 7 hz ) , 1.04 ( 3h , s ) , 1.08 ( 3h , 
 s ) , 1.15 ( 4h , m ) , 1.23 ( 2h , m ) , 1.49 ( 3h , m ) , 1.63 ( 5h , m ) , 1.77 ( 5h , 
 m ) , 1.82 ( 5h , s ) , 1.89 ( 5h , m ) , 1.97 ( 3h , s ) , 2.02 ( 3h , m ) , 2.08 ( 4h , 
 t ) , 2.22 ( 2h , m ) , 2.29 ( 1h , m ) , 2.37 ( 1h , m ) , 2.63 ( 4h , m ) , 3.05 ( 2h , 
 s ) , 3.24 ( 2h , d , j = 9 hz ) , 3.50 ( 8h , m ) , 3.71 ( 1h , 
 m ) , 3.96 ( 1h , m ) , 4.06 ( 1h , m ) , 4.99 ( 1h , m ) , 5.09 ( 1h , d , j = 11 hz ) , 5.19 ( 1h , d , j = 16 hz ) , 5.46 
 ( 1h , m ) , 5.86 ( 1h , m ) , 6.04 ( 1h , m ) , 6.19 ( 1h , m ) , 6.55 ( 1h , s ) , 6.69 
 ( 1h , m ) , 7.43 ( 1h , d , j = 7 hz ) , 8.03 ( 1h , d , j = 7 hz ) , 8.12 ( 1h , d , j = 7 hz ) , 8.66 
 ( 1h , s ) ; hresims m / z 1281.5838 [ m 
 + h ] ( calculated for c70h90n4o13s2na , 1281.5838 ) .
green solid in 
 64% yield ; h nmr ( cd3od ) ,  0.96 ( 3h , 
 d , j = 7 hz ) , 1.14 ( 3h , d , j = 6 
 hz ) , 1.22 ( 9h , s ) , 1.29 ( 3h , s ) , 1.42 ( 2h , m ) , 1.47 ( 2h , m , 1.54 ( 2h , 
 m ) , 1.66 ( 3h , m ) , 1.76 ( 3h , m ) , 1.83 ( 3h , m ) , 1.88 ( 3h , s ) , 1.92 ( 2h , 
 m ) , 2.01 ( 1h , s ) , 2.03 ( 2h , s ) , 2.07 ( 1h , m ) , 2.29 ( 5h , m ) , 2.52 ( 3h , 
 s ) , 2.69 ( 1h , t , j = 8 hz ) , 3.06 ( 1h , t , j = 8 hz ) , 3.12 ( 1h , m ) , 3.18 ( 1h , dd , j = 10 and 3 hz ) , 3.27 ( 2h , m ) , 3.35 ( 2h , s ) , 3.37 ( 1h , t ) , 3.46 ( 1h , 
 dd , j = 12 and 4 hz ) , 3.50 ( 1h , m ) , 3.62 ( 1h , m ) , 
 3.74 ( 2h , m ) , 3.86 ( 2h , m ) , 3.95 ( 1h , m ) , 4.08 ( 1h , d , j = 3 hz ) , 5.87 ( 0.3h , t , j = 7 hz ) , 5.92 ( 0.7h , 
 t , j = 7 hz ) , 6.23 ( 1h , m ) , 6.35 ( 2h , m ) , 7.00 ( 1h , 
 d , j = 4 hz ) , 7.41 ( 0.3h , s ) , 7.43 ( 0.7h , s ) , 8.04 
 ( 0.3h , d , j = 10 hz ) , 8.21 ( 0.7h , d , j = 10 hz ) ; hresims m / z 945.5728 
 [ m + h ] ( calculated for c53h76bf2n4o8 , 945.5719 ) .
white solid in 41% 
 yield ; h nmr ( cd3od ) ,  0.97 ( 3h , d , j = 7 hz ) , 1.05 ( 3h , s ) , 1.13 ( 3h , s ) , 1.20 ( 3h , s ) , 1.29 
 ( 2h , s ) , 1.37 ( 3h , m ) , 1.51 ( 4h , m ) , 1.64 ( 5h , m ) , 1.77 ( 8h , m ) , 1.88 
 ( 5h , m ) , 2.05 ( 5h , m ) , 2.16 ( 1h , m ) , 2.26 ( 4h , m ) , 2.38 ( 1h , m ) , 2.69 
 ( 1h , m ) , 2.92 ( 1h , dd , j = 13 and 5 hz ) , 3.25 ( 3h , 
 m ) , 3.34 ( 3h , m ) , 3.55 ( 1h , m ) , 3.72 ( 1h , m ) , 3.98 ( 1h , t , j = 3 hz ) , 4.08 ( 1h , m ) , 4.31 ( 1h , dd , j = 8 and 5 hz ) , 4.49 ( 1h , dd , j = 8 and 5 hz ) , 5.09 
 ( 1h , d , j = 11 hz ) , 5.19 ( 1h , d , j = 17 hz ) , 5.45 ( 1h , m ) , 5.90 ( 1h , m ) , 6.04 ( 1h , t , j = 11 hz ) , 6.34 ( 1h , d , j = 10 hz ) , 6.68 ( 0.4h , 
 t , j = 9 hz ) , 6.72 ( 0.6h , t , j = 
 9 hz ) , 8.06 ( 0.4h , d , j = 10 hz ) , 8.22 ( 0.6h , d , j = 10 hz ) ; hresims m / z 897.5406 [ m + h ] ( calculated for c49h77n4o9s , 897.5406 ) . synaptosomes were prepared 
 as previously described .
binding of [ h]-brevenol to rat brain 
 synaptosomes was measured independently using a rapid centrifugation 
 technique as previously described .
all buffers , 
 reagents , and plasticware were at ice temperature throughout each 
 experiment except during centrifugation .
all experiments were performed 
 in standard binding medium with 1 mg / ml bsa without detergent . binding of the fluorescent 
 brevenal conjugates was determined on rat brain synaptosomes using 
 a rapid filtration technique as described previously .
all buffers , reagents , and plasticware were at ice temperature 
 throughout each experiment .
all experiments were performed in standard 
 binding medium with 1 mg / ml bsa and 0.02% alkamuls detergent .
saturation binding experiments and corresponding calculation of equilibrium 
 dissociation constants ( kd ) were conducted 
 and statistically analyzed as previously described , with the exception that the fluorescent ligands in these 
 experiments were brevenal conjugates . briefly , a 96-well assay plate 
 was prepared in which 50 g ( 50 l at 1 mg / ml ) of synaptosomes 
 was suspended in assay buffer plus 5 l of etoh ( to determine 
 total binding ) or 5 l of etoh containing 10 m brevenal 
 ( to determine nonspecific binding ) .
this concentration of unlabeled 
 brevenal was sufficient to displace the fluorescent brevenal ligands 
 bound to the receptors , leaving the nonspecifically bound molecules 
 on synaptosomes .
stock solutions of 500 nm bodipy fl - brevenal , coumarin - brevenal , 
 or texas red - brevenal were prepared in assay buffer and serially diluted 
 ( 1:2 ) in the wells until the final concentration was below 1 nm .
the inhibition of binding of the fluorescent ligands was determined 
 in the presence of various competitors ranging in concentration from 
 1  10 to 1  10 m. serial dilutions ( 1:10 ) of unlabeled brevenal ( control ) , biotinylated 
 brevenal , natural brevetoxin derivatives ( pbtx-1 , pbtx-2 , pbtx-3 , 
 and pbtx-9 ) , veratridine , aconitine , and deltamethrin were prepared 
 in etoh .
serial dilutions ( 1:10 ) of tetrodotoxin and scorpion venom 
 were prepared in pbs .
the appropriate concentration of the competitor 
 solution was added ( 5 l ) to each well with 50 g of synaptosomes , 
 the fluorescent ligand ( 10 nm final concentration bodipy fl - brevenal 
 and texas red - brevenal , 100 nm final concentration of coumarin - brevenal ) , 
 and assay buffer .
competition binding experiments and the calculation 
 of equilibrium inhibition constants ( ki ) were then conducted as previously described .
data were normalized to 100% total binding so as to compare 
 the differences in binding curves and/or inhibition constants among 
 the three fluorescent brevenals ( each which yielded data in varying 
 ranges of relative fluorescent units ) and to the radioligand assay 
 ( which yielded data in counts of radioactivity per minute ) .
rat brain protein was isolated 
 into cytosolic , membrane , nuclear , and cytoskeletal fractions using 
 a compartmental protein extraction kit ( millipore ) .
protein content 
 of each fraction was assayed using a modified lowry protein assay 
 ( bio - rad ) .
fractions were treated with the biotin - brevenal conjugate 
 at a concentration of 510 g / mg of protein for over 
 1 h on ice , followed by 1 mg of myone streptavidin t1 dynabeads ( invitrogen 
 life technologies ) for 30 min to equilibrate binding . bound protein 

was removed with a magnet and then denatured via boiling and the addition 
 of laemmli sample buffer ( bio - rad ) .
brevenal 
 receptor protein samples were electrophoresed with sds - page running 
 buffer ( 25 mm tris base , 190 mm glycine , 0.1% sds ) on 7.5% mini - protean 
 tgx polyacrylamide gels ( bio - rad ) .
gels were stained with a coomassie 
 brilliant blue r-250 40% meoh solution ( bio - rad ) for 12 h 
 and then destained with a 40% meoh/10% glacial acetic acid solution 
 for 24 h. for western blot analysis , electrophoresed samples 
 were transferred to immobilon - p transfer membranes ( millipore ) using 
 transfer buffer ( 20 mm tris base , 150 mm glycine , 25% methanol ) .
membranes 
 were reacted with an affinity purified primary rabbit anti - pan voltage 
 gated sodium channel polyclonal antibody ( millipore ) .
the anti - pan 
 antibody was chosen because it recognizes a conserved region among 
 various vssc alpha subunits so as to detect any that might be present 
 in the sample .
blots were washed with pbs - tween and incubated in the 
 presence of a goat anti - rabbit igg antibody conjugated to horseradish 
 peroxidase ( millipore ) .
bound enzyme was detected with the super signal 
 west pico chemiluminescence substrate system ( thermofisher scientific ) . 

blots were exposed to kodak biomax light film ( carestream health ) 
 for 5 min and developed .
ki values determined in the fluorescent and radioligand 
 assays for 
 brevenal and biotin - brevenal were compared using student s t - test sas v9.1.3 software ( sas institute inc . ) .
results 
 are presented as the mean  sem and were considered statistically 
 significant if a p - value of less than 0.05 was obtained .
 the marine dinoflagellate karenia brevis produces 
 a family of neurotoxins known as brevetoxins . 
 brevetoxins elicit their 
 effects by binding to and activating voltage - sensitive sodium channels 
 ( vsscs ) in cell membranes . 
 k. brevis also produces 
 brevenal , a brevetoxin antagonist , which is able to inhibit and/or 
 negate many of the detrimental effects of brevetoxins . 
 brevenal binding 
 to vsscs has yet to be fully characterized , in part due to the difficulty 
 and expense of current techniques . in this study , we have developed 
 a novel fluorescence binding assay for the brevenal binding site . 
 
 several fluorescent compounds were conjugated to brevenal to assess 
 their effects on brevenal binding . 
 the assay was validated against 
 the radioligand assay for the brevenal binding site and yielded comparable 
 equilibrium inhibition constants . 
 the fluorescence - based assay was 
 shown to be quicker and far less expensive and did not generate radioactive 
 waste or need facilities for handling radioactive materials . 
 in - depth 
 studies using the brevenal conjugates showed that , while brevenal 
 conjugates do bind to a binding site in the vssc protein complex , 
 they are not displaced by known vssc site specific ligands . as such , 
 brevenal elicits its action through a novel mechanism and/or currently 
 unknown receptor site on vsscs . 
Results and Discussion
Experimental Section